Copyright International Rice Research Institute 2003

Transcription

1 The International Rice Research Institute (IRRI) was established in 1960 by the Ford and Rockefeller Foundations with the help and approval of the Government of the Philippines. Today IRRI is one of 16 nonprofit international research centers supported by the Consultative Group on International Agricultural Research (CGIAR IRRI receives support from several CGIAR members, including the World Bank, European Union, Asian Development Bank, International Fund for Agricultural Development, Rockefeller Foundation, and agencies of the following governments: Australia, Belgium, Canada, People s Republic of China, Denmark, France, Germany, India, Islamic Republic of Iran, Japan, Republic of Korea, The Netherlands, Norway, Philippines, Portugal, Sweden, Switzerland, Thailand, United Kingdom, United States, and Vietnam. The responsibility for this publication rests with the International Rice Research Institute. Copyright International Rice Research Institute 2003 Mailing address: DAPO Box 7777, Metro Manila, Philippines Phone: +63 (2) , , to 53 Fax: +63 (2) , , irri@cgiar.org Home page: Riceweb: Riceworld: Courier address: Suite 1009, Pacific Bank Building 6776 Ayala Avenue, Makati City, Philippines Tel. (63-2) , , , Suggested citation: Virmani SS, Mao CX, Hardy B, editors Hybrid rice for food security, poverty alleviation, and environmental protection. Proceedings of the 4th International Symposium on Hybrid Rice, Hanoi, Vietnam, May Los Baños (Philippines): International Rice Research Institute. 407 p. Cover design: Juan Lazaro IV Print production coordinator: George R. Reyes Layout and design: Ariel Paelmo Figures and illustrations: Ariel Paelmo Cover photo: Rody Toledo (Photo shows hybrid rice in Vietnam.) ISBN

2 Contents FOREWORD v Innovations in breeding methodologies Recent progress in breeding super hybrid rice in China 3 L.P. Yuan Advances in hybrid rice research and development in the tropics 7 S.S. Virmani Breeding strategies to enhance heterosis in rice 21 P.S. Virk, G.S. Khush, and S.S. Virmani Two-line hybrid rice breeding in and outside China 31 Tong-Min Mou, Lu Xing-Gui, N.T. Hoan, and S.S. Virmani Opportunities for and challenges to developing and using hybrid rice technology 53 for temperate countries T. Tsuchiya, A. Bastawisi, Z.Y. Yang, H.P. Moon, J.A. Mann, and H. Ikehashi Improving grain quality in hybrid rice 69 F.U. Zaman, B.C. Viraktamath, and S.S. Virmani Technological refinement in hybrid seed production Opportunities for and challenges to improving hybrid rice seed yield and seed purity 85 C.X. Mao and S.S. Virmani Hybrid rice for mechanized agriculture 97 M. Walton Biotechnological tools in hybrid breeding and seed production Improving hybrid rice through anther culture and transgenic approaches 105 S. Balachandran, G. Chandel, M.F. Alam, J. Tu, S.S. Virmani, K. Datta, and S.K. Datta Advances in understanding the genetic basis of heterosis in rice 119 Qifa Zhang and Zhikang Li Molecular approaches for fixing the heterozygosity of hybrid rice 135 Xuezhi Bi, J. de Palma, R. Oane, G.S. Khush, and J. Bennett iii

3 Management of hybrid rice Physiological bases of heterosis and crop management strategies 153 for hybrid rice in the tropics S. Peng, J. Yang, R.C. Laza, A.L. Sanico, R.M. Visperas, and T.T. Son Technological dissemination strategies Public, private, and NGO-sector partnership for developing and 173 promoting hybrid rice technology M. Ilyas-Ahmed, Ish Kumar, B.C. Viraktamath, J.S. Sindhu, and Y. Yogeswara Rao Policy and institutional support to expedite the development and use 191 of hybrid rice technology M.A. Sombilla, B. Ba Bong, B. Mishra, L.S. Sebastian, and S.B. Siddique International Task Force on Hybrid Rice 209 S.S. Virmani, Dat Van Tran, and J.S. Sindhu Hybrid rice: how to go forward? 219 Dat Van Tran Country reports Hybrid rice research and development in Bangladesh 235 A.W. Julfiquar, M. Jamil Hasan, A.K. Azad, M. Anwar Hossain, and S.S. Virmani Hybrid rice achievements and development in China 247 Ma Guohui and Yuan Longping Hybrid rice research and development in Egypt 257 A.O. Bastawisi, H.F. El-Mowafi, M.I. Abo Yousef, A.E. Draz, I.R. Aidy, M.A. Maximos, and A.T. Badawi Hybrid rice development and use in India 265 B. Mishra, B.C. Viraktamath, M. Ilyas Ahmed, M.S. Ramesha, and C.H.M. Vijayakumar Hybrid rice research and development in Indonesia 287 Suwarno, N.W. Nuswantoro, Y.P. Munarso, and M. Direja Hybrid rice research and development in the Democratic People s Republic of Korea 297 Ri Tae Sik and Rim Yun Uk Hybrid rice research and current status in Korea 313 S.J. Yang, Y.C. Song, and H.P. Moon Hybrid rice breeding in Russia 321 I.K. Gontcharova and S.V. Gontcharov Hybrid rice research and development in Myanmar 329 Khin Than Nwe, Myint Yee, Hmwe Hmwe, Myint Aung, and Aye Aye Myint Hybrid rice research and development in Japan 337 T. Takita Hybrid rice research and development in Sri Lanka 341 S.W. Abeysekera, S.N. Jayawardena, K.D.S. Kiriwaththuduwage, and D.S. de Z. Abeysiriwardena Hybrid rice development and use in Vietnam 357 Nguyen Tri Hoan and Nguyen Huu Nghia Hybrid rice technology and achievements in Iran 373 G.A. Nematzadeh, M. Sattari, A. Valizadeh, A. Alinejad, and M.Z. Nori Hybrid rice development and use in the Philippines, E.D. Redoña, F.M. Malabanan, M.G. Gaspar, J.C. de Leon, and L.S. Sebastian iv

4 Observations and recommendations 403 v

5 Foreword Hybrid rice technology was successfully developed in China during Since then, it has been used on 50% of the rice area (30 million ha) in China and elsewhere it is being developed in about 20 countries worldwide. About 800,000 ha are now covered with rice hybrids in Vietnam, India, the Philippines, Bangladesh, Indonesia, Myanmar, and the United States. This technology enables farmers to produce more rice per unit area per unit time and contributes to increased grain yields, farmers income, and rural job opportunities through hybrid seed production. It has already contributed significantly toward improving food security and environmental protection in China and its prospects outside China are good. Progress made in China and elsewhere until 1996 in the development and dissemination of hybrid rice technology was published in 1988, 1994, and 1998 in the proceedings of the hybrid rice symposia held in China (1986), at IRRI (1992), and in India (1996), respectively. Progress made on the subject since 1996 was presented and discussed at the 4th International Symposium on Hybrid Rice held in Hanoi, Vietnam (14-17 May 2002), which was attended by 187 participants from 19 countries and three international agencies (IRRI, the Food and Agriculture Organization of the United Nations, and the Asia-Pacific Seed Association). Selected papers covering important research areas (breeding methodologies, biotechnological applications, seed production, agronomic management, and technology dissemination of hybrid rice) along with country reports appear in this book. The Hanoi symposium was cosponsored by IRRI; the Ministry of Agriculture and Rural Development, Government of Vietnam; FAO; and the China National Hybrid Rice Research and Development Center. The major source of funds for the symposium was the IRRI-ADB project Sustaining Food Security in Asia through the Development of Hybrid Rice Technology. Additional financial support in cash or in kind was provided by RiceTec Inc. (USA), S.M. Sehgal Family Foundation (New Delhi, India), Xangfan Chia Tai Agriculture Development Company Ltd. (China), ICAR-UNDP Hybrid Rice Project (India), and the PETRRA Subproject on Hybrid Rice in Bangladesh. We are grateful to all these agencies for their support for holding vii

6 the symposium and publishing this book. We hope that this will become a valuable source of information for hybrid rice researchers, seed production and technology transfer agencies, and graduate students in plant breeding and seed technology. RONALD P. CANTRELL Director General International Rice Research Institute viii

7 Innovations in breeding methodologies

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9 Recent progress in breeding super hybrid rice in China L.P. Yuan China s current population is near 1.3 billion, with less than 0.1 ha of arable land for each person. This population is expected to reach 1.6 billion and crop land will decrease to about 0.07 ha per capita. Because of this population growth pressure and reduction in arable land, to feed all the Chinese people in the new century, a super rice breeding program was set up by China s Ministry of Agriculture in 1996, with the yield targets for hybrid rice listed in Table 1. With morphological improvement plus the use of intersubspecific (indica/ japonica) heterosis, several pioneer two-line super hybrid rice varieties had been developed by 2000, which attained the phase I yield standard of single-season rice. There were more than 20 demonstration locations with 6.7 ha or 67 ha each, where their average yield was more than 10.5 t ha 1 in The average yield was 9.6 t ha 1 in commercial production (235,000 ha) in 2000 and 9.2 t ha 1 (1.2 million ha) in (The average rice yield has been 6.3 t ha 1 nationwide recently.) One combination, P64S/E32, had a record yield of 17.1 t ha 1 in an experimental plot (720 m 2 ) in Efforts now focus on breeding phase II super hybrid rice and good progress is being made. Three two-line indica/japonica hybrid combinations outyielded the check (CK, the pioneer super hybrid rice variety) by 6 18% in replicated trials at our center in A three-line indica/japonica hybrid, II-32A/Ming 86, yielded t ha 1 at a Table 1. Yield standard of super rice in China. Hybrid rice a Phase % First Second Single increase cropping cropping season 1996 level Phase I ( ) More than 20% Phase II ( ) More than 40% a In t ha 1 at 2 locations with 6.7 ha each in 2 consecutive years. 3

10 demonstration location with 7 ha in Fujian Province in 2001, and it also produced a new record yield (17.95 t ha 1 ) in an experimental plot (800 m 2 ) in Yunnan Province in Based on progress in 2001, some promising combinations were prepared for demonstration at multiple locations with 7 8 ha each in Among them, the best one is P88S/0293, which yielded 12.3 t ha 1 on average in Longshan County, Hunan. This combination yielded 12.4 t ha 1 in Hainan Province again in 2003 and had a record yield in the province. In addition, three combinations are performing very well in our experimental plots (plot size m 2 ) and their estimated yield is around 13 t ha 1. A breakthrough has been achieved in breeding the first cropping of super hybrid rice. A newly developed short-growth-duration two-line indica/japonica hybrid (HY-S/F49) was demonstrated near Changsha in The area under demonstration was 7 ha and its average yield was 9.1 t ha 1, which outyielded CK 1 (three-line intervarietal hybrid) and CK 2 (inbred variety) by 20% and 40%, respectively. There are two demonstration locations with 7 8 ha each in 2003 and their estimated yield is around 10 t ha 1. Technical approaches Crop improvement practices have indicated, up to now, that there are only two effective ways to increase the yield potential of crops through plant breeding, that is, morphological improvement and the use of heterosis. However, the potential is very limited when using morphological improvement alone and heterosis breeding will produce undesirable results if it is not combined with morphological improvement. Any other breeding approaches and methods, including high technology such as genetic engineering, must be incorporated into good morphological characters and strong heterosis; otherwise, there will be no actual contributions to a yield increase. On the other hand, the further development of plant breeding for a high yield target must rely on progress in biotechnology. Morphological improvement A good plant type is the foundation for super high yield. Since Dr. Donald proposed the concept of ideotype in 1968, many rice breeders have proposed models for super high-yielding rice. Among these is the new plant type proposed by Dr. Khush at IRRI. Its main features are (1) large panicles, with 250 spikelets per panicle; (2) fewer tillers, 3 4 productive tillers per plant; and (3) a short and sturdy culm. Whether this model can realize super high yield or not has yet to be proved. Based on our studies, especially inspired by the striking characteristics of the high-yielding combination P64S/E32, which has had a record yield of 17.1 t ha 1, we have found that the super high-yielding rice variety has the following morphological features: 4 Yuan

11 1. Tall erect-leaf canopy The upper three leaf blades should be long, erect, narrow, V-shaped, and thick. Long and erect leaves have a larger leaf area, can accept light on both sides, and will not shade each other. Therefore, light is used more efficiently. Narrow leaves occupy a relatively small space and thus allow a higher effective leaf area index. A V-shape makes the leaf blade stiffer so that it is not prone to be droopy. Thick leaves have a higher photosynthetic function and are not easily senescent. These morphological features signify a large source of the assimilates that are essential to super high yield. 2. Lower panicle position The tip of the panicle is only cm above the ground during the ripening stage. Because the plant s center of gravity is quite low, this architecture enables the plant to be highly resistant to lodging. Lodging resistance is also one of the essential characters required for breeding a super high-yielding rice variety. 3. Bigger panicle size Grain weight per panicle is around 5 g and the number of panicles is about 300 m 2. Theoretically, yield potential is 15 t ha 1. Grain yield = biomass harvest index. Nowadays, the harvest index (HI) is very high (above 0.5). A further raising of the rice yield ceiling should rely on increasing biomass because further improvement of the HI is limited. From the viewpoint of morphology, to increase plant height is an effective and feasible way to increase biomass. However, this approach will cause lodging. To solve this problem, many breeders are trying to make the stem thicker and sturdier, but this approach usually results in a decrease in HI. Therefore, it is difficult to obtain a super high yield in this way. The plant model of a taller canopy can combine the advantages of a higher biomass, higher HI, and higher resistance to lodging. Raising the level of heterosis Heterosis in rice has the following general trend indica/japonica > indica/javanica > japonica/javanica > indica/indica > japonica/japonica according to our studies. Indica/japonica hybrids possess a very large sink and rich source, the yield potential of which is 30% higher than that of intervarietal indica hybrids being used commercially. Therefore, efforts have focused on using indica/japonica heterosis to develop super hybrid rice. However, many problems exist in indica/japonica hybrids, especially their very low seed set, which must be solved to use their heterosis. With wide compatibility (WC) genes and using intermediate-type lines as parents instead of typical indica or japonica lines, several intersubspecific hybrid varieties with stronger heterosis and normal seed set have been successfully developed. Biotechnology This is another important approach for developing super hybrid rice. So far, two very promising results have been obtained in this research area. Recent progress in breeding super hybrid rice in China 5

12 Prospects 1. The use of favorable genes from wild rice Based on molecular analysis and field experiments, two yield-enhancing QTLs from wild rice (Oryza rufipogon L.) were identified. Each of the QTLs contributed to a yield advantage of 18% over the high-yielding check hybrid Weiyou64 (one of the most elite hybrids). By means of molecular markerassisted backcrosses and field selection, an excellent R line (Q611) carrying one of these QTLs was developed. Its hybrid, J23A/Q611, outyielded the check hybrid by 35% in a replicated trial for the second rice crop in Its yield potential on a large scale is now being evaluated. Preliminary data show that its estimated yield is 13 t ha 1 in experimental plots and 11 t ha 1 in farmers fields planted as a second rice crop. 2. Using genomic DNA from barnyardgrass (Echinochloa crus-galli) to create a new source of rice The total DNA of barnyardgrass was introduced into a restoring line (R207) by the spike-stalk injection method and variants occurred in the D 1. From these variants, new elite stable R lines have been developed. The most outstanding one is RB207-1, and its agronomic characters such as number of spikelets per panicle and grain weight are much better than those of the original R207. Particularly, its hybrid, GD S/RB207-1, has a good plant type and very strong heterosis. Its estimated yield was more than 15 t ha 1 in our experimental plot in The yield standard of phase II super rice (12 t ha 1 ) can be achieved by By reaching this target, 2.25 t ha 1 more rice can be produced, which will increase grain by 30 metric tons annually and feed 75 million more people when it is commercialized up to 13 million ha. The development of science and technology will never stop. Rice still has a large yield potential, which can be exploited by advanced biotechnology. It was exciting to learn that C 4 genes from maize have been successfully cloned and transferred into the rice plant by the HK Chinese University. Using this transgenic plant as a donor to introduce C 4 genes into super hybrid rice parents is under way. If this approach is successful, the yield potential of rice could be further increased by a large margin. Relying on this progress, the phase III super hybrid rice breeding program is proposed, in which the yield target is 13.5 t ha 1 on a large scale by Notes Author s address: China National Hybrid Rice Research & Development Center. Citation: Virmani SS, Mao CX, Hardy B, editors Hybrid rice for food security, poverty alleviation, and environmental protection. Proceedings of the 4th International Symposium on Hybrid Rice, Hanoi, Vietnam, May Los Baños (Philippines): International Rice Research Institute. 407 p. 6 Yuan

13 Advances in hybrid rice research and development in the tropics S.S. Virmani During the past five years, hybrid rice technology in the tropics has entered the commercialization phase in India, Vietnam, the Philippines, Bangladesh, and Indonesia. Almost all the commercial rice hybrids are derived from the cytoplasmic male sterile (CMS) system. An IRRI CMS line, IR58025A, has been widely used in the tropics. Even in China, this line has been used in Jiangxi and Hunan provinces to improve the grain quality of local hybrids. IRRI s hybrid rice breeding program has focused on developing improved CMS lines possessing cytoplasmic and genetic diversity, improved grain quality, a higher outcrossing rate, and resistance to diseases and insects. Most widely used wild abortive cytoplasm has been analyzed for its effect on grain quality and resistance to/tolerance of biotic and abiotic stresses. There was no negative effect on the traits except for grain chalkiness, which needs to be studied further. As reported earlier, there was no lack of restorer lines among elite indica lines bred in the tropics. Marker-aided selection (MAS) using the sequence-tagged site marker RG140 with PvuII digestion linked with the Rf3 gene on chromosome 1 was useful for increasing screening efficiency for restorers. Progress was also made in developing stable TGMS lines possessing a low critical sterility point and good outcrossing. Several public and private hybrids were released in national agricultural research and extension systems and new elite hybrids were identified. Average seed yields were further improved in the national programs by further fine-tuning of the seed production technology, training, the choice of appropriate locations/seasons, and, above all, the experience of seed growers. A significant increase occurred in private-sector participation in the hybrid rice seed industry during the past five years. About 40 private companies are actively involved in hybrid rice research and/or seed production and marketing in tropical Asia. During 2001, private companies were producing 90% of the hybrid rice seeds in India. Hybrid rice in the tropics covered about 770,000 ha in Major constraints identified during the commercialization phase of the technology in the tropics were inconsistent performance, inferior grain quality, an inadequate level of disease/insect resistance of hybrids, the inadequate supply and high cost of hybrid seeds, and inadequate policy support. Future opportunities include the development of hybrids possessing stronger heterosis and more stable yield performance, improved grain quality, an adequate level of dis- 7

14 ease/insect resistance, and higher seed yields. Both conventional and molecular methods will be used in hybrid breeding and in assessing seed purity. Agronomic management of hybrid rice in the tropics will also be improved to improve hybrid yield and stability in performance. Hybrid rice, which made a significant impact in China in the 20th century, should contribute significantly to food security and environmental protection in the tropical rice-growing countries in the 21st century. Tropical rice hybrids have been bred at IRRI and elsewhere for the past 20 years. The rationale for their development is to increase rice productivity per unit area per unit time beyond the level of semidwarf high-yielding inbred rice varieties (HVYs) to meet the future challenges of food security and environmental protection in the major rice-growing countries in the tropics. This technology also involves active participation of the seed industry for production, processing, and marketing of hybrid rice seeds, which is skill-oriented and labor-intensive, but more profitable than inbred rice cultivation and seed production. Therefore, promotion of hybrid rice technology also helps to promote the seed industry (in the public, private, and/or NGO sector), which, in turn, creates rural employment opportunities. The progress made over the years in developing and using hybrid rice technology for the tropics has been published from time to time (Yuan and Virmani 1988, Virmani 1994, 1998). Commercialization of hybrid rice technology in the tropics began in 1994, after which some public and private rice hybrids were released in India, Vietnam, the Philippines, Bangladesh, and Indonesia (Table 1). IRRI-bred rice parental lines and hybrids played a key role in the release of these hybrids. In the Red River Delta of Vietnam, some Chinese rice hybrids (such as Nhi You 838, Shan You 63, Bo You 63, etc.) were adaptable though they possessed inferior grain quality. These were commercialized with the help of the government, which facilitated the purchase and distribution of hybrid seeds by Vietnamese seed companies in collaboration with some Chinese seed companies. IRRI s contributions in Vietnam have been primarily in the area of strengthening national capabilities to breed locally adaptable hybrids with good grain quality and to produce hybrid seeds locally. The yield advantage of some of the released rice hybrids over the best inbred HYVs in on-farm trials in these countries (Table 2) indicates their yield superiority, ranging from 20% to 30% (mean 25%). In many of the commercial tropical rice hybrids, IRRI-bred CMS line IR58025A has been extensively used. Even in China, this line has been used in Hunan and Jiangxi provinces to improve the grain quality of local rice hybrids. In 2002, estimated hybrid rice area in India (275,000 ha), Vietnam (450,000 ha), the Philippines (30,000 ha), Bangladesh (10,000 ha), and Indonesia (5,000 ha) totaled 770,000 ha. The major constraints experienced during the commercialization of the first set of released hybrids in the five tropical countries were (1) inferior grain quality in some countries, (2) inconsistent performance, (3) inadequate level of disease/insect 8 Virmani

15 Table 1. Hybrids released for commercial cultivation in some tropical countries. Name of hybrid Country of release Year of release APHR-1 India 1994 APHR-2 India 1994 MGR-1 India 1994 KRH-1 India 1994 CNRH-3 India 1995 DRRH-1 India 1996 KRH-2 India 1996 Pant Sankar Dhan India 1997 PHB-71 India 1997 ADTRH-1 India 1998 CORH-2 India 1998 Narendra Sankar Dhan-2 India 1998 Sahyadri India 1998 Magat Philippines 1994 Mestizo Philippines 1997 Panay Philippines 1999 HYT-57 Vietnam 1999 Progro 6201 India 2000 BRRI Dhan Hybrid 1 Bangladesh 2001 Pusa RH 10 a India 2001 HRI 120 (6444) India 2001 RH 204 India 2001 Intani 1 (by private sector) Indonesia 2001 Intani 2 (by private sector) Indonesia 2001 Rokan (by public sector) Indonesia 2002 Maro (by public sector) Indonesia 2002 Mestizo 2 Philippines 2002 Mestizo 3 Philippines 2002 a First rice hybrid possessing basmati grain. Table 2. Yield gains of hybrids released for cultivation in four Asian countries. Country Hybrid Inbred Yield gain Yield gain (t ha 1 ) (t ha 1 ) (t ha 1 ) (%) Bangladesh a India b Philippines c Vietnam Mean a Hybrid Sonar Bangla over inbred check on sample farms; boro season (internal report, IRRI, 2001). b Hybrid Sahyadri over best inbred check (Sasyasree) across 15 locations, multilocation trials, rabi (Directorate of Rice Research, India, 2000). c Hybrid Mestizo over best inbred check across five locations, on-farm compact technology demonstration, 1998 dry season (PhilRice, Philippines, 2001). Advances in hybrid rice research and development in the tropics 9

16 resistance, (4) inadequate supply of good-quality hybrid seed, (5) high cost of seed, and (6) inadequate policy support by the government. These constraints are being tackled by the groups responsible for technology development, seed production, and technology transfer of hybrid rice. Recent advances made in tackling these challenges are presented in this paper. Developing CMS lines possessing improved grain quality and a high outcrossing rate The major complaint against the first set of released and commercialized rice hybrids, in some countries, was their stickiness and aroma inherited from CMS line IR58025A (which possesses low amylose content and mild aroma). Recently, we have developed new CMS lines at IRRI possessing diverse cytoplasm and good outcrossing (Table 3), which have intermediate to high amylose for developing nonsticky indica rice hybrids. These lines range from nonaromatic to strongly aromatic to satisfy varying aroma requirements in different countries. Considering the need to increase hybrid seed yields and reduce the seed cost, we have been selecting new CMS lines possessing a higher outcrossing rate than the widely used CMS line IR58025A in the tropics. We have developed some new CMS lines (Table 4) that have a higher outcrossing rate than IR58025A. A male sterility facilitated composite population is also being developed at IRRI (Virmani et al, unpublished) to extract parental lines possessing higher outcrossing potential. Parental lines with resistance to biotic stresses Resistance of the hybrids to biotic stresses is primarily determined by the biotic resistance of their parental lines. Therefore, CMS and restorer lines possessing resistance to major biotic stresses (such as bacterial blight BB, brown planthopper BPH, green leafhopper GLH, tungro virus) are being developed at IRRI. Since a hybrid is always heterozygous, it is critical that we identify and deploy dominant genes for resistance. Such genes are available for BB, BPH, and GLH resistance and elite lines carrying these genes should be used to develop heterotic hybrids. IRRI is also developing transgenic Bt and Xa21 parental lines (Alam et al 1999) for transgenic rice hybrids possessing resistance to stem borer and BB. Concerted efforts are being made at IRRI to develop and use CMS lines possessing diverse CMS systems to overcome the potential genetic vulnerability of the widely used wild abortive (WA) CMS system for hybrid rice breeding. Progress made during in increasing cytoplasmic diversity among elite CMS lines bred at IRRI (Fig. 1) is encouraging. We have also developed alloplasmic elite CMS lines (Table 5) possessing different CMS sources in the genetic background of widely used maintainer lines to deal with a situation when the commercially used WA-CMS system becomes vulnerable to a biotic stress. The WA cytoplasm showed a nonsignificant effect on resistance to/tolerance of biotic and abiotic stresses (Faiz 2000, 10 Virmani

17 Table 3. Recently developed IRRI CMS lines that have intermediate to high amylose. a CMS Cyto. PS PHA OCS Mat. Ht. Grain % % % L Sh. Clk. GT % Aroma line (cm) type BR TMR HR amy. IR69625A WA Bold I 22.9 Moderate IR77805A Gambiaca Medium, L 24.5 None bold IR77290A WA Long , bs 0 I 25.6 Strong slender (basmati) IR73328A Mutagenized Medium L 22.5 Moderate IR62829B IR78371A WA Long L 26.6 Slight slender IR78372A ARC Long , bs 1 L 26.2 Slight IR79156A WA Long I 26.0 Slight IR58025A WA Long L 14.8 Strong slender a Cyto. = source of cytoplasm; PS = pollen sterility %; PHA = phenotypic acceptability; OCS = outcrossing rate; Mat. = maturity (days); BR = brown rice; TMR = total milled rice; HR = head rice; L = length; Sh. = shape; Clk. = chalkiness; GT = gelatinization temperature; Amy. = amylose content. Advances in hybrid rice research and development in the tropics 11

18 Table 4. CMS lines possessing a higher outcrossing score than the commercially used CMS line IR58025A. a CMS line Source of PS (%) PHA OCS Flow. Ht. (cm) Grain type CLK Amy. (%) Aroma cytoplasm IR79156A WA Long Slight IR80152A Mutagenized IR62829B Medium Slight IR80153A Mutagenized IR62829B Long None IR80154A Gambiaca Medium Slight IR80155A Kalinga Long slender None IR78369A ARC Long None IR78378A WA Long slender (basmati type) Strong IR79128A WA Medium Moderate IR80151A WA Long Slight IR80156A Kalinga Medium None IR80157A Mutagenized IR62829B Long slender Strong IR80158A Kalinga Medium bold Slight IR58025A WA Long slender Strong a PS = pollen sterility; PHA = phenotypic acceptability; OCS = outcrossing rate; Flow. = 50% flowering; CLK = chalkiness; Amy. = amylose content. 12 Virmani

19 ARC (5%) Mutagenized IR62829B (4%) Kalinga (2%) Dissi (15%) Gambiaca (15%) Mutagenized IR62829B WA (52%) (8%) WA (91%) ARC (8%) 1997 DS 2001 WS Fig. 1. Improvement in the cytoplasmic diversity of IRRI-bred CMS lines during Source: CMS maintenance nurseries, 1997 DS and 2001 WS. Table 5. Some alloplasmic CMS lines developed in the genetic background of widely used maintainer lines, IRRI, a Name of alloplasmic CMS line possessing cytoplasm Maintainer line Wild Gambiaca Dissi ARC Kalinga Mutagenized abortive IR62829B IR62829B IR62829A IR75595A IR68885A IR58025B IR58025A IR76767A IR80109A IR68897B IR68897A IR75601A IR75596A IR70369B IR70369A IR77805A IR77809A IR80110A Virmani et al 2001). These results indicate minimal risk in using the CMS system for hybrid rice breeding. Variation in restorer and maintainer frequency There was considerable variability in the frequency of restorers for different CMS lines and among elite lines from different sources (Eusebio et al 2002). The average frequency of restorers among elite indica rice cultivars developed in the tropics was 50%, but frequencies ranged from 0% for the CMS line IR66707A (Oryza rufipogon cytoplasm) to 67% for IR68280A, 72% for IR68897A (both WA cytoplasm), and 74% for IR73328A (IR62829B mutation cytoplasm). Elite lines from Thailand and India showed lower frequency (34% and 41%, respectively) of restorers than non- IRRI lines from the Philippines (66%) and Bangladesh (57%). Generally speaking, restorer frequency in indica (50%) and indica-tropical japonica or new plant type (NPT) (40%) lines was higher than in basmati (5%) and tropical japonica (0%) lines. Maintainer frequency was high (70%) among elite tropical japonica lines, intermediate (30%) in basmati lines, low (15%) among indica-tropical japonica derivative lines, and very low (5%) in indica lines. Advances in hybrid rice research and development in the tropics 13

20 1-kb ladder IR24 IR36 R R NR R NR NR NR IR58025A 1.25 kb 1.15 kb Fig. 2. PCR amplification products using the STS primers for the marker RG140 and RG140B (R) and digestion with PvuII. Restorers such as IR24 and IR36 exhibit a 1.25-kb band, whereas a 1:15-kb band is produced in nonrestorers such as IR58025A. We have successfully incorporated DNA marker-assisted selection (MAS) for WA fertility restoration in IRRI s hybrid rice breeding program. The major gene, designated as Rf3, underwent MAS using sequence-tagged site (STS) marker RG140 with PvuII digestion. Restorers were identified by a 1.25-kb band, while nonrestorers possessed a 1.15-kb band when resolved in an agarose gel (Fig. 2). About 450 lines were characterized for restorer/nonrestorer alleles. About 50% of the lines were identified as suspected restorers, which were then testcrossed with CMS lines. Up to 80% of the suspected restorer lines were confirmed as WA-CMS restorers. This demonstrated that MAS can be performed on the parental lines to increase our screening efficiency for restorers. Work is in progress at IRRI to tag other Rf genes and/or use MAS for Rf genes reported to be tagged by other researchers (Yao et al 1997, Tan et al 1998). Composite populations to extract maintainer and restorer lines IRRI plant breeders have been actively involved in developing male sterility facilitated composite populations to extract elite maintainer and restorer lines (Virmani 1994). Several maintainer and restorer lines (Table 6) have been extracted from these populations. The maintainer lines are being converted into CMS lines, whereas restorer lines are used to select heterotic rice hybrids. The composite populations have also been shared with some national programs at their request. These make a valuable genetic resource for national agricultural research and extension systems (NARES) for extracting locally adapted elite maintainer and restorer lines. 14 Virmani

21 Table 6. Maintainers and restorers extracted from male-sterility facilitated composite populations. Line Designation Remarks Maintainer IR B Converted to CMS line (IR80156A) having Kalinga cytoplasm IR B Converted to CMS line (IR79155A) having mutagenized IR62829B cytoplasm Restorer IR69701-C R Parent of hybrid tested in advanced yield trial, 2002 DS IR69702-C R Parent of hybrid tested in yield trial, 2002 DS IR69702-C R Parent of hybrid tested in yield trial, 2002 DS IR69702-C R Parent of hybrid tested in yield trial, 2002 DS Developing thermosensitive genic male sterile (TGMS) lines and two-line rice hybrids The TGMS system is an additional genetic tool to develop tropical rice hybrids. Since 1990, breeding of such lines has been an important activity in IRRI s hybrid rice breeding program. Initially, we developed some TGMS lines (such as IR68945S, IR68949S, and IR71018S) carrying the tms2 gene from the japonica TGMS mutant Norin PL12 from Japan (Maruyama et al 1991) and shared these with NARES. But these were found to be unstable because of their high critical sterility points (CSP). We had also induced a new mutant in indica rice (Virmani and Voc 1991), which was found to carry the tms3 gene (Borkakati and Virmani 1996). This mutant had a low CSP but fertility reversion was poor. Subsequently, by modifying our selection procedure (Sanchez et al 2002), we selected new TGMS lines (IR S, IR73834S, IR68301S, IR S, IR S) that have a low CSP and are stable over tropical environments. Use of these lines in numerous testcrosses resulted in a higher frequency (48%) of heterotic hybrids than in the CMS-derived testcrosses (16%) at IRRI. Some heterotic two-line hybrids for the tropics are in replicated yield trials at IRRI. We have also developed a nuclear male sterility facilitated composite population (Virmani et al, unpublished) to extract TGMS lines. This population could be a valuable source for NARES to extract locally adapted TGMS lines. Adaptability of rice hybrids to unfavorable conditions IRRI-Egypt collaborative trials on hybrid rice indicated that the standard heterosis of rice hybrids was significantly higher under saline soil conditions than under normal soil conditions in Egypt (Fig. 3). IRRI physiologists (A. Ismail, personal communication) have also observed that hybrids showed higher salinity tolerance than inbred lines. In India, Indo-American Seeds, a private company, has developed and marketed rice hybrids for saline soil conditions. Advances in hybrid rice research and development in the tropics 15

22 Standard heterosis (%) Normal (Sakha) Saline (El-Sinw) IR68888A/ Giza 178R IR69625A/ Giza 178R IR70368A/ Giza 178R IR69625A/ Giza 181R IR70368A/ Giza 181R IR68885A/ Giza 182R Hybrid IR68888A/ Giza 182R IR69625A/ Giza 182R IR69625A/ Giza 175R Fig. 3. Standard heterosis of some hybrids in saline and normal soils in Egypt, Mean IRRI scientists (George et al 2002, G. Atlin, personal communication) have also observed better adaptation of rice hybrids in aerobic rice cultivation conditions, in which rice is cultivated as a dryland crop without standing water and water is applied as needed to keep the soil moisture at saturated conditions. These observations have led us to start critically evaluating rice hybrids along with elite inbred rice varieties under aerobic conditions in which water-use efficiency is expected to be higher. It is interesting to note that hybrid rice shows higher heterosis (>30%) and is being accepted more readily by irrigated rice farmers in eastern Uttar Pradesh and Bihar, where yields are lower (4 5 t ha 1 ) than in Punjab, Haryana, and Andhra Pradesh, where irrigated rice yields are higher (5 7 t ha 1 ). These results indicate that prospects for hybrid rice need to be seriously explored in certain unfavorable rice-growing conditions where the seed rate is low. Hybrid rice seed production and participation of the seed industry Since 1994, when rice hybrids were commercialized in the tropics, hybrid rice seed has been produced primarily in national programs using the seed production guidelines provided by IRRI and China. Several in-country and international courses have been conducted at IRRI and in China to transfer this information to the NARES and seed industry. IRRI s hybrid rice training manual (Virmani and Sharma 1993) is now online ( and is also available on a CD to facilitate the spread of the hybrid rice seed production guidelines worldwide. 16 Virmani

23 Table 7. Number of public, private, and NGO enterprises involved in some Asian countries in Public Country Private NGOs seed Cooperatives, Total companies enterprises associations Bangladesh India Indonesia Philippines Sri Lanka 2 2 Vietnam Total Hybrid rice seed yields in NARES have averaged 0.75 to 1.5 t ha 1 and the highest seed yields ( t ha 1 ) have been reported from the tropical countries that have commercialized hybrid rice. As indicated earlier, IRRI s hybrid rice research also emphasizes developing parental lines with a higher outcrossing potential and improving management in hybrid seed production plots to increase hybrid seed yields. For this purpose, active collaboration with the seed industry is extremely useful. Over the years, the development of hybrid rice technology has attracted active participation of the seed industry to develop the hybrids and/or produce and market hybrid seeds. Currently, about 60 seed companies in the public, private, and NGO sectors are participating in this enterprise (Table 7). The public seed industry is active in Vietnam, while the private seed industry is more active in India, the Philippines, and Indonesia; some NGO-based seed companies (in Bangladesh and India) and some farmer cooperatives (in the Philippines) also participate actively. Collaboration with the seed industry has improved with the formation of a special interest group on hybrid rice under the Asia Pacific Seed Association (APSA). In this annual forum, issues related to the commercialization of hybrid rice technology are discussed. Future opportunities Hybrid rice researchers around the world are pursuing the development and use of this technology with greater enthusiasm. To date, there are more believers in this technology than nonbelievers. Outside China, 20 other countries are committed to developing and using rice hybrids since they believe that this technology would help increase their rice productivity, farmers income, food security, and environmental protection. Seed production and marketing opportunities associated with this technology have encouraged the seed industry (in the public, private, and NGO sector) to invest in the development and use of hybrid rice. From the technology development viewpoint, clear opportunities exist to further enhance heterosis in rice using NPT lines (developed in tropical japonica and Advances in hybrid rice research and development in the tropics 17

24 indica-tropical japonica backgrounds) in crosses with elite indica CMS and TGMS lines. Since the restorer frequency among NPT lines is lower than in indica lines, the two-line hybrid rice breeding system based on TGMS lines would be very useful. Heterosis can also be enhanced by identifying and using heterotic groups and heterotic gene blocks in rice. Grain quality of future rice hybrids can be improved to the desired level by using parental lines with the desired grain quality and critically evaluating the derived heterotic rice hybrids (in comparison with the check varieties) before their release for commercialization. Hybridity per se is not the cause of the poor grain quality of some of the hybrids commercialized so far. Like grain quality, disease/insect resistance of future rice hybrids can be improved by selecting suitable parental lines possessing the desired resistance genes. Biotechnological tools such as molecular markers and transformation techniques are already being used (Liu et al 2002, Alam et al 1999) and their use can be intensified to incorporate desired disease/insect resistance in hybrid rice. The viability of hybrid rice technology largely depends on hybrid rice seed yields in commercial seed production plots. Seed yields have been increasing with experience and the choice of a better location, season, and management of commercial seed production plots. The development of seed parents possessing an inherently higher outcrossing rate is a distinct possibility and progress is being made in this direction. With higher seed yields, the price of hybrid rice seeds will go down (Janaiah and Hossain 2001) and hybrid rice can become viable even in situations (e.g., direct seeding) where it is uneconomical now because of the high seed rate requirement. Experimental evidence has been collected on the adaptability of hybrid rice to unfavorable ecosystems, such as salinity-prone areas, water-limited aerobic rice, and moderately stressed rainfed lowland areas. The prospects for hybrid rice for those areas need serious exploration. Private-sector participation in rice research and development has increased in recent years primarily because of the development of hybrid rice technology. Rice hybrids also provide seed companies with a protection mechanism for their proprietary products (including some transgenics) in the developing countries where the enforcement of intellectual property rights is not yet satisfactory. The wider-scale adoption of hybrid rice technology would certainly increase private investment in rice research and development in the tropics. The promotion of any new technology requires policy support from a national government. Therefore, governments of the countries interested in developing and using hybrid rice should identify critical policy intervention points and provide the required policy support for the development and dissemination of this technology. Opportunities for international collaboration also exist for countries involved in developing and using hybrid rice technology to increase their efficiency in carrying out this task. During the past four years, IRRI, in collaboration with FAO, APSA, and China, has established an international network on hybrid rice in Asia with funding from the Asian Development Bank under the umbrella of the Irrigated Rice Re- 18 Virmani

25 search Consortium. The scope of this collaboration should be broadened beyond Asia to make it a truly global effort. In conclusion, hybrid rice made a significant impact in China in the 20th century. It should contribute significantly toward food security, environmental protection, and poverty alleviation in tropical rice-growing countries in the 21st century. References Alam MF, Datta K, Abrigo E, Oliva N, Tu J, Virmani SS, Datta SK Transgenic insectresistant maintainer line (IR68899B) for improvement of hybrid rice. Plant Cell Rep. 18: Borkakati RP, Virmani SS Genetics of thermosensitive genic male sterility in rice. Euphytica 88:1-7. Eusebio W, Casal C, Parado B, Bartolome V, Virmani S, McLaren G Variations in frequency of maintainers and restorers in testcrosses of the hybrid rice breeding program. Philipp. J. Crop Sci. 27(Suppl. 1):80. Faiz FA Effect of wild abortive cytoplasm inducing male sterility on biotic/abiotic resistance/tolerance and agronomic and grain quality in some basmati rice hybrids. Ph.D. thesis. Central Luzon State University, Muñoz, Nueva Ecija, Philippines. 181 p. George T, Magbanua R, Laza M, Atlin G, Virmani SS Magat, a wetland semidwarf hybrid rice for high-yielding production on irrigated dryland. Int. Rice Res. Notes 27(1): Janaiah A, Hossain M Adoption of hybrid rice technology in India: an economic assessment of early farm-level experiences. In: Peng S, Hardy B, editors. Rice research for food security and poverty alleviation. Proceedings of the International Rice Research Conference, 31 March-3 April 2000, Los Baños, Philippines. Los Baños (Philippines): International Rice Research Institute. p Liu GJ, Vera Cruz CM, Sogawa K Disease and insect management in hybrid rice. In: Abstracts of papers presented at the 4th International Symposium on Hybrid Rice, May 2002, Hanoi, Vietnam. p 14. Maruyama K, Araki H, Kato H Thermosensitive genic male sterility induced by irradiation. In: Rice genetics II. Manila (Philippines): International Rice Research Institute. p Sanchez DL, Virmani SS Identification of thermosensitive genic male sterile lines with low critical sterility point for use in commercial hybrid rice production. Philipp. J. Crop Sci. 27(Suppl. 1):32. Tan XL, Vanavichit A, Amornsilpa S, Trangoorung S Genetic analysis of rice CMS- WA fertility restoration based on QTL mapping. Theor. Appl. Genet. 97(5/6): Virmani SS Prospects of hybrid rice in the tropics and sub-tropics. In: Virmani SS, editor. Hybrid rice technology: new developments and future prospects. Manila (Philippines): International Rice Research Institute. p Virmani SS Hybrid rice research and development in the tropics. In: Virmani SS, Siddiq EA, Muralidharan K, editors. Advances in hybrid rice technology. Los Baños (Philippines): International Rice Research Institute. p Virmani SS, Faiz FA, Dela Cruz QD, Ali AJ Has WA cytoplasm any genetic liability on rice hybrids? Agronomy Abstracts. American Society of Agronomy Meeting, Charlotte, North Carolina, USA. Advances in hybrid rice research and development in the tropics 19

26 Virmani SS, Sharma HL Manual for hybrid rice seed production. Manila (Philippines): International Rice Research Institute. Virmani SS, Voc PC Induction of photo- and thermo-sensitive male sterility in indica rice. Agron. Abstr Yao FY, Xu CG, Yu SB, Li JX, Gao YJ, Li XH, Zhang QF Mapping and genetic analysis of two fertility restorer loci in the wild-abortive cytoplasmic male sterility system of rice (Oryza sativa L.). Euphytica 98(3): Yuan LP, Virmani SS Organization of a hybrid rice breeding program. In: Hybrid rice. Manila (Philippines): International Rice Research Institute. p Notes Author s address: Plant breeder and deputy head, Plant Breeding, Genetics, and Biochemistry Division, International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines. Citation: Virmani SS, Mao CX, Hardy B, editors Hybrid rice for food security, poverty alleviation, and environmental protection. Proceedings of the 4th International Symposium on Hybrid Rice, Hanoi, Vietnam, May Los Baños (Philippines): International Rice Research Institute. 407 p. 20 Virmani

27 Breeding strategies to enhance heterosis in rice P.S. Virk, G.S. Khush, and S.S. Virmani Indica germplasm has served as a rich reservoir for the identification of heterotic combinations in rice. However, the yield potential of indica parental lines is about t ha 1 under tropical conditions. Further improvement in the yield potential of parental lines and broadening of their genetic base should lead to the identification of hybrid combinations with even higher heterosis. To develop elite lines with higher yield potential, breeding work on the development of the new plant type (NPT), using tropical japonica (TJ) germplasm, began in 1989 at IRRI. Since then, major progress has been made in developing high-yielding NPT lines. Two high-yielding NPT lines have been released as varieties in China from this program. To further improve the new plant type for wider acceptability and its ability to withstand harsh tropical environments, elite indica lines were crossed with NPT lines. NPT lines derived from IJ (indica/japonica) indica elite lines possess higher yield, the desired grain quality, and resistance to several diseases. They also have strong stems and hence don t lodge even after heavy rains and strong winds. We are continuing to improve the NPT lines for grain quality and for resistance to various diseases and insects. Improved NPT lines with high yield potential, developed from the crosses between indicas and tropical japonicas, offer new opportunities in heterosis breeding in rice. We are now investigating whether the present level of heterosis (15% to 20%) observed in indica indica crosses can be further increased by exploiting the newly developed germplasm. In fact, enhanced heterosis (>40%) observed at IRRI in indica/tropical japonica crosses encouraged us to develop CMS lines in the genetic background of elite TJ lines possessing wide compatibility (WC) genes. A very low frequency of restorer lines among elite TJ lines did not allow us to use them as the male parent. In addition, TJ CMS lines showed two major weaknesses: degeneration of panicles and low outcrossing rate. Consequently, we are emphasizing developing TGMS lines in the genetic background of elite TJ and indica/tj derivative lines. The latter appear to be better adapted, and higher yielding in the tropics. They also possess acceptable indica grain type. Heterosis between indica (TGMS)/(indica/TJ) derivative lines is being studied to identify heterotic combinations. We have also identified six putative heterotic groups in rice that are likely to help us in using heterotic combinations. 21

28 In China, more than 50% of the rice area is under hybrid rice cultivation. Five other countries (India, Philippines, Vietnam, Indonesia, and Bangladesh) have also released rice hybrids for commercial cultivation. These hybrids show 15 20% standard heterosis and are based on indica germplasm. Although japonica hybrids are also cultivated commercially in China, the area under such hybrids is limited, primarily because of lower standard heterosis (less than 10%). The current level of standard yield heterosis is economically viable, but, to make hybrid rice technology more attractive, rice hybrids with a higher level of heterosis should be produced. Genetic diversity between parents is generally related to the magnitude of heterosis. Therefore, to raise the level of heterosis, Chinese and Japanese scientists proposed using indica/japonica crosses (Maruyama 1988, Ikehashi 1991). Our earlier studies at IRRI indicated a higher level of heterosis for yield in tropical japonica/ indica crosses than in indica/indica crosses. This encouraged us to explore the possibility of using tropical japonica germplasm for hybrid development. We shall also highlight the progress made in improving tropical japonica germplasm and its potential use for developing tropical rice hybrids with enhanced heterosis. Tropical japonica germplasm Breeding work on the new plant type (NPT) began at IRRI in Around 2,000 tropical japonica entries from the IRRI gene bank were field-evaluated to identify donors for traits such as low tillering, large panicles, thick stems, a vigorous root system, and thick dark green leaves. These germplasm entries came from Indonesia and were popularly called bulus. All the donors were very tall in stature. We also identified two short-statured japonicas, Shen Nung from China and MD2 from Madagascar, with the sd-1 gene. Therefore, all the tall donors were crossed with these semidwarf lines. Breeding lines with short stature and the aforementioned target traits were selected. The selected lines were intercrossed and the plants with the desired ideotype were selected. The first batch of NPT lines had large panicles, few unproductive tillers, thick stems, and large and dark green flag leaves. However, these NPT lines failed to deliver the big harvest we expected because many grains failed to develop. Fortunately, we traced this flaw to certain donor parents and were able to overcome this problem by including only parents with a very high percentage of filled grains in the subsequent hybridization programs. Several high-yielding elite NPT lines are now available in the tropical japonica background. These NPT lines were field-evaluated in replicated yield trials during the 1998 dry season. The best-yielding entry, IR , yielded 8.84 t ha 1 vis-à-vis 8.07 t ha 1 for check variety IR72. Since then, many NPT elite lines yielding higher than IR72 have been developed at IRRI. In fact, two varieties, Dianchao 1 and Dianchao 3, based on IRRI-bred NPT germplasm, have been released in China. These NPT lines perform very well in areas where disease pressure is low and the preference is for sticky and bold-grain types. 22 Virk et al

29 Table 1. Level of heterosis of inter- and intravarietal group hybrids evaluated during the 1993 wet season and 1994 dry season (adopted from Khush et al 1998). Group % Heterosis wet season dry season Tropical japonica/indica Indica/indica Tropical japonica/tropical japonica 7 13 Enhancing heterosis using NPT lines Genetic diversity between parents is generally related to the magnitude of heterosis. We produced hybrids between tropical japonica (TJ) NPT and indica (I) lines to obtain hybrids with enhanced heterosis. In fact, our earlier work showed that various groups of hybrids yielded in the order TJ/I > I/I > TJ/TJ (Table 1, Khush et al 1998). However, many of the TJ/I F 1 s showed a varying degree of spikelet sterility because of interaction at the S-5 locus, which causes female gametes carrying the japonica allele to be eliminated (Ikehashi and Araki 1984). However, varieties with wide compatibility (WC) genes carry a neutral allele at the S-5 locus (Ikehashi and Araki 1984, 1986). Our experience showed that the WC gene is widely distributed in TJ germplasm and won t be a constraint to the development of parental lines for hybrid breeding. Developing NPT parental lines for hybrid rice breeding In the hybrid rice program at IRRI, numerous A, B, and R lines and some thermosensitive genic male sterile (TGMS) lines are already available in the indica background. We began a breeding program in 1996 to identify maintainer (B) and restorer (R) lines in the TJ germplasm. However, we failed to identify any restorers in the TJ germplasm. On the other hand, maintainer frequency of the TJ lines was 70% vis-à-vis 5% in indicas. The apparent lack of restoration ability in the TJ germplasm indicated that these lines couldn t be used as pollen parents of an I/TJ hybrid. Hence, we started to convert TJ lines into cytoplasmic genic male sterile (CMS) lines. In fact, we have produced male sterile lines in the TJ background. However, the phenotypic acceptability of such CMS lines is rather low and, more importantly, their outcrossing rate is very low (Table 2). We have also observed a continued degeneration of the panicles in certain TJ genotypes even in the advanced backcross generations (Fig. 1). In addition, TJ lines don t possess the desired level of disease or insect resistance and they possess short and bold grains. It appears that the original TJ CMS lines will also be of limited use for developing commercial TJ/I hybrids. However, the original NPT lines are being improved for these limitations (see next section). Breeding strategies to enhance heterosis in rice 23

30 Table 2. Tropical japonica CMS lines with WC genes and their phenotypic acceptability and outcrossing rate. TJ CMS line Cytoplasm Phenotypic Outcrossing acceptability a rate b IR72083A ARC IR72795A WA a On a 1 9 scale, where 1 = excellent and 9 = very poor. b On a 1 9 scale, where 1 = excellent and 9 = very poor. IR A, BC 1 (NPT) Degenerated panicle IR B (NPT) Normal panicle Fig. 1. Comparison between panicles of a new plant type (NPT) line and its backcross derivative with a CMS line. Further improvement of NPT lines The results of comparative studies between NPT lines and high-yielding indicas indicated that NPT lines did not have sufficient biomass to deliver a big harvest. An increase in tillering capacity was needed to increase biomass production. Second, most of the NPT lines lacked resistance to tropical diseases and insects as the parents used for developing these lines were susceptible. Also, there were no donors for resistance to brown planthopper, green leafhopper, and tungro virus in the tropical japonica germplasm. Third, farmers and consumers in the tropical rice-growing countries prefer varieties with long and slender grains and with intermediate amylose content. Although a small number of long-grain tropical japonica donors were available, these were found to be poor combiners. Considering the above limitations of the tropical japonica germplasm, modern high-yielding indica varieties/elite lines were included in the hybridization program. 24 Virk et al

31 Table 3. Field performance of improved new plant type lines during 2001 dry season at IRRI. Yield Yield Grain Days to advantage advantage Designation yield maturity over best over best (t ha 1 ) check (%) check (t ha 1 ) IR IR IR IR IR IR IR IR IR IR IR IR IR IR PSBRc18 (check) IR72 (check) This was necessary, as explained above, to increase the biomass, incorporate genes for resistance to tropical diseases and insects, and change the grain appearance and quality. During the 2001 dry season at IRRI, 14 NPT lines outyielded the best indica check variety by 12% to 30% in the replicated yield trials (Table 3). Most of the improved NPT lines possess long and slender grains and intermediate amylose content. They also have a varying degree of resistance to blast, bacterial blight, tungro, and brown planthopper. Prospects for using improved NPT lines for hybrid rice breeding Preliminary results showed that improved NPT lines (I-TJ derivatives) are as good restorers as indicas. Also, their maintainer frequency is almost three times that of indicas. It shows their great potential for use as pollen parents in hybrid rice breeding. Unlike original TJ lines, they also possess long and slender grains with intermediate amylose content. Their level of resistance to various diseases and insects is also more like that of indicas. All these features make them an excellent candidate for developing commercial rice hybrids. Experiments are under way to determine the heterosis level of hybrids between I-TJ NPT and indica lines. Breeding strategies to enhance heterosis in rice 25

32 Table 4. Some selected lines with the thermosensitive genic male sterile gene. Line TGMS gene Generation Pedigree a IR76762 tms 3 F 6 IR /IR (I/TJ) IR76763 tms 3 F 6 IR /IR (I/TJ) IR76764 tms 3 F 6 IR /IR (I/TJ) IR77275 tms 2 F 5 Norin PL12/IR (I/TJ) IR77277 tms 2 F 5 IR /IR (I/TJ) IR77284 tms 3 F 5 IR /IR (I/TJ) IR tms 2 (?) Stable line ID24/IR64 (I/I) IR73834 tms 2 (?) Stable line ID24/IR58025B (I/I) IR68301 tms 3 Stable line IR /IR68 (I/I) a I = indica, TJ = tropical japonica. Two-line hybrids using TJ NPT lines Our earlier results showed that the usage of original TJ lines in the development of commercial I/TJ rice hybrids would be limited. Therefore, we started a breeding program to incorporate the TGMS system in the TJ lines. Crosses were made between indica TGMS lines and TJ NPT lines possessing the WC gene to incorporate the TGMS gene into them. Advanced male sterile lines with tms 2 and tms 3 genes are now available (Table 4). Currently, we are evaluating two-line hybrids in replicated yield trial nurseries. We are also incorporating TGMS genes in the I use TJ NPT lines that would be useful in developing future two-line hybrids. Prospects for using heterotic groups to develop highly heterotic combinations Heterotic groups have been identified and used to breed superior hybrids of maize, rye, faba bean, and oilseed rape (Melchinger and Gumber 1998). In rice, Zhang et al (1995) also indicated the possible existence of heterotic groups. At IRRI, Xu et al (2003) conducted systematic studies to identify heterotic groups in a set of 104 diverse accessions of rice from 18 countries and six tester lines, representing the commercialized parental lines of hybrids developed in China and India and at IRRI. Two hundred ninety-two crosses, obtained in the 2000 dry season using the line tester scheme, were evaluated in the succeeding wet season using an augmented experimental design (Kempthorne 1957). Potential panicle weight (PPWg), calculated as the product of the total number of spikelets and grain weight, was used as a representative trait for heterotic grouping because, by experience, panicle weight was found to be highly correlated with yield. A line tester two-way table for mid-parent heterosis was constructed consisting of lines in rows and testers in columns. Euclidean distance was calculated and used as a similarity measurement for each pair of parental lines. A dendrogram was constructed using the clustering method of UPGMA with the software NTSYSpc (Rohlf 1993). Putative heterotic groups (PHGs) were identified by defining a critical cut, which would include most of the tested lines in 26 Virk et al

33 an optimum number of clearly separated groups (clusters) such that the separated clusters (groups) would show maximum intergroup and minimum intragroup variation. To make the identified PHGs more meaningful, each of the PHGs identified at each critical cut was limited to at least three or more lines. One-way ANOVA was then performed on the mid-parent heterosis to determine within- and between-group variances as well as the percentage of the between-cluster variation over the total variation. Dendrogram analyses results (Fig. 2), based on a 0.4 cut, resulted in six putative heterotic groups that covered 85 (81.7%) of the lines, minimum intracluster variance, and a high intercluster variance. The six PHGs consisted of two large ones (PHG 3 and PHG 4), two intermediate ones (PHG 1 and PHG 5), and two small ones (PHG 2 and PHG 6). Since panicle weight, like all quantitative traits, is known to show significant genotype environment interaction, and these results are based on a single-environment experiment, these grouping results are only putative and need to be tested further in multiple target environments. Additional details of this study are given in another paper (Xu et al) in this publication. References Ikehashi H Genetics of hybrid sterility in wide hybridization in rice (Oryza sativa L.). In: Bajaj YPS, editor. Biotechnology in agriculture and forestry. 14. Rice. Berlin (Germany): Springer Verlag. p Ikehashi H, Araki H Varietal screening of compatibility types revealed in F 1 fertility of distant crosses in rice. Jpn. J. Breed. 34: Ikehashi H, Araki H Genetics of F 1 sterility in remote crosses of rice. In: Rice genetics. Manila (Philippines): International Rice Research Institute. p Khush GS, Aquino RC, Virmani SS, Bharaj TS Using tropical japonica germplasm to enhance heterosis in rice. In: Virmani SS, Siddiq EA, Muralidharan K, editors. Advances in hybrid rice technology. Proceedings of the 3rd International Symposium on Hybrid Rice, November 1996, Hyderabad, India. Manila (Philippines): International Rice Research Institute. p Kempthorne O An introduction to genetic statistics. London (UK): John Wiley and Sons, Inc. Maruyama K Strategy and status for developing hybrid rice. Iden. (Heredity) 42(5): (In Japanese.) Melchinger AE, Gumber RK Overview of heterosis and heterotic groups in agronomic crops. In: Lamkey KR, Staub JE, editors. Concept and breeding of heterosis in crop plants. Madison, Wis. (USA): Crop Science Society of America. p Rohlf FJ NTSYSpc. Numerical taxonomy and multivariate analysis system. New York: Applied Biostatistics Inc. Zhang QF, Gao YJ, Saghai Maroof MA, Yang SH, Li JX Molecular divergence and hybrid performance in rice. Mol. Breed. 1: Breeding strategies to enhance heterosis in rice 27

34 93027 Amol 3 (Sona) Karnal Local Babaomi Oang 16 Yun jian 7 BG304 Cisadane Chuitanaya Khazar FR 13A Yu-Qui-Gu Bhavani IR50 Zhong 123 CDR22 ASD 16 B4122 P8BRC 28 ASD 18 OM997 Dhan 4 PR106 CR203 Zao-Xian-14 Basmati 370 C71 OM1723 TKM9 BR11 BR24 JP 5 UPR IR64 Domaiah Nipponbare Doddabyranellu MR77 Jiang-Xi-Si-Miao Pokhreli MR185 MR84 TEQING Haoannong MR106 Jhona 349 Manawthukha At354 Gouind Basmati Basmati 385 IR72 Shen Nong Hua-Gen-Xian 74 MR167 Zhong 143 Yunhui 290 BG300 BG41-1 Chonghui448 Shive Theue Yin Hyu C8 94 PSBRC 66 Feng-Ai-Zan IR Pusa Basmati Hmibyeo Khao Daeng IR6 Yun Hui 11 Doddi X 21 Pahenle Binain Madhukar BG90-2 Cuil Cisanggarung Giza 159 TB 154-E-TB-2 M401 Theahtatyin TGM829 Zhong-Yu-Zao 81 MR159 R644 Chhomrong Milagrosa Dian Tun 502 OMI 706 X22 Ai-Zi-Dao C70 Pahkmawpeum Mavagg Gayabyeo Milyang 23 IR68897B Budda Iksan438 Q5 Co43 IR58025B Swarma X Fig. 2. Cluster dendrogram of 104 accessions of rice based on UPGMA using Euclidean distance as a similarity measurement for mid-parent heterosis data of potential panicle weight. Six putative heterotic groups were identified as 1 to 6 at the 0.4 cut level. 28 Virk et al

35 Notes Authors address: Plant Breeding, Genetics, and Biochemistry Division, International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines. Citation: Virmani SS, Mao CX, Hardy B, editors Hybrid rice for food security, poverty alleviation, and environmental protection. Proceedings of the 4th International Symposium on Hybrid Rice, Hanoi, Vietnam, May Los Baños (Philippines): International Rice Research Institute. 407 p. Breeding strategies to enhance heterosis in rice 29

36 Two-line hybrid rice breeding in and outside China Tong-Min Mou, Lu Xing-Gui, N.T. Hoan, and S.S. Virmani The two-line system for hybrid breeding is more efficient than the three-line system for exploiting heterosis in rice to increase the yield per unit area. Three PGMS genes and five TGMS genes have been identified by scientists from China, Japan, IRRI, Vietnam, and India. The PGMS genes pms 1, pms 2, and pms 3 were located on chromosomes 7, 3, and 12, respectively. The TGMS genes tms 1, tms 2, tms 3, tms 4 (t), and tms 5 were located on chromosomes 8, 7, 6, 9, and 2, respectively. The molecular markers linked with these genes have also been found. In China, more than ten EGMS lines were used to breed commercial two-line hybrid rice. Thirty two-line rice hybrids were released in various rice-growing regions up to The area planted to two-line hybrid rice was 1.54 million ha in EGMS lines are multiplied in three ways: in the autumn season, in the winter season, and under cool-water irrigation. Yields of seed multiplication were 2 5 t ha 1. A technological package for seed production of two-line hybrid rice has been developed. The area of seed production was around 13,000 ha across China in The average yield was 2.5 t ha 1, which was similar to that of threeline hybrid rice seed production. The two-line hybrid rice breeding program at IRRI focuses on developing the technology for the tropics by deploying the TGMS system. TGMS genes tms 2 (from Norin PL12) and tms 3 (from IR32364S mutant) have been primarily deployed for breeding new TGMS lines. ID24, a TGMS line introduced from India, has also been used. TGMS lines IR S and IR73834S, derived from ID24 mutant, have stable sterility. Identification of such lines can be done in the field by evaluating them during the wet season when the mean temperature is 1 2 o C lower than in the dry season. These lines can be multiplied by growing them under high-altitude conditions. To facilitate incorporation of the tms 2 gene, a simple sequence repeat (SSR) marker, RM11 located on chromosome 7, was identified and found to be useful in identifying heterozygous fertile plants in F 2 populations and F 3 F 4 progenies for generation advance in selected crosses. The tms genes are transferred in indica and indica/tj derivative lines from IRRI and elite lines from collaborating countries (Bangladesh, the Philippines, and Sri Lanka). TGMS lines are also being developed at IRRI through a shuttle breeding procedure for some temperate countries such as China, Iran, and Egypt. Using the genetic male sterility facilitated recurrent selection procedure, a 31

37 composite population is also being developed at IRRI for extracting TGMS lines. This population will be shared with national agricultural research and extension systems to enable them to extract locally adapted TGMS lines. In Vietnam and India, several TGMS lines with a relatively low critical sterility point were developed. Some of them are being evaluated in the field and used for developing two-line hybrid rice. Some two-line rice hybrids were tested in multilocation trials. Other countries, such as the Philippines, Bangladesh, Sri Lanka, Iran, Egypt, and Indonesia, were evaluating EGMS lines introduced from IRRI. In the tropics, TGMS lines can be multiplied by growing them under high-altitude conditions. Experimental seed production of two-line hybrid rice was carried out at IRRI and in the Philippines, Vietnam, and India. Two-line hybrid rice would be commercialized in 3 5 years in tropical countries. Future strategies and constraints to developing two-line hybrid rice worldwide are discussed in this paper. The discovery of the rice semidwarf gene (sd-1) and applications in rice breeding resulted in a quantum increase in yield per unit area in the 1960s. The discovery of the cytoplasmic genic male sterility (CMS) system and corresponding three-line hybrid rice resulted in another quantum increase in yield in China in the 1970s. The growth rates of world rice production have continuously increased slightly more than those of population since 1961, mainly because of those innovations. Unfortunately, this trend was reversed for the first time in Rice production grew more slowly than population (Tran 2001) probably because few new attractive innovations have been invented or applied since the 1980s. Two-line rice hybrids, new plant type rice, and genetically modified rice will be attractive methods for increasing rice yield potential in the first decade of the 21st century. Compared with the three-line system (CMS) for exploiting heterosis in hybrid rice breeding, the two-line system (environmental genic male sterility, EGMS) has some major advantages. First, sterility traits are controlled by recessive nuclear genes. Any genotype with good combining ability can be used as a male parent. The frequency of obtaining heterotic hybrids in testcrosses is relatively higher than in the three-line system. Second, it is suited for developing hybrids in japonica and basmati types in which the frequency of CMS restorers is very low. Third, it is especially suited for developing subspecific hybrids, including indica/japonica, indica/tropical japonica, and tropical japonica/temperate japonica, because there is no restriction of the restoring-maintaining relationship in the EGMS system. This paper will review the progress of two-line hybrid rice breeding since the third International Symposium on Hybrid Rice held in Hyderabad, India, in Mapping and molecular markers of EGMS genes Rice mutants with EGMS genes may not have all the desirable agronomic attributes required in cultivars. Often, there is a need to transfer EGMS genes to elite lines with 32 Tong-Min Mou et al

38 desirable agronomic traits. Linkage with morphological or molecular markers facilitates the transfer of these genes and thus increases the efficiency of the breeding process. In most cases, EGMS genes are not linked to any easily identifiable morphological characters. Until flowering, EGMS mutants resemble their normal counterparts or wild types. This character of sensitivity to environmental factors is expressed only under certain specific ranges or conditions of the environmental factors. Under such situations, molecular markers are handy and useful. Scientists in China, Japan, IRRI, India, and Vietnam did intensive work on the mapping of EGMS genes in rice mutants in the past decade. Three PGMS genes and five TGMS genes and linked molecular markers have been identified. PGMS genes Zhang et al (1994), using the bulked extremes and recessive class in the F 2 population of 32001S Minghui 63, mapped the genes for photoperiod genic male sterility (PGMS) in rice. Two chromosome regions, each containing a PGMS locus, were identified on a published restriction fragment length polymorphism (RFLP) linkage map. One locus was designated pms 1, on chromosome 7, and another was designated pms 2, on chromosome 3. The effect of pms 1 was reported to be two to three times larger than that of pms 2. The locus pms 1 is located between markers RG477 and RG511. It is located at 3.5 recombination units from RG477 and 15.0 recombination units from RG511. The locus pms 2 is located between markers RG191 and RG348. It is about 7.0 cm from RG191 and 10.6 cm from RG348 (Fig. 1A,B). Wang et al (1996), using bulked segregant analysis in the F 2 of NK58S NK58F, identified a random amplified polymorphic DNA (RAPD) marker linked to one of the PGMS genes on chromosome 7 and named it PGMS 0.7. Sequencing results showed PGMS 0.7 to have 718 bp. Mei et al (1999) identified another PGMS gene in F 2 populations of crosses NK58S NK58 and NK58S 1514 using bulked segregant analysis. This gene was named pms 3. It was found that pms 3 is linked to the markers C751 and R2708 (Fig. 1C). TGMS genes Wang et al (1995) used bulked segregant analysis of the F 2 population of the cross 5460S Hong Wan 52 to identify the RAPD marker linked to the TGMS gene tms l. They found that one single-copy fragment (size = 1.2 kb) amplified by primer OPB- 19, and subsequently named TGMS 1.2, cosegregated with the TGMS gene tms l and was mapped on chromosome 8 with a genetic distance of about 19.3 cm from RZ562 on one side and 5.0 cm from RG978 on the other side (Fig. 2A). TGMS 1.2 has been sequenced and changed to a sequence-tagged site (STS) marker, which is available for public use. Yamaguchi et al (1997) reported the linkage of molecular markers with the TGMS gene tms 2. They estimated the locus of tms 2 using RFLP markers in an F 2 population derived from a cross between Norin PL12 and aus variety Dular. They concluded that tms 2 was located between the markers R643A and R1440, with a distance of 0.2 cm from R643A on chromosome 7 (Fig. 2B). Further studies by Two-line hybrid rice breeding in and outside China 33

39 A B C Chromosome 12 Chromosome 7 cm RG146B 5.4 RG Chromosome RG678 WG719 RG30 CDO533 RG477 pms 1 RG511 RZ272 cm R348 pms 2 RG191 (RG226) RG450 (RG117) RG335 RG128 pms 1 pms 2 cm pms 3 R2708 pms 3 C751 RZ261.F3 V4 C2 RG543 CDO344 CDO459 G2140 RG9 AU RA27+RA Fig. 1. Location of PGMS genes on linkage map in rice. Lopez et al (2000) showed that SSR markers RM2 and RM11 flanked tms 2. These two markers select the TGMS plant with 96.6% accuracy. Subudhi et al (1997) employed bulk segregant analysis in conjunction with the RAPD technique in an F 2 population of the cross IR32364 TGMS IR68 to identify the molecular markers linked to the TGMS gene tms 3. Fertile and sterile bulks were constructed following the classification of F 2 plants into true-breeding sterile, fertile, and segregating fertile plants based on F 3 family studies. From the survey of 389 arbitrary primers in bulked segregant analysis, four RAPD markers were identified, in which three, OPF , OPB19 750, and OPAA7 550, were linked to tms 3 in the repulsion phase and one, OPAC3 640, was linked to tms 3 in the coupling phase. The tms 3 gene was flanked by OPF and OPAC3 640 on one side and by OPAA7 550 and OPB on the other side (Fig. 2C). Subsequently, using a mapping population available at IRRI, OPAC3 640 was mapped to the short arm of chromosome 6. No RFLP markers from this region, however, showed linkage to tms 3 owing to a lack of polymorphism between the parents. Lang et al (1997, 1999) developed a polymerase chain reaction (PCR)-based marker for the tms 3 gene. The sequence information from RAPD markers was used to design several pairs of primers for PCR amplifications. One of the RAPD markers, OPF , could be converted into two co-dominant STS markers tightly linked to the tms 3 gene. The primers F 18F/F 18RM and F18 FM/F18RM were linked to tms 3 at a distance of 2.7 cm. The efficacy of marker- 34 Tong-Min Mou et al

40 A B C D E Chromosome 2 AnnongS-1/Nanjing 11 cm ACCTG7 3.6 Chromosome cm 8 RM Chromosome 9 Y2724R RG20 Chromosome 7 AS2/N cm AACAG cm 2.4 R1788(D24362) Chromosome 6 EACT/MCAG 0.0 tms RG333 IR32364TGMS/IR68 RM RZ R643A(23948) cm 3.4 R349 RZ516 TGMS 1.2 tms RM257 R tms 5.7 RZ AGCTG12 1 tms (t) 1.7 RZ2 4 RG RG1 2.4 OPF EAA/MCAG 18.5 R1440(D24156) 7.2 RZ OPAC tms RG (t) 23.1 TS OPAA7 R646(D23951) OPA12 RM211 RZ649 OPB tms 1 (Wang et al 1995) tms 2 (Yamaguchi et al 1997) tms 3(t) (Subudhi et al 1997) tms 4(t) (Reddy et al 2000) tms 5 (Jia et al 2000) Fig. 2. Location of TGMS genes on linkage map in rice. Two-line hybrid rice breeding in and outside China 35

41 assisted selection for this trait was calculated as 84.6%. A polymorphism survey of 12 prospective elite lines to be introgressed with the TGMS gene indicated that these PCR markers for tms 3 can now be used in selecting TGMS plants at the seedling stage in segregating populations in environments independent of a controlled-temperature regime. Dong et al (2000), using bulked segregant analysis of the F 2 population of the cross TGMS-VN1 CH1, identified the molecular markers linked to the TGMS (tgms-vn1) gene and subsequently determined its chromosomal location on the linkage map of rice. From the survey of 200 amplified fragment length polymorphism (AFLP) primer combinations, four AFLP markers (E2/M5-600, E3/M16-400, E5/ M12-600, and E5/M12-200) linked to the TGMS gene were identified. The marker E5/M showed polymorphism in RFLP analysis and was closely linked to the TGMS gene at a distance of 3.3 cm. This marker was subsequently mapped on chromosome 2. The results of further experiments with SSR markers showed that RM27 was linked with tgms-vn1. Reddy et al (2000) identified a TGMS gene by using RAPD, AFLP, and microsatellites through bulked segregant analysis of the F 2 population from the cross between SA2 (TGMS mutant) and N22 (male parent), which was linked with microsatellite marker RM257 at 6.2 cm on chromosome 9. It was tentatively designated tms 4 (t)(fig. 2D). Jia et al (2001) used an F 2 population from the cross between AnnongS-1 and Nanjing11 to construct a genetic linkage map. A new TGMS gene, known as tms 5, was identified on the short arm of chromosome 2. It was found that tms 5 was located between markers R349 and RM71 and it was closely linked to the marker RM174 (Fig. 2E). Table 1 summarizes the linkage of molecular markers with the EGMS genes reported in rice and their location on the chromosomes. There are two TGMS genes, tms 5 and tgms-vn1, on chromosome 2. It is essential to further analyze their allelic relationship. Two-line hybrid rice breeding in China Breeding of EGMS lines The EGMS mutants identified or induced seldom possess all the desirable characteristics, such as good plant type, higher yield potential, higher outcrossing, good combining ability, resistance to major pests and diseases, and desirable grain quality, required for their commercialization. Hence, there is a need for specific breeding of EGMS lines by transferring EGMS genes from the mutants to elite agronomic backgrounds before using them in rice improvement programs. The PGMS rice mutant Nongken 58S (Shi 1981) and TGMS mutant AnnongS-1 (Li and Deng 1990), identified as spontaneous mutants, were used as the major gene donors in the EGMS line breeding program in the past two decades in China. Since the major attribute to be selected in EGMS breeding is sterility, the F 2 and subsequent segregating generations are to be grown under appropriate environ- 36 Tong-Min Mou et al

42 Table 1. Linked molecular markers and chromosomal location for EGMS genes in rice. EGMS gene Linked molecular markers Chromosomal Reference Materials studied a location PGMS gene pms l RG 477 and RG Zhang et al (1994) 32001S b Minghui 63 PGMS gene pms 2 RG 191 and RG Zhang et al (1994) 32001S Minghui 63 PGMS gene pms 3 R2708 and C Mei et al (1999) Nongken 58S Nongken 58 TGMS gene tms l RAPD marker Wang et al (1995) 5460S Hong Wan 52 TGMS gene tms 2 RFLP markers R643A and R Yamaguchi et al (1997) Norin PL12 Dular SSR markers RM11 and RM2 Lopez et al (2000) Norin PL12 KDML105 TGMS gene tms 3 RAPD markers OPF , 6 Subudhi et al (1997) IR32364 TGMS IR68 OPAC3 640, OPAA7 550, and OPB TGMS-VN1 CH1 TGMS gene tgms-vn1 RM27, E5/M Dong et al (2000) SA2 (TGMS mutant) N22 TGMS gene tms 4 (t) RM257, TS200 9 Reddy et al (2000) AnnongS-1 Nanjing11 TGMS gene tms 5 RM174, R394, and RM71 2 Jia et al (2001) a Nongken 58, 5460S, and AnnongS-1 originated from China, Norin PL12 from Japan, IR32363TGMS from IRRI, TGMS-VN1 from Vietnam, and SA2 from India. b 32001S was a derivative from Nongken 58S. Two-line hybrid rice breeding in and outside China 37

43 mental conditions (i.e., low temperature/long photoperiod, high temperature/short photoperiod) to identify segregants carrying male sterility genes and characterizing sterility/fertility expression (Mou et al 1998). To advance the generations, the selected segregants carrying male sterility genes are grown by using ratooned plants in environmental conditions conducive to inducing fertility. Anther culture can advance the generations and the stability of sterility (Li et al 1995). More than 100 EGMS lines have been developed in China since Most of the EGMS lines (apart from japonica PGMS lines) developed before 1989 were not used in two-line hybrid rice breeding because of their higher critical sterility points (CSP) of temperature for fertility alteration. The EGMS lines with a CSP of higher than 24 o C (mean) or o C (minimum-maximum) are not safe for seed production because of temperature fluctuations in the seasons of seed production. Breeders have focused on selecting EGMS lines with a relatively low CSP, 23.5 o C (mean) or o C (minimum-maximum) under appropriate conditions (specific natural areas or controlled conditions) since Eleven EGMS lines have been used for largescale seed production in China (Table 2). Peiai64S, a PTGMS line bred by the National Hybrid Rice Research and Development Center, was used extensively in breeding two-line rice hybrids. Almost half of the more than 30 combinations released up to 2001 were derived from Peiai64S (Table 3). Breeding of two-line rice hybrids Intensive efforts in China since 1982 to develop two-line rice hybrids gave promising results. First, japonica two-line hybrid N5047S/R9-1, which was developed in the Hubei Academy of Agricultural Sciences, was demonstrated in farmers fields in During the past two decades, more than 100 two-line hybrids developed in different provinces in China have been evaluated in regional and national trials. Based on extensive trials, more than 30 hybrids were approved and released for commercialization (Table 3) in different rice-growing regions in China. In the Yangtze valley, including Jiangsu, Anhui, Jiangxi, Hunan, Zhejiang, Hubei, and Sichuan provinces and the southern regions of Henan and Shanxi provinces, six japonica two-line rice hybrids have been approved and released for the second cropping season. Their yields were usually t ha 1 compared with that of the conventional japonica varieties. Four indica two-line hybrids used for the first cropping season and six for the single or second cropping season, respectively, were approved and released in this region. Yields of about 5 8% higher than those of three-line hybrids were obtained. In southern China, including Guangdong, Guangxi, Fujian, and Hainan provinces, 11 combinations were approved and released for double cropping. The area under twoline rice hybrids in China has increased steadily from 5,000 ha in 1991 to 1.54 million ha in 2000 (Table 4). Seed multiplication of EGMS lines The multiplication of EGMS lines is different from that of CMS lines. EGMS lines are multiplied at appropriate locations and in favorable seasons. Three methods have been used for multiplying EGMS lines in China. 38 Tong-Min Mou et al

44 Table 2. EGMS lines in large-scale seed production of released two-line hybrids in China. EGMS lines Subspecies a Type of Origin CSPT c CSPP d Developed by e Hybrids reaction b of gene (mean C) (h) released N5088S J PGMS NK58S Hubei AAS S J PGMS NK58S Anhui AAS 4 Pei-ai 64S I PTGMS NK58S Hunan HRRC 14 Shuguang612S I PTGMS NK58S Sichuan AU 1 SE21S I TGMS NK58S 23.0 Fujian AAS 2 W9593S I PTGMS NK58S Hubei AAS 1 HS-3 I PTGMS NK58S Fujian AU 1 Xiang125S I TGMS AnnongS Hunan HRRC S I TGMS AnnongS Hunan HRRC 1 TianfengS (F131S) I TGMS AnnongS Ganzhou AAS, Jiangxi 1 810S I TGMS AnnongS An-jiang AU, Hunan 2 a J = japonica, I = indica. b PGMS = photoperiod-sensitive genic male sterility, PTGMS = photo-thermosensitive genic male sterility, TGMS = thermosensitive genic male sterility. c CSPT = critical sterility point of temperature. d CSPP = critical sterility point of photoperiod. e AAS = Academy of Agricultural Sciences, HRRC = Hybrid Rice Research Center, AU = Agricultural University. Two-line hybrid rice breeding in and outside China 39

45 Table 3. Two-line rice hybrids released up to 2001 in China. Hybrid Pedigree Type a Year Growing region/crop of release Ejingza No. 1 N5088S/R187 J 1995 Yangtze valley /second Huajingza No S/1514 J 1995 Huajingza No. 2 N5088S/65396 J you S/Wanhui 9 J you S/99 J you S/Xiushui 04 J 1994 Peiliangyou Teqing Pei-ai 64S/Teqing I 1994 Yangtze valley/single or second Peiliangyou 288 Pei-ai 64S/R288 I 1996 Pei Liangyou Yuhong Pei-ai 64S/Yuhong No.1 I 1997 Liangyou Peijiu Pei-ai 64S/9311 I 1999 Liangyou 932 W9593S/Shengyou No. 2 I 2001 Liangyou 681 Shuguang612S/881 I 1999 Xiangliangyou 68 Xiang125S/D68 I 1998 Yangtze valley/first 8-liangyou100 Annong810S/D100 I 1998 Tian Liangyou402 TianfengS/R402 I 1998 An Liangyou S/Zao25 I 1998 Peiza Shanqing Pei-ai 64S/Shanqing I 1997 South China/first and second Jinliangyou 36 HS-3/946 I 2000 Peiza Shuangqi Pei-ai 64S/Shuangqizhan I 1998 Liangyou2163 SE21S/Minghui63 I 2000 Liangyou2186 SE21S/Minghui86 I 2000 Fu Liangyou63 FJS-1/Minghui63 I 2000 Pei Liangyou275 Pei-ai 64S/275 I 1999 Pei Liangyou99 Pei-ai 64S/Gui99 I 1998 Peiza Maosan Pei-ai 64S/Maosan I 2000 Peiza Maoxuan Pei-ai 64S/Maoxuan I 2000 Peiza67 Pei-ai 64S/G67 I 2000 Yunguang No. 8 N5088S/Yunhui11 J 2000 West China/single a I = indica, J = japonica. 1. Autumn season in central China. The sterility/fertility alterations in most japonica PGMS lines are mainly controlled by daylength and are partially regulated by temperature during the sensitive stages. The results of dynamic observations for fertility expression indicated that, when the photoperiod is shorter than h and the mean temperature is lower than 28 o C from mid-august to mid-september in central China, japonica PGMS lines could reproduce by self-pollination. Seed multiplication yields of PGMS lines N 5088S and 7001S in normal years reached 3 4 t ha 1 with good management of seed plots. But the indica PTGMS and TGMS lines could not reproduce because their fertility expression was mainly controlled by temperature, and the temperature from mid-august to mid-september in the region was usually higher than the CSP of PTGMS and TGMS lines. 40 Tong-Min Mou et al

46 Table 4. Area under twoline hybrid rice in China. Year Area (000 ha) , , , Winter season on Hainan Island. Rice can grow year-round in the southern region of Hainan Island. Temperature and daylength are lower and shorter in the winter season. The experimental results of interval sowing showed that most EGMS lines could reproduce by self-pollination. Japonica PGMS lines are fertile when they flowered from August to May. Self-seed setting (bagged) was 10 70%. PTGMS lines were fertile from November to March and self-seed setting was 10 60%. TGMS lines were fertile from December to March and self-seed setting was 10 60%. The multiplication yields of EGMS lines could reach 5 6 t ha 1 with appropriate management. Cultivation technology should include choosing appropriate seeding time for each line, increasing cropping density, applying fertilizer in appropriate growth stages, and protecting against pests and diseases. 3. Cool-water irrigation. The results of experiments on EGMS lines showed that appropriate cool-water irrigation could induce the EGMS lines to become fertile under higher air temperature (Table 5). Seed setting was higher than with other methods and yields were more stable. To obtain high yield under multiplication in cool-water irrigation fields, it is necessary to Select appropriate seed plots with sufficient cool water: The water temperature should be higher than C and lower than the CSP of special EGMS lines. Use cool water in the correct development stage: Cool-water irrigation should start from the young panicle differentiation of the stamen and pistil primordia to meiotic division of the pollen mother cell stage. Duration of coolwater irrigation should be days. Have an adequate water depth: A cool-water depth of cm is essential to obtain high multiplication yield. Make tillers grow uniformly: Good crop management should be planned and conducted during the vegetative growth phase. The uniform growth of tillers would be helpful in responding to cool-water treatment. Proper fertilizer should be applied so that rice plants can grow well. Two-line hybrid rice breeding in and outside China 41

47 Table 5. The experiment of Peiai64S multiplication with cold-water irrigation (1995). Treatment Seeding Irrigation Time Fields near to Medium Fields far from Depth of time duration of 10% reservoir fields reservoir water in flowering fields (cm) Water Yield Water Yield Water Yield temp. (t ha 1 ) temp. (t ha 1 ) temp. (t ha 1 ) ( o C) ( o C) ( o C) I 17 April July 4 August II 25 April July 6 August III 10 May 20 July-5 August 9 August Tong-Min Mou et al

48 Spray proper quantity of GA 3 and make supplementary pollination: Some sterile panicles, which were not treated by cool water because of different development, would receive pollen from the fertile panicles. Seed production of two-line hybrid rice In a comparison of two-line hybrid rice with three-line hybrid rice in hybrid seed production techniques, the most important aspects to be considered in two-line hybrid rice are the flowering time coinciding with the stable sterility-inducing period at a given location for a certain EGMS line. The photoperiod- and temperature-sensitive stage of an EGMS line generally lasts for 20 days from 25 to 5 days before heading. The variance in growth development among the plants in a field is around 10 days. The difference in sowing time and transplanting among farmers in an area may be 10 days or more. Hence, the sterility-inducing period should be longer than 40 days to ensure the seed purity of hybrid rice. The length of the sterile period was decided by temperature and daylength in the area and the CSP of EGMS lines. In central China, such as Hubei, Hunan, Anhui, and Jiangxi, the stable sterile periods of W9593S and Peiai64S, two indica PTGMS lines that have a CPS of 23.5 o C and 13.0 h, were about 2 months from late July to late September. The favorable flowering time for seed production was mid- to late August, when the temperature was intermediate and rain was scarce. Hybrid rice breeders in China have divided the rice-growing area into four regions for seed production of two-line hybrid rice based on temperature and daylength (Table 6). The PTGMS lines with a CSP of o C are most adaptable for hybrid seed production in all four regions. The second adaptable EGMS lines are TGMS lines that can be used for seed production in regions I, II, and III. PGMS lines have narrow suitable regions (III and IV) for seed production. Two-line hybrid rice breeding outside China Development of tropical TGMS lines IRRI scientists have successfully transferred the genes conferring the TGMS trait of Norin PL12 from Japan (tms 2 ), IR32364 (TGMS) (tms 3 ) from IRRI, and ID 24 from India, which contains a gene allelic to tms 2 and additional genetic factor(s) that affect its fertility/sterility expression (Reddy et al 2000), into indica- and indica-tropical japonica derivative lines from IRRI and elite lines from collaborating countries such as Bangladesh, the Philippines, Sri Lanka, Iran, and Egypt. The genes are transferred using the pedigree selection method. The TGMS donor is crossed with elite breeding lines and agronomically acceptable plants in the F 2 generation are harvested. During sterility-inducing conditions (maximum temperature above 30 C), phenotypically desirable fertile plants from F 3 to F 5 families segregating for sterility are selected. At F 5 to F 6, sterile plants possessing good agronomic traits are ratooned and transferred to the phytotron (26/21 C) to revert to fertility. Seeds of these suspected TGMS lines are harvested and planted in sterility-inducing conditions to confirm the transfer of TGMS genes (Lopez and Virmani 2000). Molecular marker-aided selection is Two-line hybrid rice breeding in and outside China 43

49 Table 6. Regions for hybrid seed production in China. Region Provinces Season of seed Ranges of mean Daylength EGMS Lines now being used included production temperature ( o C) (h) lines for seed production I South part Sanya city, Lingshui Early May-late TGMS, PTGMS Peiai64S, SE21S, of Hainan and Dongfang counties Oct. Xiang125S, 1356S, TianfengS(F131S), 810S II Southern China Guangdong, Guangxi, Mid-July-mid TGMS, PTGMS Peiai64S, SE21S, Hainan, and Fujian Sep. Xiang125S, 1356S, provinces; south parts TianfengS(F131S), of Jiangxi, Hunan, and 810S Zhejiang provinces III Central and Hubei, Anhui, and Mid-July-late PGMS, PTGMS, N5088S, 7001S, eastern China Jiangsu provinces; Aug. TGMS Peiai64S, SE21S, Zhongqing city; north Xiang125S, 1356S, parts of Jiangxi, Zhejiang, TianfengS(F131S), and Hunan provinces; 810S, Shuguang south part of Henan 612S, W9593S, Province HS-3 IV Chengdu- Sichuan, south part Mid-July-mid PGMS, PTGMS Shuguang612S, Hanzhong basin of Shanxi Aug. W9593S, N5088S, 7001S, Peiai64S 44 Tong-Min Mou et al

50 being adapted to increase the efficiency of transferring the tms 2 gene located on chromosome 7. The SSR marker RM11, which is closely linked to tms 2 (Lopez et al 2000), was found to be useful in identifying heterozygous fertile plants in F 2 and F 3 F 4 progenies for generation advance. To obtain hybrid seeds with a high degree of purity, self-fertility of the TGMS line caused by fluctuating temperature during two-line hybrid rice seed production should be minimized, if not eliminated. This is achieved by developing TGMS lines with a low critical sterility point (CSP). Previously, TGMS lines were evaluated at IRRI in April-May (average maximum temperature during the critical stage is above 31 C) by sowing them in January-February (Lopez and Virmani 2000). This procedure resulted in the development of TGMS lines with a high CSP. Such TGMS lines tended to revert to fertility at temperature fluctuations below 30 C during the critical stage of panicle development; thus, these could not be used for commercial hybrid seed production. The sensitivity of some TGMS lines to fluctuating temperature was confirmed by Viraktamath and Virmani (2001). Studies conducted at the IRRI phytotron showed that sudden interruption with 27 C for 2 hours under the sterilityinducing regime of 32/24 C or a 4-hour interruption of 27 C for at least 1 day during the critical stage resulted in partial fertility in IR , a TGMS line with Norin PL12/IR36 backgrounds. The average maximum temperature during the wet season at IRRI is C, with occasional fluctuations below 30 C during cloudy and/or rainy weather. Evaluating TGMS lines for sterility in the wet season was considered helpful for eliminating those lines with a high CSP. Therefore, since 2000, TGMS lines have been evaluated for sterility in the wet season at IRRI. TGMS lines with a low CSP can be multiplied by sowing them in October-November, with flowering in December to February, at IRRI or by planting them in higher altitudes. Two promising low-csp TGMS lines (Table 7), which are completely sterile at 28 C maximum temperature, have been developed. To improve TGMS line breeding efficiency, a shuttle-breeding program on two-line hybrid breeding for the tropics also began at Huazhong Agricultural University, Wuhan, China, in collaboration with IRRI. Some TGMS breeding lines bred under this program are also being evaluated at IRRI. To share diverse background breeding materials, a TGMS composite population using the genetic male sterility system is being developed at IRRI. The procedure is summarized in Figure 3. The composite population will be maintained by growing it in isolation at a fertility-inducing temperature for TGMS. The seeds from phenotypically acceptable genetic male sterile plants caused by random outcrossing with the fertile plants (tms 2 tms 2 Msms) are harvested to compose the next cycle of a TGMS composite population. Phenotypically acceptable fertile plants are handled through pedigree selection to extract new improved TGMS lines from this population. This population will be shared with NARES to enable them to extract locally adapted TGMS lines. Two-line hybrid rice breeding in and outside China 45

51 Table 7. Characteristics of promising low-csp TGMS lines. TGMS line IR S IR73834S Pedigree ID24/IR64 ID24/IR58025B Days from sowing to heading Phenotypic acceptability a 3 5 Seed setting b Outcrossing potential (%) Grain Length c 5 3 qualityshape d 5 1 traits Chalkiness e 5 0 Gelatinization temperature HI/I L Amylose content (%) a On a scale of 1 9, 1 = excellent and 9 = unacceptable. b On a scale of 1 9, 1 = highly fertile (>90% spikelet fertility) and 9 = completely sterile (0% spikelet fertility). c On a scale of 1 7, 1 = extra long (more than 7.5 mm), 7= short (5.5 mm or less). d On a scale of 1 9, 1 = slender, 9 = round. e On a scale of 0 9, 0 = none, 9 = large (more than 20%). Characterization of tropical TGMS lines India, Vietnam, and the Philippines have introduced some TGMS lines from IRRI, China, and Japan. Sterility and the main agronomic traits were evaluated in local environmental conditions. After being systematically characterized in Hyderabad, Coimbatore, and Pantnagar, India, seven promising TGMS lines were identified under Indian conditions (Table 8) (Krishnaiah et al 2001). In Vietnam, more than 10 promising TGMS lines were identified and evaluated for agronomic traits. Results showed that the stable periods of complete male sterility of five TGMS lines lasted for more than 4 months from May to October (Table 9). 11S was a practicable TGMS line for commercializing seed production. The temperature-sensitive stage of 11S was from 13 to 19 days before heading and its CSP was 25 C (Hoang et al 2000). PhilRice-Los Baños in the Philippines evaluated 22 TGMS lines. Some of them showed complete sterility in the wet season of 1999 and dry season of 2000 (Table10) (Dela Rosa et al 2001). Two-line hybrid rice breeding Two promising low-csp TGMS lines (IR S and IR73834S) at IRRI are being used to make testcrosses for preliminary evaluation of two-line hybrids. Preliminary observations of two-line hybrids derived from the two TGMS lines showed much higher frequency (77%) of the heterotic combinations than three-line hybrids (20 30%). In Vietnam, more than ten two-line rice hybrids were tested in the national trials in TM4 (11S/MH86) was a promising combination, whose yield in the national trials was similar to that of the check (three-line, highest-yielding hybrid from China). Grain quality was better than that of three-line hybrids. Procedures for F 1 seed production were established and seed yield reached 2.2 t ha 1. TM4 will be released in the near future (Hoang et al 2000). Experiments to determine the sowing 46 Tong-Min Mou et al

52 Season Activity Condition 1 Crosses made between IR58025Bms and TGMS lines possessing tms 2 2 F 2 grown and harvested 3 F 2 families grown Desirable fertile F 2 plants selected Fertility-inducing conditions 4 Selected F 3 families grown Completely sterile F 3 families selected Sterility-inducing conditions 5 Remaining seed from selected sterile F 3 families grown F 3 families segregating for fertility/sterility selected Fertile plants (tms 2 tms 2 MsMs, tms 2 tms 2 Msms) Fertility-inducing conditions 6 F 4 bulk families grown Sterile plants (tms 2 tms 2 msms) crossed with fertile/partially fertile plants (tms 2 tms 2 MsMs, (tms 2 tms 2 Msms) Fertile/partially fertile plants used as male parent harvested Fertility-inducing conditions 7 F 1 s and male parents planted side by side in single rows Fertile-sterile ratio of 1 tms 2 tms 2 Msms:1 tms 2 tms 2 msms in F 1 and 3 tms 2 tms 2 Msms:1 tms 2 tms 2 msms in corresponding male parent selected Crosses made between sterile and fertile plants from plots with 1:1 fertile-sterile ratio Fertility-inducing conditions 8 Remaining F 1 seed and crosses made from previous season planted Sterile plants (tms 2 tms 2 msms) crossed with fertile plants (tms 2 tms 2 Msms) through natural crossing and handcrossing Fertility-inducing conditions (26/21 C in phytotron) Fig. 3. Development of a TGMS composite population using genetic male sterility at IRRI. Two-line hybrid rice breeding in and outside China 47

53 Table 8. Promising TGMS lines identified in India. Location TGMS CSP a CFP Remarks line ( o C) ( o C) Hyderabad DRITG Good outcrossing DRITG Good outcrossing DRITG Good outcrossing MLTG Good, early, higher outcrossing Coimbatore TS Good outcrossing Pantnagar UPRI Good plant type UPRI Good plant type a CSP = critical sterility point, CFP = critical fertility point. time for TGMS multiplication were carried out at VASI, Vietnam, from 1996 to 1998 for TGMS7 and TGMS11. The results suggested that the best season for multiplying TGMS lines was December and the crop flowered in April. This time, temperature increased to about 25 C and humidity and sunlight also increased. TGMS yield ranged from 1,876 to 3,657 kg ha 1. The results of multiplication in mountainous areas (Sapa or Dalat) in the summer season showed that yields were t ha 1. Constraints and future outlook The two-line hybrid rice breeding approach has been developed successfully over two decades in China. Intensive efforts have been made in developing different kinds of rice EGMS sources and lines through mutations, screening of existing elite varieties, and hybridizations outside China. Although great progress was made in the late 20th century, we still face some constraints: The average yield potential in commercial two-line combinations in China is not significantly higher than in three-line hybrid rice. The yields and costs of seed multiplication and production have been similar to those of three-line hybrid rice up to now in China. Training and experience of researchers are insufficient in two-line hybrid rice breeding in tropical countries. Low-CSP TGMS lines are lacking in tropical countries. To exploit the advantages of two-line hybrid rice over three-line hybrid rice, research on the following areas should be strengthened: Developing intersubspecific rice hybrids to enhance the heterosis of twoline rice hybrids. Further improving the package of seed multiplication and production technology to increase yield and decrease costs for farmers benefit. Marker-aided selection can be used to expedite the breeding of EGMS lines. More intensive efforts should focus on developing low-csp TGMS lines in the tropics for stable expression of sterility in seed production seasons. 48 Tong-Min Mou et al

54 Table 9. Duration of male sterility of TGMS lines under natural conditions at Hanoi, Vietnam. TGMS Date of starting and ending of male sterility of some TGMS lines Days (no.) VNTGMS6S 10 May-30 Sept. 15 May-30 Sept. 18 May-30 Oct. 16 May-30 Sept. 1 May-9 May 140 VNTGMS7S 10 May-15 Oct. 15 May-10 Oct. 18 May-5 Oct. 16 May-30 Sept. 1 May-10 Oct. 140 VNTGMS8S 10 May-15 Oct. 15 May-10 Oct. 18 May-5 Oct. 15 May-30 Sept. 1 May-5 Oct. 135 VNTGMS11S 10 May-15 Oct. 15 May-10 Oct. 18 May-5 Oct. 15 May-30 Sept. 5 May-5 Oct. 134 T24S 20 May-5 Oct. 16 May-30 Sept. 5 May-30 Sept. 136 Two-line hybrid rice breeding in and outside China 49

55 Table 10. Sterility expression of several promising TGMS lines at PhilRice-Los Baños, Philippines. a TGMS line 1999 WS 2000 DS 2000 WS IR32364 CS PS/PF PF/F NorinPL12 CS ID 24 CS PS/PF IR CS/PS CS PS/F IR PS/CS PS/CS PS/CS TGMS 6-Maligaya CS TGMS 7-Maligaya CS TGMS 8-Maligaya CS TGMS 9-Maligaya CS a CS = complete sterility, PS = partial sterility, PF = partial fertility, F = fertile. References Serious efforts should be made to develop EGMS lines in japonica and basmati rice in which restorer genes for CMS lines are either absent or infrequent among the elite inbred lines for developing commercial hybrids. Dela Rosa AM, Masajo TM Development of TGMS based two-line hybrids at PhilRice- Los Baños and UPLB. In: Redoña ED, Gaspar MG, editors. Hybrid rice in the Philippines: progress and new horizons. Proceedings of the 2nd National Workshop on Hybrid Rice, November PhilRice, Maligaya, Muñoz, Nueva Ecija, Philippines. Dong NV, Subudi PK, Luong PN Molecular mapping of a rice thermosensitive genic male sterile (TGMS) gene by AFLP, RFLP, and SSR techniques. Theor. Appl. Genet. 100: Hoang TM, Nghien TN, Lam QD, Nguyen TT, Vu TT, Pham TT, Tran DQ Development of two-line rice hybrids. In: Bui BB, editor. Rice research and development in Vietnam for the 21st century. Proceedings of the conference on rice research and development in Vietnam for the 21st century: aspects of Vietnam-India cooperation, Cantho, Vietnam, September Jia JH, Zhang DS, Li CY, Qu XP, Wang Q, Weng ML, Wang B Constructing a genetic linkage map and mapping a new thermosensitive genic male sterile gene (tms 5 ) in rice. (In press.) Krishnaiah K, Viraktamath BC, Ilyas Ahmed M, Vijayakumar CHM, Ramesha MS Recent developments in hybrid rice research in India. In: Peng S, Hardy B, editors. Rice research for food security and poverty alleviation. Proceedings of the International Rice Research Conference, 31 March-3April 2000, Los Baños, Philippines. Los Baños (Philippines): International Rice Research Institute. p Lang NT, Subudhi PK, Virmani SS, Huang N, Brar DS Development of PCR based markers for thermosensitive genetic male sterility gene tms 3 (t) in rice. Rice Genet. Newsl. 14: Tong-Min Mou et al

56 Lang NT, Subudhi PK, Virmani SS, Brar DS, Khush GS, Huang H Development of PCR based markers for thermosensitive genetic male sterility gene tms 3 (t) in rice (Oryza sativa L.). Hereditas 131: Li, BH, Deng HF Discovery of and preliminary studies on AnnongS-1. In: Ministry of Agriculture of the People s Republic of China, editor. Proceedings of PGMS and TGMS line breeding in rice and heterosis application of between subspecies varieties. Beijing: p Li WM, Chen QF, Hao JM Breeding strategy of indica PGMS line. J. Fujian Agric. Univ. 24(4): Lopez MT, Tojinda T, Vanavichit A, Tragoonrung S Introgression of thermosensitive male sterility gene to aromatic Thai rice. Poster presented during the 4th International Rice Genetics Symposium, October 2000, IRRI, Los Baños, Laguna, Philippines. Lopez MT, Virmani SS Development of TGMS lines for developing two-line rice hybrids for the tropics. Euphytica 114: Mei MH, Chen L, Zhang ZH pms3 is the locus causing the original photoperiodsensitive male sterility mutation of Nongken 58S. Sci. China (Series C) 42(3): Mou MT, Li CH, Yang GC, Lu XG Breeding and characterizing indica PGMS and TGMS lines in China. In: Virmani SS, Siddiq EA, Muralidharan K, editors. Advances in hybrid rice technology: Proceedings of the Third International Symposium on Hybrid Rice, November 1996, Hyderabad, India. Los Baños (Philippines): International Rice Research Institute. p Reddy OUK, Siddiq EA, Sarma NP, Ali J, Hussain AJ, Nimmakayala P, Ramasamy P, Pammi S, Reddy AS Genetic analysis of temperature-sensitive male sterility in rice. Theor. Appl. Genet. 100: Shi MS Preliminary report of breeding and utilization of late japonica natural double purpose line. J. Hubei Agric. Sci. 7:1-3. Subudhi PK, Borkakati RP, Virmani SS, Huang N Molecular mapping of a thermosensitive genetic male sterility gene in rice using bulked segregant analysis. Genome 40: Tran DV Closing the yield gap for food security. In: Peng S, Hardy B, editors. Rice research for food security and poverty alleviation. Proceedings of the International Rice Research Conference, 31 March-3 April 2000, Los Baños, Philippines. Los Baños (Philippines): International Rice Research Institute. p Viraktamath BC, Virmani SS Expression of thermosensitive genic male sterility in rice under varying temperature situations. Euphytica 122: Virmani SS, Voc PC Induction of photo- and thermo-sensitive male sterility in indica rice. Agron. Abstr Wang B, Xu WW, Wang JZ, Wu W, Zheng HG, Yang ZY, Ray JD, Nguyen HT Tagging and mapping the thermosensitive genic male sterile gene in rice (Oryza sativa L.) with molecular markers. Theor. Appl. Genet. 91: Wang B, Wang JZ, Xu WW, Tagging and mapping rice photoperiod sensitive genic male sterile gene with molecular markers. Plant Genome IV. San Diego, 57. Yamaguchi Y, Ikeda R, Hirasawa H, Minami M, Ujikara A Linkage analysis of thermosensitive genic male sterility gene, tms 2, in rice (Oryza sativa L.). Breed. Sci. 47: Two-line hybrid rice breeding in and outside China 51

57 Zhang KM, Zhou CX, Liu JB A summery of the research on the multiplying technique by cold-water irrigation of TGMS rice with low critical sterile temperature in Hybrid Rice 2: Zhang Q, Shen BZ, Dai XK, Mei MH, Saghai-Maroof MA, Li ZB Using bulked extremes and recessive class to map genes for photoperiod sensitive genic male sterility in rice. Proc. Natl. Acad. Sci. USA 91: Zhou TB, Xiao HC, Lei DY, Duan QZ The breeding of indica photoperiod male sterile line. J. Hunan Agric. Sci. 6: Notes Authors addresses: Tong-Min Mou, National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan , China; Lu Xing-Gui, The Institute of Crop Breeding and Cultivation, Hubei Academy of Agricultural Sciences, Wuhan , China; N. T. Hoan, Hybrid Rice Research Center, VASI, Hanoi, Vietnam; S.S. Virmani, International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines. Citation: Virmani SS, Mao CX, Hardy B, editors Hybrid rice for food security, poverty alleviation, and environmental protection. Proceedings of the 4th International Symposium on Hybrid Rice, Hanoi, Vietnam, May Los Baños (Philippines): International Rice Research Institute. 407 p. 52 Tong-Min Mou et al

58 Opportunities for and challenges to developing and using hybrid rice technology for temperate countries T. Tsuchiya, A. Bastawisi, Z.Y. Yang, H.P. Moon, J.A. Mann, and H. Ikehashi Despite the adverse climate for cereal breeding under the sufficient supply of cereals, breeding of hybrid rice was reestablished through the 1990s, and a series of new hybrids has been released and have shown a remarkable performance in Japan, the United States, and the temperate region in China, thus demonstrating an enormous potential for hybrid rice in the area. Superior experimental hybrids have been developed in Egypt and Korea. In that development, the private seed industry is playing a key role, as has been shown in India and China, while public institutes are still in a leading position in Egypt and Korea and for japonica hybrids in China. Even in Japan, where incentives for cereal production have been very low, a few private companies are trying to establish hybrid rice breeding in addition to Mitsui Chemicals Inc., which released hybrids to the market. Initiatives by the private sector seem to be decisively important in the development of hybrids. Strong demand for high rice grain quality is also common in the countries and the region being considered. The new series of hybrids is now much closer to the high quality standards than preceding ones. Hybrid rice technologies are not simple because of the coexistence of the two subspecies and the lack of heterogeneity in japonica types in temperate countries. Breeding methods are diversified because both indica and japonica types or intermediate types are planted in the countries. There is a strong preference for japonica-type rice except for the United States. The japonica characteristic is also essential for cultivation in cooler climates, though adaptability to such climates is not strongly required in Egypt and the southern states of the U.S. While the broad diversity in the U.S. hybrid programs makes it easy to derive intersubspecific hybrids, which may account for the early success in high-yielding hybrids, the development of a heterotic genetic background is a central issue in the seed production system for japonica-type hybrids. 53

59 Egypt is a unique country for rice with both indica and japonica types. The favorable rice climate characterized by a clear sky, high solar radiation, low relative humidity, and intermediate temperature and the controlled irrigation system allow Egypt to be one of the leading rice-producing countries, with a national average of 9.3 t ha 1 in Rice cultivation is limited to million hectares in Egypt, with one rice crop a year. Water is a restriction for rice cultivation. With high-yielding hybrids, rice area can be reduced without reducing total production. In China, japonica hybrid rice is planted to about 230, ,000 ha, about 3 4% of the total japonica rice area. Japonica rice is preferred by the people in the southeast coast, while the japonica hybrid is more preferred in South China than in North China, where the grain quality of typical japonica inbred rice is more preferred. Recently, because of the changed diet preference, the japonica rice-eating population is increasing with expanding markets in South China. Accordingly, some seed companies in North China have embarked on seed production of japonica hybrid rice and the products of japonica hybrids will be supplied to the southern markets. Japonica hybrid rice breeding has mostly been conducted in public institutes, but it is now also being done in the private sector and by seed companies. In Korea, 5 million t of rice are produced annually on a polished rice basis from 1.5 million ha. This yield is the highest in the world, with an average of 6.5 and 9 t ha 1, respectively, for japonica-type and super-high-yielding rice in Although the area for rice is decreasing, surplus rice increases yearly because of declining consumption and increasing productivity per unit area. Thus, more emphasis is placed on better marketing and eating quality than on yield. Hybrid rice is studied for food security in the public sector in view of the decrease in rice cultivation areas in the future. Increasing productivity per unit area is recognized to be important. One way to achieve this is to develop hybrids. In Japan, more than 10 million t of rice are produced annually on a brown-rice basis, and more than 700,000 t are imported under the WTO treaty. Domestic rice consumption is decreasing year by year, while domestic rice production adjusts to consumption by reducing the area of rice cultivation. Accordingly, farmers income from rice production is declining. Hybrid rice is expected to help progressive farmers recover their income through its high yield if it is accepted for good taste and grain quality. Mitsui Chemicals Inc. (MCI) is presently the only private company that sells hybrid seed of rice in Japan, although hybrid rice breeding has been attempted by only a few private companies and public institutions. In the U.S., consumption and production of rice have increased at 5% annually for the past several decades. In 1994, 8.9 million t of rice were harvested from 1.33 million ha. Rice production in the U.S. accounts for only a few percent of world production, but a significant part of world trade. Private commercial seed and agricultural chemical producers have played key roles in the rice industry by supplementing public research and extension systems. The first commercial rice hybrid in the U.S. was released by RiceTec, Inc. in This hybrid was sold on about 5,000 ha that year, and on average outyielded the competing varieties by 18 20%. Two more improved hybrids are envisaged for marketing in Tsuchiya et al

60 Early attempts at hybrid rice breeding in each country In Egypt, hybrid rice research began in 1982 in testing Egyptian rice varieties for restoring ability to some Chinese cytoplasmic male sterile (CMS) lines, and none of them showed restoring ability (Maximos and Aidy 1994). During the 1980s and early 1990s, rice hybrids from IRRI and foreign companies were evaluated and found to be either inferior to local inbred late-maturing varieties or poorly adapted with unacceptable grain quality (Bastawisi et al 1998). Hybrid rice breeding was reorganized in 1995; since then, a series of new hybrids has been identified with locally developed restorers and CMS lines from IRRI. In the temperate region of China, japonica-type restorer line C57 was developed by a bridging cross of indica(ir8)/japonica//japonica and a three-line system was completed in Since then, the production of japonica hybrid rice has been intensively promoted. A series of japonica CMS lines and related rice hybrids, such as Li-You 57, Xiu-You 57, etc., were released for commercial production. Because the restorer line C57 has the indica genetic background from IR8, hybrids with C57 as a restorer line showed a significant yield increase, more than 15% over that of improved inbred japonica varieties. After the initial success, a period of stagnation occurred because of seed mixtures and grain quality problems. But, since the mid- 1990s, japonica hybrid rice breeding has been reorganized as mentioned in the following section. In Korea, research on hybrid rice started in the early 1970s and then a national program of hybrid breeding began in the early 1980s. Since then, the Korea-IRRI collaborative research project has played a significant role. Hybrid breeding was active in 1985 when the wild abortive (WA) CMS system was introduced. Several tongil-type cultivars and lines such as Milyang 23 were identified as maintainers, while most tongil-type cultivars showed good restoring ability for WA CMS. Japonica CMS lines were developed by transferring BT-CMS into elite Korean cultivars and breeding lines in 1994, and japonica restorer lines were developed by backcrossing with some success (Heu et al 1985, Heu and Koh 1990, Virmani et al 1990). In Japan, a program of hybrid rice development at MCI began in In the initial stages, more than 200 varieties of hybrids were introduced from China and evaluated. Some of the Chinese hybrids showed high yields, with increases of 20 30% over those of conventional Japanese cultivars, but they were not acceptable to the market because of their cooking and grain quality. Then, MCI reorganized the program in 1990 with a clear focus on acceptable grain quality. Meanwhile, at public institutes and research centers of agricultural cooperatives, hybrids were tested to use the heterosis of indica-japonica crosses. There were some encouraging results, but those hybrids were not released because of instability of pollen fertility under cool weather. Besides, some genetic studies were continued at public institutes to identify temperature-sensitive genic male sterility and wide-compatibility genes to solve the sterility of indica-japonica hybrids. Experimental hybrids with high heterosis were also developed at a national center (Takita 1999, Takita et al 2001). Opportunities for and challenges to developing and using hybrid rice technology for temperate countries 55

61 In the U.S., hybrid rice development began in An agreement between the Chinese Government, through the Hunan Hybrid Rice Research Center (HHRRC), and Occidental Petroleum, the owner of Ring Around Products (RAP), allowed RAP to initiate development efforts. A 10-year germplasm agreement gave RAP access to the HHRRC parent-line materials, specifically sources of wild-abortive (WA) cytoplasm and restorer lines. This agreement ended in 1990, with no commercial hybrids being developed. In 1987, RAP entered into a joint commercialization effort with RiceTec Research, a small seed company within Farms of Texas, which was called Hybrid Rice Inc. (HRI). But, serious purity problems with the parental lines and even more serious problems with the hybrids prevented the success of that venture, and it was abandoned in From that experience, however, Farms of Texas gained insights into and interest in hybrid rice and initiated breeding programs using RAP germplasm. When HRI was disbanded, all crosses and progeny using RAP germplasm were destroyed, but RiceTec still had access to two A/B lines then in the public sector, V20 and ZH97. A significant number of IRRI restorer lines (IR8, IR24, etc.) were also introduced by Dr. H. Beachell into RiceTec s variety breeding program in In fact, the restorer line for RiceTec s first commercial hybrid (XL6) was identified from a cross involving IR24 and U.S. materials. RiceTec, Inc. was formed in 1990 from the dissolution of Farms of Texas and had two component parts: a very small breeding program with four staff and a consumer rice business focused on specialty rice products (now Rice Select ). Hybrid breeding efforts were slow in the first two years, but in 1993 they were intensified by the addition of a new specialist and with an agreement with Professor Yuan and HHRRC, which gave RiceTec, Inc. access to CMS germplasm, technical assistance from HHRRC scientists, and exclusive marketing rights in certain countries in the Western Hemisphere. Then, RiceTec released the first commercial rice hybrid in the U.S. in In the U.S., hybrid rice efforts in the public sector have been limited and focused primarily on basic science. Dr. Neil Rutger continues his work on apomixis and the generation of genetic male sterile lines. Little progress has been reported on apomixis, but genetic sterile lines are available for release. Louisiana State University has reported the development of environmental genic male sterile (EGMS) lines by its tissue culture group, but no information is available on further research on those lines. Marketing of hybrids and their yield gains since the mid-1990s Despite the rather cool climate for increased cereal production under sufficient supplies of grains and the declining demand for rice, hybrid rice breeding has been reorganized in the temperate countries and in northern China. A new series of hybrids with much improved grain quality close to that of market standards has been released and has showed a remarkable level of yield increase over standard varieties. In Egypt, breeding of hybrid rice intensified after 1995 and 24 hybrid combinations were evaluated in yield trials. Out of these, two hybrids, SK Tsuchiya et al

62 (IR69625A/Giza 178 R) and SK 2046 (IR69625A/Giza 181 R), have been selected that showed at least a 15% yield increase over the best local inbred lines, with an advantage of 1.5 t ha 1. By 2005, these hybrids will be available to Egyptian farmers. In China, after the initial success of hybrid rice with the use of japonica-type restorer line C57 and the BT-type CMS source, a series of japonica CMS lines such as Liming A, Xiuling A, and the related rice hybrids, such as Li-You 57, Xiu-You 57, etc., were developed and released for commercial production. Up to the mid-1980s, the japonica hybrid rice-growing area in North China reached 130, ,000 ha annually. Then, because of the mixture of parental lines and poor grain quality, the total area of japonica hybrids decreased by the early 1990s. In the mid-1990s, japonica hybrid rice breeding made new progress and a new indica-inclined japonica restorer line, C418, with good combining ability was developed and contributed to a new series of R lines with tallness and lodging resistance and stable and high outcrossing characters. Recently, 10 hybrids with C418 as a restorer line have been released by national or provincial variety committees in different places of China, and they have showed a 10 15% yield increase over inbred varieties. In Korea, the breeding program for tongil-type hybrid rice was reduced because of problems with cooking quality. Still, WA-type CMS lines, maintainers, and restorers are being developed. Forty CMS lines have been developed during the past 6 years by transferring WA-CMS and COA-CMS sources into leading Korean cultivars and breeding lines. Of these 20 lines are tongil-type, derived from indica/japonica hybridization, and 17 lines have the genetic background of japonica. Several experimental hybrids outyielded elite cultivars by about 4 34% until The most promising rice hybrid was a combination of IR58025A/Cheongcheongbyeo, which yielded 12.4 t ha 1. Suweon-Jabjong 1 and Suweon-Jabjong 2 are the first developed hybrid rice lines at the National Crop Experiment Station. The hybrids showed field resistance to major diseases and insects (Table 1). Commercial rice hybrids are not yet available to farmers because of poor grain quality and labor-intensive seed production systems. However, hybrid rice research restarted in 2000 to attain 10 t ha 1 of milled rice as a long-term project for food security in the 21st century. In Japan, Mitsui Chemicals Inc. (MCI) reorganized its hybrid rice program in 1990 and screened five restorer lines (R1 to R5) from those introduced from China on the basis of relatively low amylose content (Table 2). Many combinations between Japanese cultivars (F) as female parents and the five male Chinese R lines (R1 R5) were tested as shown in Figure 1. Then, the company released two commercial hybrids, MH2003 and MH2005, for the first time in Japan. They showed a remarkable yield advantage and good taste (Table 3, Tsuchiya et al 1997). MH2003 showed an extremely high yield, with an average of t ha 1 and maximum of t ha 1 in each year from 1996 to The increase rates were 36% to 51% on average and sometimes reached 82% to 120%, nearly two times the national average. MH2005 also recorded a high yield, with an average of t ha 1 and maximum of t ha 1. The taste score of MH2005 for each production year was the same as that of Koshihikari, the most preferred rice in Japan. Grain yield and taste are listed in Table 4. MH2003 and MH2005 have the characteristic traits of Opportunities for and challenges to developing and using hybrid rice technology for temperate countries 57

63 Table 1. Super-high-yielding hybrid rice developed by using cytoplasmic genetic male sterility. Designation Combination Growth Culm Grain yield (milled) Remarks b duration a length (d) (cm) Potential Index to (t ha 1 ) standard variety Suweon- SR16284A/ Both are Jabjong 1 Yongmoonbyeo mutiresistant to BL, BB, Suweon- SR16283A/ SV, and BPH Jabjong 1 Taebaegbyeo a Growth duration indicates the growing days from transplanting to harvest. b BL = blast, BB = bacterial blight, SV = stripe virus, BPH = brown planthopper. Table 2. Traits of five restorer lines from China. Heading a Yield Good Amylose (t ha 1 ) grain (%) (%) R1 1 Aug R2 28 Jul R3 6 Aug R4 18 Jul R5 25 Jul 45.0 a Sowing date 17 Apr., transplanting 8 May. modern Japanese cultivars: late maturity, long culm, long panicle, fewer panicles, significantly high yield, and a satisfactory taste similar to that of Koshihikari (Nakamura et al 1997). Some public research centers continued to breed experimental rice hybrids by combining the genetic background of indicas and japonicas. One such experimental line, THR1, showed a mean yield increase of 26% against the standard for (Takita 1999). But such lines have not yet been marketed. In the U.S., the first commercial rice hybrid, XL6, was released by RiceTec in XL6 was a cross between a Chinese indica A line and a RiceTec-derived R line, which contained IR24 in its parentage. This hybrid was sold on about 5,000 ha that year and on average outyielded competing varieties by 18 20%. From an agronomic perspective, it was not successful because of high lodging in about one-third of fields and poor milling yields in most fields. In 2002, RiceTec was releasing two new hybrids, XL7 and XL8, for U.S. farmers. XL7 shows a yield advantage over Cocodrie, the current best commercial variety, of 1.2 t ha 1 (Fig. 2). XL8 has shown an advantage of 1.8 t ha 1 over Cocodrie (Fig. 3). Both hybrids have lower milling yields than Cocodrie and the commercial advantage is thus lower than that indicated 58 Tsuchiya et al

64 No. of varieties >90% <50% Percentage of good grain of HR tested F/R1 F/R2 F/R3 F/R4 F/R5 F R Combination Amylose percentage of HR tested No. of varieties F/R1 F/R2 F/R3 F R Combination <13% >20% Taste evaluation of HR tested based on actual consumption No. of varieties F/R1 F/R2 F/R3 Combination Fig. 1. The yield, taste, and appearance of hybrid rice by combination. Scale < Check by just the yield advantage. The commercialization of XL6, XL7, and XL8 marks the beginning of the hybrid rice era in the U.S. While at this point only one commercial enterprise is marketing hybrids, the expectation exists that other companies will soon begin their own programs. Public-sector programs are interested, but may not have the resources to invest in the technology. RiceTec has established a large and talented group of scientists and is committed to making hybrid rice a viable and valuable product for U.S. farmers. Opportunities for and challenges to developing and using hybrid rice technology for temperate countries 59

65 Table 3. General traits of MH2003 and MH2005. Trait MH2003 MH2005 Growth period (d) Culm length (cm) Panicle length (cm) Panicle number Grain yield (t ha 1 ) Appearance a 4 3 Eating value b Taste score c Blast resistance gene Pi-a, Pi-I Pi-a, Pi-I a Appearance is ranked on a scale of 1 (excellent) to 9 (very bad). b Eating value means the value in the evaluation by actual eating. c Taste score was mechanically measured by the Taste Meter SATAKE TB-1A. Table 4. Grain yield and taste were measured for MH2003 and MH2005 produced on farms located in the Kanto to Kyusyu area. Year MH2003 MH2005 Check a Yield Increase Taste Yield Increase Taste Yield Taste (t ha 1 ) rate (%) score b (t ha 1 ) rate (%) score b (t ha 1 ) score 1996 Max Av Max Av Max Av Max Av Max Av a For the check, yield (t ha 1 ) means nationwide and the taste score showed the results for Koshihikari. b Taste score was measured mechanically by the Taste Meter SATAKE TB-1A. Constraints and solutions in breeding hybrids for diverse rice agronomy and markets As shown above, hybrid rice has been brought to markets for the first time in temperate countries besides China. However, there is still a long list of challenges for breeders to advance hybrids to wider agronomic conditions and diverse markets. In Egypt, some hybrid combinations have short, medium, and long grain with a moderate degree of acceptance for grain quality. Consumers prefer short grain with low amylose content. So, short-grain indica types need to be improved for grain quality. Thus, hybrids with a japonica genetic background are envisaged. 60 Tsuchiya et al

66 Grain yield (t ha 1 ) Research Development Farm tests University Overall tests 33 XL7 Cocodrie No. of tests No. of tests Fig. 2. In 144 replicated head-to-head comparisons over 3 years, XL7 has a 1.2 t ha 1 yield advantage over Cocodrie Grain yield t (ha 1 ) Research Development Farm tests University Overall tests XL8 Cocodrie No. of tests Fig. 3. In 114 replicated head-to-head comparisons over 3 years, XL8 has a 1.8 t ha 1 yield advantage over Cocodrie. In China, the size of markets for hybrids with japonica-type taste is narrow and seems to be restricting the participation of private seed producers. The increasing consumption in southern China of japonica-type rice will pave the way for seed producers, as the new series of hybrids with C418 is highly promising. In Korea, as mentioned before, the development of hybrid rice technology has not been encouraged as a research priority because of low quality in comparison with Korean japonica and tongil-type varieties. Also, japonica restorer lines showed partial fertility restoration and limited yield heterosis of 5 10%. In the research, TGMS showed an inadequate response to natural conditions, while the breeding of commercially usable hybrids requires long initial stages. Opportunities for and challenges to developing and using hybrid rice technology for temperate countries 61

67 In Japan, selection for market acceptability has been the focus in the MCI hybrid project. Rigorous selection was practiced for restorers from China and only five lines were selected on the basis of low amylose content and grain quality. More than 400 hybrids were tested over a period of three years between the five restorers, R1 R5, and Japanese cultivars as female parents. Among the hybrids, hybrids resulting from combinations of F/R1 showed superior potential in yield, taste, and grain characteristics (Fig. 1). Specifically, some hybrids showed high yields of more than 11 t ha 1. One had an amylose content of less than 18% and scored more than 2.0 for eating value, based on its taste traits. The grain quality indicated by the percentage of good grain had a strong correlation with that of restorer lines (Oka et al 1994). Most of the hybrids derived from the F/R1 combinations showed more than 90% good grain, even though the good grain ratio of R1 is 83.6%. From the F/R1 combinations, two elite rice hybrids were registered as Mitsuhikari 2003 (MH2003) and Mitsuhikari 2005 (MH2005) in the variety registration report. In the U.S., greater emphasis has to be placed on grain quality and on features required for mechanized agriculture. U.S. quality standards are strict, requiring specific length/width ratios, amylose content, and amylographic characters, and low chalk and good grain appearance. Hybrids must come close to meeting the criteria established by varieties. In addition, lodging tolerance is more an issue in drill-seeded conditions than in transplanted situations, partly because of the different plant systems, and partly because of the higher plant stands in U.S. farmers fields as compared with ones in China or Vietnam. Indica germplasm tends to be annual in nature and to move most or all stored carbohydrates into the seeds. This results in lodging potential and under U.S. farmer conditions becomes completely unacceptable. U.S. adapted lines are generally tropical japonicas, have good lodging tolerance, and thus provide opportunities for improvement in lodging potential of hybrids. In the commercial yield trials harvested at nine locations from 29 o 30 N (El Campo, Texas) to 36 o 35 N (Essex, Missouri), XL6 had an average lodging index of 35, equivalent to 35% of the crop being lodged flat on the ground. XL7 and XL8, released 2 years later, had average scores of 24 and 6, respectively. The best U.S. varieties show scores of about 2 4% lodging. Milling is a more difficult issue, but XL8 in particular has a higher milling potential than either XL6 or XL7. The issues of milling and lodging are related in the sense that nitrogen is positively related to milling yield and negatively related to lodging. The development of hybrids such as XL8, which have lodging resistance, allows for the application of higher N rates, and thus offers potential for improved milling. Hybrid rice seed production The production of hybrid seeds is a central issue of hybrid rice breeding, ranging from synchronization of both parents to outcrossing ratios of parental types. In Egypt, CMS maintenance and F 1 seed production for experimental research began in The initial work for purification of parental lines started in Three hybrid combinations were used for training the researchers and technicians 62 Tsuchiya et al

68 involved in seed production. For hybrid rice seed production, some problems such as synchronization of flowering and purification of parental lines are being solved. In China, since the total area for japonica hybrid rice is small compared with that of indica hybrids, seed companies cannot obtain large profits from japonica hybrid rice seed production, so most of the japonica hybrid seeds were produced by research institutes or state-owned farms on a very limited scale. The poor cooperation between breeding institutes and seed companies caused the slowing down of japonica hybrid rice development. In Korea, two new CMS lines, IR62829A (IRRI) and SR16282A (Korea), were evaluated for seed production with different row ratios. IR62829A had the highest seed production, with 2.2 t ha 1 with a row ratio of 1:1 for CMS and maintainer lines. SR16282A had a seed yield of around 1.5 t ha 1. These results indicate a fairly high level of hybrid seed production. In Japan, MCI has attained success in the on-farm production of rice hybrids MH2003 and MH2005 and the seeds harvested are already available to be sold to farmers. A great deal of labor is required to attain success using the general methods of seed production mainly developed in China. For example, there is transplanting, flag-leaf clipping, gibberellic acid (GA 3 ) spraying, supplementary pollination, roguing of contamination, and separate harvesting of the males and females, etc. However, labor cost is very high in Japan. To attain higher seed production, reduced production costs, and lower seed price, several improvements are necessary in a way different from the Chinese system. MCI has succeeded in building a mechanical seed production system and in achieving a harvest efficiency of 1 to 1.5 t ha 1 on-farm, and is making full use of convenient agricultural machinery, except for supplementary pollination and roguing work. In other words, farmers are actually using sowing machines, transplanters, small combines, sprayers for GA 3, and multipurpose managing machines. In the U.S., the mechanized cultural system used also adds to the problems that challenge U.S. hybrid breeders. Whereas a Chinese breeder works in a system in which average hybrid seed usage is kg ha 1, the U.S. system uses kg ha 1 seed. While in Asia rice is transplanted and seed production fields might be 1 5 ha in size, the U.S. seed production field is drill-seeded and from 10 to 1,000 ha. In a Chinese production field, the distance across a female bay is m; in the U.S., the bay widths for females vary from 6 to 10 m and are combine-harvested. So, while breeding techniques are usually similar to those employed by breeders in China or India, the list of absolute requirements is much longer, and thus the breeding techniques and the time frames for selection are often expanded. Breeding methods In Egypt and the southern states in the U.S., where both indica and japonica types are planted, introduced CMS lines from the wild rice origin (WA CMS) have been used for hybrid rice seed production. Both japonica- and indica-type CMS lines are tested Opportunities for and challenges to developing and using hybrid rice technology for temperate countries 63

69 in Korea, where the breeding of tongil types provides a unique background for breeding strategies. In Egypt, some improved CMS lines introduced from IRRI were screened out, while restorer lines were chosen from indica-type breeding lines. Earlier work had helped to identify a large number of maintainer/restorer sources from the breeding lines for the development of parental lines. Sources for hybrids are evaluated at various stages of testing in different nurseries source nursery, testcross nursery, backcross nursery, and combining ability nursery and in a series of yield tests. Among them, japonica hybrids still show little heterosis even in japonica/tropical japonica crosses. To solve this problem, we attempted to introduce indica/japonica crosses by transferring the wide compatibility (WC) gene to one of them. Thus, a parental line improvement program began that included restorer development and the development of lines with the WC gene for use in indica/japonica hybrid development. Further breakthroughs should be made by breeding CMS lines and restorers by testing combining ability. Breeding methods in the U.S. follow to a large degree those in China, but some selection criteria are different or have added importance because of a different customer base. WA cytoplasm is most common in U.S. hybrids, but other cytoplasm exists at least in breeding populations. Most of the early A lines used by RiceTec had South China indica backgrounds, but that is being modified over time to meet quality standards. R lines were initially based on IRRI restorer lines, but are being modified to meet U.S. quality and agronomic standards. Standard pedigree breeding methods are used for most of the U.S. breeding efforts. In total, efforts range from markerassisted breeding to population development and recurrent selection, but most of the efforts and, to date, all of the hybrids have been carried out with pedigree breeding. RiceTec is involved in the International Rice Micro-Satellite Initiative (IRMI), a consortium involving Cornell University, IRRI, China, and Japan, and is thus focused on making molecular markers an important part of its breeding efforts. In China, a very famous restorer, C57, was obtained by IR8/japonica type// japonica type by Professor Yang, who named this method a bridging cross. Many restorers that were developed on the basis of C57 retained some indica genetic background and revealed a higher level of heterosis in their crosses to japonica-type CMS lines. As mentioned before, much-improved restorers have been developed on the basis of C57. The WC genes have been introduced into the improved restorers. On the basis of C57-derived restorers and through the introduction of WC genes, many restorer lines with a more or less indica-type genetic background and improved quality were developed, some of which were evaluated in MCI s trials. In Korea, both WA and BT CMS lines are used to develop hybrid seed. So far, 19 CMS lines have been produced by WA and BT CMS (Table 1). The WA-CMS of V20A, Zhen Shan 97A, and V5-20A was transferred into tongil-type maintainers, while some japonica-type CMS lines were developed by transferring the WA-CMS of V20A, Reimei A, and BT-CMS into some elite japonica cultivars. The developed lines were found to show stable sterility. Restorer lines were selected from testcross nurseries based on the fertile reaction of the F 1 plants. Breeding lines and cultivars 64 Tsuchiya et al

70 developed locally were crossed with Z97A, V20A, and other tongil-type A lines. Similarly, some restorers were identified as japonica A lines, which were developed by backcrossing japonica types. Hybrids, on the basis of IR58028A, showed good results with yields of 9.1 and 7.6 t ha 1. This is a 12 34% increase in yield over that of the standard variety Namcheonbyeo, which yields 6.8 t ha 1. Further trials continue to develop new CMS lines with diverse cytoplasmic and nuclear genetic backgrounds by transferring the CMS factor(s), restoring-ability gene(s), and wide compatibility gene(s) to Korean tongil-type varieties. Also, maintainers and restorers are to be developed with new plant types and a tropical japonica background. An anther culture technique is used to conserve and propagate the yield vigor in F 1 hybrids. In Japan, after selecting the best hybrids among the Japanese cultivars as the female parent and the selected Chinese restorers, the female parents were converted into male sterile lines (CMS lines) by means of nucleus substitution (Akagi et al 1989, 1991). This is an indispensable process before producing a large quantity of hybrid rice seeds for establishing large-scale rice production tests and subsequent propagation on farms, although the backcross method is the conventional form of nucleus substitution. MCI has developed more than 30 new CMS lines by using protoplast fusion for japonica-type rice hybrids. The CMS lines for MH2003 and MH2005 were also derived by this method and are named MHA23 and MHA25, respectively. Experiences with the use of indica/japonica hybrids In the northern provinces of China and Japan, indica types are not adapted to the cool climate nor are they acceptable because of their cooking quality. Thus, hybrids with BT cytoplasm have been tested in the japonica background. As a single restorer gene is responsible for the CMS cytoplasm, the development of CMS lines and restorer lines is relatively simple. But it is not easy to find a highly heterotic combination for hybrids under the narrow genetic background of japonica rice. This is also a problem in Egypt, where the preference for the japonica type has to be considered in breeding. Another important point is that practically no restorer gene exists among the japonica types. If restorer genes are introduced from indica types, there is a problem of hybrid sterility, which appears to a varying degree in any indica/japonica crosses. Hybrid sterility is caused by heterozygous genotypes at some particular loci. In the typical indica-japonica crosses, sterility is basically caused by an allelic interaction at the S-5 locus near the pigmentation gene C on chromosome 6. A large portion of female gametes with a japonica-type allele are aborted in the genotype of S-5-i/S- 5-j, resulting in semisterile panicles (Ikehashi 2000). The problem of hybrid sterility in indica/japonica crosses has been overcome by two methods. One is to introduce the Rf gene from indica types while the indica hybrid sterility gene S-5-i is replaced by the japonica-type allele. As mentioned above, an excellent restorer (C57) was obtained by IR8/japonica type//japonica type by Professor Yang with his bridging cross method. In that three-way cross, the progeny may have had a fertile genotype of S-5-i/S-5-j and a semisterile genotype of S-5-i/S-5-j. The former type was selected together with the Rf gene of IR8. Opportunities for and challenges to developing and using hybrid rice technology for temperate countries 65

71 Another approach to solving hybrid sterility is to use a neutral allele S-5-n at the S-5 locus, which is known as the WC gene. Any indica or japonica lines with the S-5-n showed nearly normal levels of fertility in their crosses to indica or japonica lines, because the genotypes S-5-n/S-5-i or S-5-n/S-5-j are relieved of gamete abortion. Some japonica-type CMS lines with the WC gene were developed so that any indica types that possess the Rf gene are used as restorers. Several indica/japonica hybrids with high heterosis have been developed. But a serious problem with them is their lower levels of pollen fertility because of indica/japonica hybrid sterility, as there are several loci at which indica/japonica heterozygotes lead to partial abortion of male gametes. Such an abortion of pollen by hybrid sterility is shown less in indica/javanica crosses. The level of pollen fertility is especially affected by cool temperature at the time of gamete formation. Thus, the indica/japonica hybrids lacked stability in cool climate. But the introduction of the WC gene into indica or japonica breeding lines allowed extended genetic bases for hybrid rice breeding. Many restorers with some indica background and WCG have been developed in China. For instance, an excellent restorer line, Chenghui 448, has been developed by using the WCG of Lemont to produce superior hybrids such as D62A/ Chenghui 448 in Sichuan Province, China (Ren Guangjun et al 1999). The idea of a bridging cross is essentially the same as the idea of introducing the WC gene from javanicas. It is now clear that restorers with some indica-type genetic background and the Rf gene can be developed by introducing the WC gene and using repeated selection for japonica-type adaptabilities, and that a single combination of unimproved indica and japonica-type hybrids may not produce stable commercial hybrids. The two-line system In Egypt, the two-line system with either thermosensitive genic male sterility (TGMS) or photoperiod-sensitive genic male sterility (PGMS) has been tested by using materials provided by IRRI. Efforts are also being made to develop TGMS lines in the background of locally adapted varieties. Selected lines from 21 cross combinations in different generations were raised and promising selections were made for further evaluation and selection. In Korea, IRRI-bred TGMS lines will be evaluated at the national experiment station and the TGMS gene(s) will be transferred to local materials. In the U.S., there are few reports of the identification of TGMS. Contrastingly, China has a series of two-line commercial hybrids that contain the WC gene. The indica-japonica bridging technique has been used to create usable variation and to overcome the genetic barriers from indica-japonica hybridization, and then to introduce indica-japonica germplasm into heterosis breeding. By using this technique, two basic parental lines C418 from the F 5 of WanLun 422/Milyang 23 and Guangzhan 63S from N 422S/Guangzhan 63 have been selected. Guangzhan 63S belongs to the indica-inclined mid-growth-duration PTGMS line. It has stabilized sterility, a high outcrossing rate, good grain quality, and strong resistance to pests. This PTGMS line has a lower critical sterile-inducing temperature and shorter critical sterile-inducing daylength (photoperiod). Meanwhile, Guangzhan 63S has passed 66 Tsuchiya et al

72 the evaluation of experts from different provinces organized by the Anhui provincial agricultural bureau in Training of specialists In Egypt, training and human resource development are emphasized. With the help of FAO, training on hybrid rice technology, breeding, and seed production started in Training on CMS maintenance and F 1 seed production for experimental research began in Three hybrid combinations were used for training the researchers and technicians involved in seed production. In 2002, rice production specialists, rice growers, extension officers, and farmers will be trained on hybrid rice seed production and cultivation. In other countries, the participation of the private sector has practically solved the problem of training specialists as well as many other aspects of hybrid rice development. References Akagi H, Sakamoto M, Negishi T, Fujimura T Construction of rice cybrid plants. Mol. Gen. Genet. 215: Akagi H, Taguchi T, Fujimura T Stable inheritance and expression of the CMS traits introduced by asymmetric protoplast fusion. Theor. Appl. Genet. 91: Bastawisi AO, Aidy IR, El-Mowafy HF, Maximos MA Resesarch and development for hybrid rice technology in Egypt. In: Virmani SS, Siddiq EA, Muralidharan K, editors. Advances in hybrid rice technology. Manila (Philippines): International Rice Research Institute. p Heu MH, Cho YH, Kim HY Development of hybrid rice facilitated by cytoplasmic genetic male sterility: the yield heterosis of Korean-bred male sterile lines which have CMS-WA male sterility cytoplasm. Agric. Res. Seoul Natl. Univ. 10(suppl): Heu MH, Koh HJ Development of hybrid rice facilitated by cytoplasmic genetic male sterility: breeding CGMS and restorer lines in japonica rice. Korean J. Breed. 22(2): Ikehashi H Hybrid sterility in rice: its genetics and implication to differentiation of cultivated rice. In: Nanda JS, editor. Rice breeding and genetics. Science Publishers, Inc., USA. p Maximos MA, Aidy IR Hybrid rice research in Egypt. In: Virmani SS, editor. Hybrid rice technology: new developments and future prospects. Manila (Philippines): International Rice Research Institute. p Nakamura A, Oka M, Sano T, Arai N, Sawada R, Matsumura T, Akagi H, Tsuchiya T, Fujimura T, Samoto S Breeding of an elite hybrid rice line MH2005, with extremely high yield and good quality. Jpn. J. Breed. 47(suppl.1):177. (In Japanese.) Oka M, Sano T, Arai N, Matsumura T, Nakamura A, Sawada R, Tsuchiya T, Takahashi M, Fujimura T Appearance of brown rice on hybrid rice in the case of Japanese varieties as female parent. Jpn. J. Breed. 44(suppl.1):284. (In Japanese.) Ren Guangjun, Lu Xianjun, Li Qingmao, Zhang Chi Breeding and utilization of wide compatibility rice restorer line Chenghui 448. Chinese J. Rice Sci. 13(2): (In Chinese with English summary.) Opportunities for and challenges to developing and using hybrid rice technology for temperate countries 67

73 Takita T High yielding ability of japonica-indica hybrid rice. Tohoku J. Crop Sci. 42: (In Japanese.) Takita T, Terashima K, Yokogami N, Kataoka T Stable high yielding ability of japonicaindica hybrid rice. Paper presented at the Fourth International Symposium on Rice Genetics, October 2000, International Rice Research Institute, Los Baños, Philippines. Tsuchiya T, Matsumura T, Nakamura A., Oka M, Fujimura T, Samoto S, Taguchi T Characters of elite hybrid rice and practical use in the farmer s field. Jpn. J. Crop Sci. 66(extra issue1): (In Japanese.) Virmani SS, Young JB, Moon HP, Kumar I, Flinn JC Increasing rice yields through exploitation of heterosis. Paper presented at the International Rice Research Conference, Aug 1990, Seoul, Korea. Notes Authors addresses: T. Tsuchiya, Bio-Product Marketing Dept., Mitsui Chemicals, Inc., Kasumigaseki, Chiyodaku, Tokyo , Japan; A. Bastawisi, Rice Research and Training Center, ARC, Sakha, Kafr El-Sheikh, Egypt; Z.Y. Yang, North China Hybrid Rice Research Center, Shenyang, China; H.P. Moon, National Yeongnam Agricultural Experiment Station, Milyang, Korea; J.A. Mann, RiceTec, Inc., Alvin, Texas, USA; H. Ikehashi, College of Bioresource Sciences, Nihon University, Kameino 1,866, Fujisawa , Japan. Citation: Virmani SS, Mao CX, Hardy B, editors Hybrid rice for food security, poverty alleviation, and environmental protection. Proceedings of the 4th International Symposium on Hybrid Rice, Hanoi, Vietnam, May Los Baños (Philippines): International Rice Research Institute. 407 p. 68 Tsuchiya et al

74 Improving grain quality in hybrid rice F.U. Zaman, B.C. Viraktamath, and S.S. Virmani Grain quality preferences in rice vary from region to region and country to country. In most countries, long-grain indica rice, which is soft and nonsticky on cooking, is preferred; in others, low-amylose japonica rice, which is soft and sticky on cooking, is liked. Premium basmati rice is yet another group possessing some specific characteristic features, such as the presence of aroma, tenderness, and linear elongation on cooking. In the case of hybrids, quality considerations assume greater significance as their produce is formed by F 2 seed generation. Most of the hybrids commercialized in the tropics are based on IR58025A. Although these hybrids yielded 15 20% higher than high-yielding popular varieties, their grain quality did not find acceptability by consumers in some parts of India and Bangladesh. In some regions of India, the Philippines, and Vietnam, hybrids derived from IR58025A were widely accepted. This led to the development of hybrids to meet specific regional quality requirements. With this in view, as a first step, efforts have begun at IRRI and other centers to critically analyze the quality characteristics of all available CMS lines, maintainers, restorers, and their hybrids. This has helped to identify some new CMS and TGMS lines combining desirable quality characteristics for the development of hybrids. Simultaneously, the directed development of parental lines combining good grain quality, including basmati characteristics, has begun in India. Efforts in this direction have helped in the development of the first basmati highquality hybrid, Pusa RH-10. Among other hybrids released in India so far, ADT RH-1 possesses good grain quality. In China also, greater emphasis is being given to the improvement of grain quality of hybrids. Indica/japonica hybrids have shown high heterosis, but their grain quality seems to be a serious impediment. This has prompted the use of indica/japonicaderived lines to overcome quality problems. Studies on the effects of nucleocytoplasmic interaction on quality traits reveal that they vary from cross to cross. Cytoplasmic effects on chalkiness, head rice recovery, and amylose content need to be given due consideration when choosing parental lines for the development of hybrids. However, further studies to overcome the problem are required. 69

75 Grain quality in rice is of primary importance everywhere, more so because it is mainly consumed as whole rice, although preferences vary from region to region and country to country. The market price of rice is decided by its quality, which in turn determines economic return to farmers. The main attributes deciding quality are (1) high milling and head rice recovery, (2) grain shape and appearance, and (3) cooking and eating characteristics. In most countries long-grain indica rice, which is soft and nonsticky on cooking, is preferred; in others, low-amylose short-grain japonica rice, which is soft and sticky on cooking, is liked. Premium basmati rice is yet another group possessing some specific characteristic features, such as the presence of aroma, tenderness, and nonsticky linear elongation on cooking. For hybrid rice, quality considerations assume still greater significance as its commercial produce is formed by F 2 seed generation. Early commercial hybrids were mainly based on IR58025A. Although they yielded 15 20% more than prevalent high-yielding varieties, their grain quality did not find acceptability by consumers in some parts of India and Bangladesh. In some regions of India, the Philippines, and Vietnam, hybrids derived from IR58025A were widely accepted. This warranted the development of hybrids to meet specific regional quality requirements. Indica/japonica hybrids have shown quite high heterosis, but their grain quality seems to be a major impediment. This has prompted the use of indica/japonica-derived lines to overcome quality problems. Early studies on the effects of nucleo-cytoplasmic interaction on quality traits reveal that they vary from cross to cross. Hence, it may not be a serious problem, yet cytoplasmic effects on grain chalkiness, head rice recovery, and amylose need to be further analyzed. With this in view, many research efforts have been made focusing on parental lines at various hybrid rice research centers, including IRRI-Philippines and IARI-India. In this paper, progress in grain quality improvement in hybrid rice has been reviewed and problems needing further attention have been identified. Achievements in the improvement of hybrid rice grain quality The impression that hybrids ought to be poor in quality is not true. Success with new hybrids in India and at IRRI has shown beyond a doubt that, if parental lines are chosen carefully, grain quality as good as that of any good-quality conventional rice variety can be achieved. The release of Pusa RH-10, developed at IARI-India, is one such good example. It combines the most desirable basmati quality characteristics, considered to be a highly complex constellation of quality traits. The quality of early hybrids involving IR58025A was unacceptable in many parts of India and Bangladesh, mainly because of a lack of awareness of the quality of parental lines. Using parental lines with diverse quality traits, especially starch characteristics, showed segregation for some grain quality characteristics. This was revealed by Khush et al (1988) from their study using hybrids involving diverse parental lines. They concluded that genetic heterozygosity did not impair grain quality as long as both parents had good grain quality. Many other reports appearing later have also revealed that hybrids as such did not show impaired grain quality and 70 Zaman et al

76 hybrids with desired grain quality can be developed by careful selection of parents (Islam and Virmani 1992, Liang et al 1998). Often, quality preferences vary from region to region. Consumers in southern India do not like hybrid combinations involving IR58025A as the female parent, mainly because of the presence of aroma; whereas, in the eastern region, there is no such problem and hybrids are increasingly becoming popular. More recently, there has been greater emphasis on choosing parental lines carefully for grain, cooking, and eating quality. This has been possible with the availability of diverse CMS and restorer lines developed at IRRI and locally in different countries and regions. Some selected CMS lines possessing varied amylose content developed at IRRI and elsewhere are listed in Tables 1 and 2. In India, IR68895 and IR70369 possessing intermediate amylose have been found to produce hybrids with good grain quality. Restorer line KMR-3 had better milled rice recovery and yield than KRH-2. Both new hybrids are free from aroma, which is disliked by consumers in Karnataka (Balasundra and Vidyachandra 2002). Of the 22 hybrids developed by both the public and private sector in India, almost all had intermediate amylose, four recorded head rice recovery of %, and eight had long slender grain. Overall, five hybrids had good grain, milling, cooking, and eating quality and were superior in yield (Shobha Rani et al 2002). Hybrids PHB 71, KRH-2, and Pusa RH-10 are already popular with farmers. Pusa RH-10, by virtue of its excellent grain quality, has had overwhelming acceptance by farmers and consumers. It matures in days and yields 6 8 t ha 1. Hybrids such as Xie you 46, Liang you peite, Xie you 2375, D you 68, Yueza 122, and 80 you 121 from China have been reported to have good grain quality. Yue feng A, a CMS line developed through maintainer reconstruction breeding for good grain quality, combines good grain appearance with low amylose content, soft gel consistency, lower gelatinization temperature, and aroma. This has been used as a parent in 13 hybrid combinations in China. RiceTec in the United States has reported that rice hybrids XL7 and XL8 possess excellent grain-milling and starch properties. In Iran, first-generation hybrids developed by using IRRI CMS line IR58025A and restorers IR60219R, IR68749R, IR62686R, and Sepidrod have medium amylose content, medium gelatinization temperature, and medium gel consistency, with good taste and cooking quality (Dorosti et al 2002). In Egypt also, a few commercially exploitable hybrids with consumer-acceptable grain quality have been identified. Characteristic features of some hybrids developed at IRRI combining high yield and good grain quality are given in Table 3. Developing basmati-quality hybrids Research efforts in this direction have been mainly made at IARI-India and IRRI- Philippines. Basmati quality is a constellation of complex traits extra-long fine grain combining starch properties to show a nearly two-times linear increase upon cooking without any swelling and to remain soft and fluffy with a good taste and typical aroma, which all required a systematic breeding of parental lines combining ideal basmati characteristics. The task was quite challenging in view of the scarce usable Improving grain quality in hybrid rice 71

77 Table 1. Grain quality traits of some selected CMS lines developed at IRRI and IARI. CMS line Cytoplasm Grain type % Brown % Total % Head Length b Grain Chalk- Gelatinization % Aroma source a rice milled rice rice shape c iness d temperature e Amylose IR58025A WA Long slender L 14.8 Strong IR69625A WA Bold I 22.9 Moderate IR77805A Gambiaca Medium bold L 24.5 None IR77290A WA Long slender (banana 0 I 25.6 Strong (basmati) shape) IR73328A Mut. IR62829 Medium L 22.5 Moderate IR78371A WA Long slender L 26.6 Slight IR78372A ARC Long (banana 1 L 26.2 Slight shape) IR79156A WA Long I 26.0 Slight Pusa 5A WA Long slender I 20.0 Slight a WA = wild abortive. b On a 1 9 scale, where 1 = very long and 9 = very short. c On a 1 9 scale, where 1 = slender and 9 = bold. d On a 1 9 scale, where 1 = translucent and 9 = very chalky. e L = low, I = intermediate. 72 Zaman et al

78 Table 2. Amylose content of basmatiquality CMS lines developed at IRRI and IARI. CMS lines Amylose content range (%) IR67684A IR68280A IR68281A IR69617A IR70372A Pusa 3A Pusa 4A Pusa 6A Pusa 8A basmati-quality source material. Testcrosses revealed that many dwarf basmati breeding lines were good maintainers. Therefore, desirable lines were selected and backcrossed for conversion into CMS lines. As a result, many basmati-quality CMS lines could be developed (Table 4). The complete absence of basmati-quality restorer lines required the development of breeding lines combining basmati quality and male fertility restoration, with sufficient diversity to produce heterotic combinations. Following a scheme illustrated in Figure 1, many restorer lines with ideal basmati quality and high yield potential have been developed at IARI. The selective mating route of this scheme has given excellent results and many high-yielding restorers combining ideal basmati characteristics are available for use. Two such restorers have been released for commercial cultivation as Pusa Sugandh- 2 and Pusa Sugandh-3 varieties. The comparative grain quality of some restorer lines is given in Table 5. Such exclusively bred parental lines have led to the development of several basmati hybrids with excellent grain quality (Table 6). Of these hybrids, Pusa RH-10 has been released for commercial cultivation by virtue of its 40% yield advantage over Pusa Basmati-1, with comparable grain quality. Several new hybrids are now in the pipeline. Indica/japonica hybrids Indica/japonica hybrids possess the highest yield potential in both sink and source. Their yield may be 30% more than that of the existing highest yield of intervarietal hybrids (Yuan 1994). However, the grain and cooking quality of commercial F 1 produce of subspecific parental lines of a diverse nature will not find acceptance in most rice-growing countries. The use of indica/japonica-derived lines with identical grain characteristics and sufficient indica/japonica genetic divergence to enhance the extent of heterosis for grain yield would be more appropriate. Tsuchiya et al (2002) Improving grain quality in hybrid rice 73

79 Table 3. Grain quality traits of some hybrids. Season a Duration IR no. Cross Yield Amy- Chalk- Grain Grain Brown Total Head Gelatiniza (kg ha 1 ) lose iness b length c shape d rice milling rice -tion (%) (%) recovery recovery temperature e (%) (%) 2001 DS Early IR76712H IR68897A/IR , I/L 3-2R d IR78386H IR68897A/IR , I/L R IR78380H IR68897A/IR , I/L R IR78390H IR68897A/IR , I/L R IR77843H IR70369A/IR , I/LI/L 2-2-R IR78383H IR68897A/IR , I/L R IR76702H IR68888A/IR , I/L 3-3-2R IR78379H IR68888A/IR , I/L 3-3R IR77265H IR68897A/IR , I/L 1R PSBRc28, HK 6, HI/I IR64616H Magat, check 6, I/L IR78388H IR58025A/IR , HI/I/L 3-2-2R Medium PSBRc52, check 6, HI./I d IR68284H Mestizo, check 7, L a DS = dry season. b On a scale of 1 9, where 1 = translucent and 9 = very chalky. c On a scale of 1 9, where 1 = very long and 9 = very short. d On a scale of 1 9, where 1 = slender and 9 = bold. e I = intermediate, L = low. 74 Zaman et al

80 Table 4. CMS lines of basmati quality developed at IRRI and IARI. CMS lines Days to Outcrossing Grain size Grain Aroma Gelatinization Amylose flowering elongation temperature (%) ratio IR67684A 87 Moderate Long 1.71 Strong Low 23.2 IR68280A 88 Moderate Long 1.92 Strong Intermediate 22.3 IR68281A 88 Moderate Long 1.64 Strong Low 19.8 IR69617A 78 Moderate Long 1.66 Strong Intermediate 19.8 IR70372A 96 Moderate Extra long 1.90 Strong Intermediate 22.0 Pusa 3A 105 Moderate Extra long 2.00 Strong Intermediate 24.0 Pusa 4A 100 Moderate Extra long 1.90 Strong Intermediate 24.0 Pusa 6A 95 High Long 1.80 Strong Intermediate 21.0 Pusa 8A 100 High Extra long 2.00 Strong Intermediate 23.0 Improving grain quality in hybrid rice 75

81 A line Partially restored basmati line F 1 (partially restored) F 2 Selection Improved fertility Diverse plant characteristics Basmati grain and cooking quality F 3, F 4, F 5 Progeny Fresh hybridization Selective mating With new source Testcross Selfing Hybrids As above Fig. 1. Basmati restorer breeding scheme. have suggested that breeding of refined restorer or maintainer lines with a more or less indica genetic background would be more practical than a direct hybrid of indica and japonica. Good progress has been made in developing indica/japonica-derived lines in India through indica/japonica hybridization locally or by involving tropical japonica lines developed at IRRI. At IARI, many indica/japonica-derived lines with improved grain quality have been produced using the parental lines given in the schematic flow chart (Fig. 2). Newly generated indica/japonica derivatives are being used to develop hybrids with enhanced heterosis. Nucleo-cytoplasmic interactions and their effects on quality characteristics in hybrids Rice hybrids are usually developed by using the cytoplasmic genetic male sterility and fertility restoration system although initial attempts are being made to deploy the environment-sensitivity genic male sterility (GMS) system to develop two-line hybrids. The first system is more stable and is widely used for developing three-line hybrids worldwide. Three-line hybrids have the sterility-inducing cytoplasm contributed by their female parents and this cytoplasm is reported to influence the expression of various characteristics of hybrids, including quality traits. Information on nucleo-cytoplasmic interactions and their effects on quality traits is limited. Such 76 Zaman et al

82 Table 5. Quality characteristics of promising basmati restorer lines. Quality trait Basmati restorer lines/check IET IET IET IET IET Pusa Basmati-1 Taraori Basmati Milling (%) Head rice recovery (%) Kernel length (mm) Kernel breadth (mm) Length/breadth ratio Kernel length after cooking (mm) Elongation ratio Aroma SS SS SS SS SS SS SS Amylose content (%) Improving grain quality in hybrid rice 77

83 Table 6. Grain quality of basmati hybrids. All had strong aroma. Characteristics Pusa Pusa Pusa Pusa Pusa Taraori RH-10 RH-8 RH-9 RH-18 Basmati-1 Basmati Milling (%) Head rice recovery Kernel length (mm) Length/breadth ratio Kernel length after cooking (mm) Elongation ratio Amylose content (%) Alkali spread value Yield advantage over PB-1 (%) N 22 Pusa 2-21 Tainan 3 N 22 Pusa 312 Gharbharan IR72 Pusa 743 Xiangnuo 4 Pusa 989 Pusa 1120 Pusa 1077 Pusa 1266 new plant types Pusa Basmati-1 Selective mating and parental line development I/J derivatives with improved grain quality Fig. 2. Development of indica/japonica (I/J) derivatives for heterosis breeding. studies have implications for hybrid rice breeding, particularly with reference to quality improvement. In general, the head rice recovery of A/R hybrids was lower than that of their corresponding B/R hybrids (Shivani 2002, Yi and Chen 1992). At the Directorate of Rice Research, India, studies revealed a reduction in head rice recovery (HRR) of A/R hybrids ranging from 6.64% (IR58025A-based hybrids) to 12.49% (IR68888A-based hybrids), which could be due to the negative effects of cytoplasm or problems in grain development. Parents with a high HRR have to be chosen to ensure a higher HRR in the hybrids. There was a slight reduction in the kernel length of A/R hybrids vis-à-vis B/R hybrids, though the differences were not significant. Nucleo-cytoplasmic effects were quite evident for water uptake. The ef- 78 Zaman et al

84 fects were cross-specific. For hybrids based on IR58025A, IR62829A, and IR68888A, the effects were negative, whereas positive effects were noticed for hybrids with IR69628A and IR68888A. For gelatinization temperature, no clear trend was observed as the effects were cross-specific and the same was the case for gel consistency. However, a significant negative effect of sterility-inducing cytoplasm was observed for amylose content. The mean reduction in amylose content ranged from 1.38% to 3.82%. This observation has significant implications for developing hybrids with better grain quality. Restorers with low amylose have to be avoided as the resultant hybrids will also have low amylose. Similarly, the maintainers identified for conversion should have intermediate amylose. Hari Prasanna (2002) did not find any difference between the cytoplasmic effect of A lines and B lines with respect to hulling, milling, and head rice recovery irrespective of the cytoplasm source. Generally, WA cytoplasm did not affect the kernel length of uncooked grain, whereas ARC cytoplasm had a significant positive effect. Further, cross-to-cross differences were seen with other cytoplasm for cooking behavior. ARC cytoplasm generally showed a negative effect (Table 7). At IRRI, grain length and L/B ratio showed a significant influence of WA cytoplasm. Grain breadth did not show any influence, whereas chalkiness appeared to be negatively influenced by WA cytoplasm (Table 8). Studies on the effects of nucleo-cytoplasmic interactions on quality traits at various centers have revealed many traits to be unaffected; in some cases, these effects either varied from cross to cross or showed inconsistency at different locations, suggesting that this causes no serious concern. Grain quality traits can easily be managed with careful parental line selection. The relative influence of cytoplasm on quality traits does not seem to be of much consequence, but further studies would help to confirm this. Improving grain quality in hybrid rice 79

85 Table 7. Effect of sterile cytoplasm on quality traits of rice hybrids made with selected restorers and A and B lines of WA and ARC cytoplasm. WA-based CMS Restorer Hulling Milling Head Kernel Kernel Kernel Kernel Elongation Av (%) rice length length breadth breadth ratio score recovery before after before after cooking cooking cooking cooking Pusa 3A & B PRR72 PRR78 PRR80 IR73885 * PRR72 PRR78 PRR80 IR58025A & B PRR72 +* ** PRR78 ** * ** ** PRR80 ** +** +** IR68897A & B IR73885 IR IR ** ** ** * ARC-based CMS +** +** ** ** ** IR68273A & B IR ** +* ** ** ** IR ** ** ** ** ** IR69715 ** IR7338A &B IR ** ** IR71604 ** ** +** +** ** IR ** APMS 2A & B IR73885 ** IR71604 ** IR * ** ** CRMS 32A IR65514 * a * = significant at the 5% level, ** = significant at the 1% level. = negative difference, + = positive difference. 80 Zaman et al

86 Table 8. Mean differences for quality traits between A and B lines and A/R and B/R crosses. Quality trait IR58025 IR70372 IR68280 A-B A/R-A/B A-B A/R-B/R A-B A/R-B/R Grain length 0.1** 0.12** 0.12** 0.16** 0.3** 028* Grain breadth 0 ns 0.01 ns 0.0 ns 0.01 ns 0.0 ns 0.0 ns Length/breadth 0.1* 0.08** 0.1* 0.07* 0.2** 0.16** Amylose content 1.4** 0.39** 1.6** 1.57** ** Gelatinization 3.2 ns 3.18** 0.6 ns 0.63 ns 2.8 ns 2.84** temperature Gel consistency 7.8** 7.8** 1.1 ns 0.93 ns 11.9* 11.9** Aroma (strong) Chalkiness 2** 1.9** 4** 5.3** 2** 2.8** Milled rice 1.4** 0 ns 2.7** Head rice 2.5** 3.6** 7.7** * = significant at the 5% level, ** = significant at the 1% level. References Balasundara DC, Vidyachandra B Promising rice hybrids for grain quality traits. 4th International Symposium on Hybrid Rice, Abstracts. p 82. Hari Prasanna K Studies on basmati quality rice hybrids. Ph.D. thesis submitted to Indian Agricultural Research Institute, New Delhi, India. Khush GS, Kumar I, Virmani SS Grain quality of hybrid rice. In: Hybrid rice. Manila (Philippines): International Rice Research Institute. p Liang M, Zaman FU, Dikshit HK Inheritance of starch content in hybrid rice grain. Life Sci. Res. (China) 2(3): Shivani D Quality considerations for development of intra- and inter-sub-specific hybrids in rice (Oryza sativa L.). Ph.D. thesis submitted to Acharya N.G. Ranga Agricultural University, Hyderabad, India. 253 p. Shobha Rani N, Subba Rao LV, Prasad GSV, Prasad ASR, Mishra B Quality characteristics of promising experimental rice hybrids. 4th International Symposium on Hybrid Rice, Abstracts. p 93. Tsuchiya T, Bastawisi A, Yang ZY, Moon HP, Mann JA, Ikehasi H Opportunities for and challenges to developing and using hybrid rice technology for temperate countries. 4th International Symposium on Hybrid Rice, Abstracts. p 7. Yi XP, Chen Y Genetical effects of different cytoplasm on rice cooking, milling, and nutrient quality in indica type rice hybrid. Chinese J. Rice Sci. 6: Yuan LP Increasing yield potential in rice by exploitation of heterosis. In: Virmani SS, editor. Hybrid rice technology: new developments and future prospects. Selected papers from the International Rice Research Conference. Los Baños (Philippines): International Rice Research Institute. p 1-6. Improving grain quality in hybrid rice 81

87 Notes Authors addresses: F.U. Zaman and B.C. Viraktamath, Directorate of Rice Research, Hyderabad, India; S.S. Virmani, International Rice Research Institute, Los Baños, Philippines. Citation: Virmani SS, Mao CX, Hardy B, editors Hybrid rice for food security, poverty alleviation, and environmental protection. Proceedings of the 4th International Symposium on Hybrid Rice, Hanoi, Vietnam, May Los Baños (Philippines): International Rice Research Institute. 407 p. 82 Zaman et al

88 Technological refinement in hybrid seed production

89

90 Opportunities for and challenges to improving hybrid rice seed yield and seed purity C.X. Mao and S.S. Virmani After success in China, more than 20 countries worldwide have started hybrid rice programs and about 40 rice hybrids have been released for commercial production. Hybrid rice seed production area and yield have increased rapidly as hybrid rice growing area expanded in many countries. Seed yield in China has reached t ha 1 and about t ha 1 outside China in commercial seed production plots. The world hybrid rice growing area should increase to million ha in 2005, of which 16.5 million ha are in China and the rest outside China. The total area for F 1 seed production and parental line multiplication should reach 123,400 ha in China and 35,000 ha outside China. Considering the Chinese experience, there are tremendous opportunities to improve hybrid rice seed yield and seed quality. The challenges to improving hybrid rice seed yield and seed quality are (1) establishing a suitable system for parental line multiplication and purification, F 1 seed production, and quality control; (2) finding the best sites and season for large-scale seed production; (3) training seed growers to increase their skills and experience; (4) refining seed production techniques locally; (5) improving field management approaches, including agronomic and pest control; (6) developing seed parent lines with a high outcrossing rate and resistance to major pests; (7) purifying existing parental lines; and (8) setting up national purity or quality standards for hybrid rice seed production. International cooperation and the extensive participation of public, private, and nongovernment organization sectors in hybrid rice seed production and marketing would contribute toward both food security and additional rural employment in rice-growing countries. It has been more than 25 years since hybrid rice was first commercialized in China in More than 20 countries outside China have tried or have been conducting hybrid rice research and/or a development program since the late 1970s. About 40 rice hybrids have been released and more than 60 public, private, and nongovernment organization (NGO) seed companies have shown interest or have been engaged in hybrid rice seed production and marketing outside China. The total growing area 85

91 Table 1. Current and projected hybrid rice area and seed requirement worldwide. Country Area (million ha) Seed requirement (t) China , ,000 India ,000 20,000 Vietnam ,600 16,000 Philippines ,000 Bangladesh ,000 Others ,000 Total , ,000 of hybrid rice outside China in 2001 was about 700,000 ha (IRRI 2002 ) and the expected area for 2005 is 2.6 million ha. That means there would be an increased demand for hybrid rice seed in the future. Hybrid rice seed production is a systematic, complex, and demanding approach compared with inbred seed production. The most important step from research to commercialization of hybrid rice is hybrid seed production. The timely supply of cheap and good-quality hybrid rice seeds from reliable channels is a prerequisite to the rapid expansion of hybrid rice growing area. In the past five years, hybrid rice area did not expand as fast as expected, mainly because of poor grain quality and lower seed yield of most existing rice hybrids. Therefore, breeders and seed growers are trying to overcome these problems. The nationwide average yield of hybrid seed production in China has been t ha 1 since the late 1990s (Mao 2001). Outside China, commercial seed yield reached t ha 1 (Virmani et al 2001). Seed yield in most countries outside China is still low and its quality is not reliable. This paper deals with the opportunities for and challenges to raising seed yield, reducing production costs, and maintaining good quality of seeds of hybrids and their parental lines for wide-scale commercialization of this technology. Opportunities for hybrid rice seed production In 2001, the hybrid rice growing area in the world was million ha (Table 1). China grew 15.5 million ha, whereas other countries had million ha. In 2005, China is expected to increase its area to 16.5 million ha because of the availability of super-high-yielding and short-duration hybrids (Yuan, personal communication) and the area in other countries could reach 2.6 million ha. These developments would require 330,000 tons of hybrid rice seed in China and 52,000 t outside China by 2004, thereby creating tremendous opportunities for producing and marketing goodquality hybrid rice seeds. 86 Mao and Virmani

92 Table 2. Current and potential seed yields in some countries producing hybrid rice. Country Current yield Highest yield Remarks (t ha 1 ) (t ha 1 ) China India Vietnam Philippines Indonesia Bangladesh Sri Lanka Experimental plots Egypt Experimental seed production Current and potential seed yield Theoretically, if the outcrossing rate of the seed parental lines can reach 50%, and crop growth is similar to that of any inbred high-yielding variety (HYV), hybrid rice seed yield could equal 35 40% of the yield of the variety. After more than 20 years efforts, nationwide seed yield in China has reached t ha 1, which is equal to 40 50% of the mean yield of inbred rice in the country. In Vietnam, the average seed yield in a several-hundred-hectare area has reached 2.2 t ha 1. About 80% of the hybrid rice seed demand in Vietnam is met by seed imports from China. Therefore, Vietnam plans to reduce seed imports from China in phases so that it can become self-sufficient in hybrid rice seeds by In India, the nationwide average seed yield has reached 1.5 t ha 1, with some record yields of t ha 1 in a small area. India plans to produce its own seed. In the Philippines, the average yield in the dry season is t ha 1 and in the wet season it is t ha 1. In many other tropical countries, the seed yield obtained from small plots is 1 2 t ha 1 (Table 2). The gap between the current average seed yield and the maximum seed yield obtained in different countries indicates opportunities for increasing seed yield. Factors affecting seed yield Because hybrid rice seed production is complex, many factors can affect seed yield as well as quality. The G E M model (i.e., genetic background of parental lines, environmental conditions, and management level during seed production) should be considered for the improvement of hybrid rice seed production. Genetic background of parental lines. The outcrossing rate of seed parental lines including CMS lines and TGMS/PGMS lines is the most important factor that can directly affect seed yield. Flowering behavior and resistance to major pests of male and female parental lines are also important factors for seed yield as well as seed quality. In recent years, some new CMS and TGMS lines with a high outcrossing rate and improved resistance have been developed at IRRI and in China, India, Vietnam, Opportunities for and challenges to improving hybrid rice seed yield and seed purity 87

93 Table 3. Record high yield of hybrid rice seed production in Hunan Province, China. County Year Yield Area Remarks (t ha 1 ) (ha) Long Hui Hilly area Tao Jiang Hilly area Wu Gang Hilly area Xu Ning Hilly area Zhi Xing Hilly area Hilly area Hilly area (the record) Hilly area Philippines, Indonesia, and the United States. In China, such lines (such as Bo A, Zhi A, II-32 A, You 1 A, etc.) have shown significantly higher outcrossing and seed yields (4 4.5 t ha 1 ); therefore, the new seed parental lines developed at IRRI and in national agricultural research and extension systems (NARES) should also help to increase seed yield when hybrids derived from these lines will be commercialized. Environmental conditions. The environmental conditions at a location, such as season and weather, soil fertility, irrigation, and pest incidence, can affect seed yield to a great extent. In China, the favorable conditions for a high outcrossing rate have been identified as daily temperature of C, relative humidity of 70 80%, diurnal difference in temperature of 8 10 C, and sunny days with a breeze (Xu and Li 1988). It was also observed that the high seed yield area in China is mostly mountainous or hilly area. The average seed yield in the mountainous provinces such as Sichuan, Hunan, Jiangsu, and Anhui was more than 3 t ha 1, but in the plains and coastal area, including Fujian, Guangdong, and Guang Xi, seed yield averaged about 2 t ha 1 or lower. Even in the same province, some locations with favorable conditions had a higher seed yield than other locations. The high seed yields (above 6 t ha 1 ) recorded in Hunan Province were all obtained in hilly areas (Table 3). In the Philippines, seed yield is higher in the dry season and lower in the wet season. In Bangladesh, results from 1999 to 2001 showed that the seed yield of the same rice hybrid (IR69690H) in the boro season was three times higher than in the T. aman season (Table 4). Recently, it was reported that the hybrid seed production yield in highland provinces in Vietnam such as Quang Nam, Kon Tum, and Tay Nguyen was higher than in other provinces. The highest yield obtained in these areas in the spring season of 2001 and 2002 was t ha 1 and 4 5 t ha 1, respectively. Management level. Hybrid rice seed production yield can be improved by accumulating experience and skills of technicians and seed growers. Extensive training and refinement on hybrid rice seed production in China and other countries have led to gradual improvement in seed production technology (Lin and Yuan 1980, Yuan 1985, Mao 1988, Yuan and Chen 1988, Mao and Zhou 1990, Xu et al 1991, Virmani and Shrma 1993, Virmani et al 1993, Zhou and Cao 1997, Mao et al 1998, Virmani 2001). In China and Vietnam, the nationwide seed yield increased several fold over 88 Mao and Virmani

94 Table 4. Seed yield (kg ha 1 ) of IR69690H in different seasons in Bangladesh ( ). Year Boro season T. aman season , , , Av 1, Source of data: Progress report of IRRI-ADB project on Development and Use of Hybrid Rice in Asia during May-December 2001 from Bangladesh. Yield (t ha 1 ) China 1.0 Vietnam Year Fig. 1. Nationwide average hybrid rice seed production yield in China and Vietnam. time (Fig. 1). Examples of technology refinement are (1) achieving synchronization in flowering of parental lines (2) producing and transplanting good-quality seedlings of parental lines, (3) using optimum spacing and row ratio, (4) using optimum fertilizer level and application time, (5) identifying a good water management strategy, (6) using a good pest management strategy, (7) applying an optimum dosage of gibberellic acid (GA 3 ), (8) practicing optimum extent of leaf clipping and supplementary pollination, and (9) practicing determining proper harvesting and seed-processing procedures. Each seed production agency has to fine-tune its seed production technology depending on the parental line, location, skills, and experience of the farmers. Improvement of the technology is possible through continuous learning and testing. Maintaining purity of parental lines and quality of F 1 seeds Nearly all steps of hybrid rice seed production and parental line multiplication are linked or can affect seed quality, including purity. The G E M model influencing seed yield also affects hybrid rice seed quality. The genetic background of the paren- Opportunities for and challenges to improving hybrid rice seed yield and seed purity 89

95 tal lines affects their stability and hence purity of the seed produced. Environmental conditions can also include the existence of volunteer seeds in the seed production plot and isolation from other rice fields. Mistakes made in management include mechanical mixture of other rice seeds during sowing, transplanting errors, inadequate and improper roguing, mechanical mixture during harvest and seed processing, lack of standards for hybrid rice seed quality control, inadequate seed quality inspection, and lack of a parental line purification system. In the major hybrid rice seed-producing countries, the seed quality control system must be established as this seriously affects the pace of adoption of the technology. In China, nearly all the parental lines and F 1 seeds are produced by the government-owned seed companies. In the 1980s, the government made a seed law and established seed quality standards of hybrid rice and its parental lines to control hybrid rice seed quality. Parental line purification is done by the provincial seed company and parental line multiplication by the prefectural seed company, whereas F 1 seed is produced mostly by the county-level seed companies. Parental line purification is done every year through paired crosses of selected typical plants of the parental lines. About 3,000 3,500 tons of hybrid rice seed are produced annually in India, 95% of it by the private sector. Since quality is directly linked to the business and reputation of the company, seed quality is controlled by the private companies and seed is sold as truthfully labeled seed. The Philippines does not have strong public and private seed companies to undertake large-scale hybrid rice seed production. IRRI and PhilRice produce and provide nucleus, breeders, and foundation seeds. Hybrid seed is produced by private companies and farmers cooperatives. A sustainable mechanism for this purpose is yet to be developed. In Vietnam, 80% of the hybrid seed is imported from China and the balance is produced in the country, mostly by government-owned seed companies. The quality control system is similar to that in China, but not as effective. In Bangladesh, all three sectors (public, private, and NGO) are involved in hybrid rice seed production, with variable seed quality. Challenges to improving hybrid rice seed yield and quality The challenges to improving hybrid rice seed yield and quality are (1) establishing an effective hybrid rice seed production system for a country or a region, (2) finding out the best location and season for large-scale seed production, (3) training seed growers and technicians to improve their skills and experience, (4) refining seed production techniques according to the environmental conditions in the country or region, (5) improving the field management of seed production, including agronomic and pest control approaches, (6) developing seed parental lines (CMS and TGMS) with a high outcrossing rate and resistance to major pests, (7) purifying the existing seed parental and pollen parental lines, and (8) setting up national purity standards for hybrid rice seed production. 90 Mao and Virmani

96 Strategies for increasing seed yield The strategies to meet the challenges to improving seed yield in the tropics are given below. Finding the best season and location for hybrid rice seed production The tropics have two major seasons dry and wet seasons. In the wet season, seed yield is usually lower than in the dry season. But, in the dry season, the temperature is very high and the relative humidity is quite low, which are not suitable for obtaining high outcrossing and seed set. If the location could be in a hilly area with relatively lower temperature and higher humidity during the heading-flowering period in the dry season, seed yield would increase. In the wet season, a place without strong typhoon and heavy rain (such as Mindanao Island of the Philippines) would be useful. Selecting seed production sites with minimum disease and insect pests In the tropics, disease and insect pests are more serious than in temperate areas. This is a major reason for loss of seed yield. Therefore, the seed production site and season should be such that there are minimal disease/insect problems. In addition, integrated pest control should be practiced to protect seed production plots from prevailing pests, especially in the later stage when supplementary pollination has been done. Achieving synchronous heading and flowering panicles within plants and between female and male parents Good synchronization of heading and flowering of panicles within and between parental lines is essential for attaining high seed yields. The practices that contribute toward achieving this are (1) raising of seedlings with multiple tillers, (2) using sparse sowing combined with early and heavy fertilization in the seedbed, (3) increasing transplanting density to minimize excessive tiller and ineffective panicle development in the field, and (4) draining out water when the expected population size of effective tillers has been reached. Providing extensive training to seed growers Compared with Chinese seed growers, seed growers in the tropics usually have less skill and experience. In China, seed growers are the owners of the seed production plots and they are effectively organized by the seed companies through a long-term contract. But, in most tropical countries, seed growers hire field laborers for some seed production activities. These laborers need to be trained to increase their skills for attaining a high seed yield. Fine-tuning seed production technology The principles of the technology for hybrid rice seed production are the same, but different hybrids require minor adjustments to attain a high seed yield in a different location and season. Seed production agencies should therefore not only adopt the Opportunities for and challenges to improving hybrid rice seed yield and seed purity 91

97 techniques introduced from China, IRRI, or other countries into their own seed production plots, but they should also modify these to suit local conditions. The seed production agencies need to set up an adaptive research group to fine-tune the technology. Strategies for hybrid rice seed quality control in the tropics The principles and general operations for hybrid rice seed quality control have been described by Yuan and Chen (1988), Virmani and Sharma (1993), Lou and Mao (1994), and others. Now, the challenge to hybrid seed production in tropical countries is the lack of an effective quality control system. Genetic purity is the most important problem for hybrid rice compared with other crops or the physical purity of rice seeds. The genetic purity and stability of the parental lines could affect the purity of F 1 hybrid seeds. Poor isolation and careless application of F 1 hybrid seed could be other causes of quality problems. The following points should be kept in mind for hybrid rice seed quality control in the tropics: Establish a system to control seed quality in the whole country or in the seed company. Issue standards for the seed quality of F 1 hybrids and parental lines. Set up seed-quality test facilities and mechanisms, including field inspection, laboratory testing, field grow-out, etc. Train seed growers to increase their responsibility and capability to carefully apply field activities in each step, especially to identify the various offtypes. Organize a specific seed production base for parental line purification and F 1 seed production using strict isolation and use pure parental line seeds every season. Use only one cropping season of the seed production plot to prevent volunteer seed mixtures. Sow, transplant, rogue, harvest, and process the seed at the appropriate time. Lessons to be learned from China China met many challenges in the late 1970s and early 80s when hybrid rice technology was expanding in the country. After two decades of effort, seed yield increased and stabilized at a high level and seed quality became reliable. The increased seed yield is the result of the gradual improvement of parental lines for outcrossing, the selection of favorable environments/locations for seed production, and improvements in the management of Chinese seed growers in the last 25 years. Besides technical improvements, policy and institutional support from the government has also been very important for improving hybrid rice seed production and quality. For example, the Chinese government passed a seed law and related regulations to discourage corruption and protect farmers interests. In the mid-1980s, China released its first standard for hybrid rice seed quality, including three-line parents and F 1 hybrid 92 Mao and Virmani

98 seeds. In the mid-1990s, when hybrid rice development reached a peak, seed growers and businessmen profited greatly from the increased seed demand. Consequently, a lot of fake and poor-quality seeds entered the market, which reduced farmers benefits and destroyed the reputation of hybrid rice. This has also happened in India and Vietnam, and probably will happen in other countries in the future when hybrid rice commercialization occurs. Therefore, good seed laws are essential to discourage fake and poor-quality hybrid seed. Future outlook Hybrid rice will be commercialized in more and more countries in the future. From the technical viewpoint, hybrid rice seed production will no longer be the major barrier to hybrid rice development in the world. The technology of hybrid rice seed production has been developed and practiced successfully not only in China but also in many other countries with temperate and tropical conditions. Technologically, there should be no serious problem for hybrid rice seed production. The 1 2 t ha 1 seed yield obtained in many countries under different conditions is economically viable. There are ample opportunities to increase seed yield beyond this level. Developing the skills of technicians and seed growers is a solid basis for further raising and stabilizing hybrid rice seed yield. Breeding approaches including the development of high-outcrossing CMS and TGMS lines and their good panicle exsertion using the eui gene should be helpful in increasing hybrid rice seed production. Molecular markers can be used to identify QTLs for high-outcrossing and to incorporate these QTLs into the CMS and TGMS lines. This should increase hybrid rice seed yields. Seed-quality control mechanisms established in China should also be established in other countries as they start adopting the technology. Molecular markers can also be used for seed purity tests replacing the time-consuming grow-out test. More and more private and NGO seed companies will be engaged in hybrid rice seed production. That means that the responsibility for large-scale seed production organized by the government will be shared and maybe the private sector will also increase its investment in hybrid rice research and development. Global cooperation on hybrid rice seed production will be strengthened when more and more public, private, and NGO seed companies join hybrid rice programs and businesses. International agencies such as IRRI, FAO, and APSA and countries such as China, India, and Vietnam with experience in developing and using the technology will play important roles for the enhancement of hybrid rice seed production. Various models will be established for hybrid rice seed production and industry in different countries and regions. Multinational seed companies will expand their business rapidly. Hybrid rice seed production therefore has a bright future for contributing to food security and increased employment through the participation of the seed industry. Opportunities for and challenges to improving hybrid rice seed yield and seed purity 93

99 References IRRI (International Rice Research Institute) Development and use of hybrid rice in Asia: terminal report of IRRI/ADB project, Los Baños (Philippines): IRRI. 51 p. Lin SC, Yuan LP Hybrid rice breeding in China. In: Innovative approaches to rice breeding. Manila (Philippines): International Rice Research Institute. p Lou XZ, Mao CX Hybrid rice in China: a success story. Bangkok (Thailand): Asia- Pacific Association of Agricultural Research Institutes, FAO Regional Office for Asia and the Pacific. 26 p. Mao CX Hybrid rice seed production in China. In: Seed health. Manila (Philippines): International Rice Research Institute. p Mao CX Improving seed production to speed up the global commercialization of hybrid rice. In: Peng S, Hardy B, editors. Rice research for food security and poverty alleviation. Manila (Philippines): International Rice Research Institute. p Mao CX, Zhou SY Hybrid rice production techniques. Beijing (China): Agricultural Publishing House. 96 p. Mao CX, Virmani SS, Kumar I Technology innovations to lower the cost of hybrid rice seed production. In: Virmani SS, Siddiq EA, Muralidharan K, editors. Advances in hybrid rice technology. Proceedings of the Third International Symposium on Hybrid Rice, November 1996, Hyderabad, India. Manila (Philippines): International Rice Research Institute. p Virmani SS Opportunities and challenges of developing and using hybrid rice technology in the tropics. In: Peng S, Hardy B, editors. Rice research for food security and poverty alleviation. Manila (Philippines): International Rice Research Institute. p Virmani SS, Sharma HL Manual for hybrid rice seed production. Manila (Philippines): International Rice Research Institute. 57 p. Virmani SS, Mao CX, Toledo RS, Hossain M, Janaiah A Hybrid rice seed production technology for improving seed industries and rural employment opportunities in Asia. Paper presented at the International Workshop on Seed and Seedling Sciences and Technology, Taiwan, China, June Virmani SS, Manalo J, Toledo R A self-sustaining system for hybrid rice seed production. Int. Rice Res. Newsl. 18:4-5. Xu SJ, Li BF Managing hybrid rice seed production. In: Hybrid rice. Manila (Philippines): International Rice Research Institute. p Xu SJ, Pan WL, Xu F Studies and application on super high yielding techniques for hybrid rice seed production. Hybrid rice (special issue). Changsha (China): China National Hybrid Rice Research and Development Center. Yuan LP A concise course in hybrid rice. Hunan (China): Hunan Science and Technology Publishing House. 168 p. Yuan LP, Chen HX Hybrid rice breeding and cultivation. Hunan (China): Hunan Science Technology Publishing House. 364 p. Zhou ZY, Cao MF Hybrid rice seed technology and principles. Hunan (China): Hunan Science and Technology Publishing House. 94 Mao and Virmani

100 Notes Authors address: Plant Breeding, Genetics, and Biochemistry Division, International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines. Citation: Virmani SS, Mao CX, Hardy B, editors Hybrid rice for food security, poverty alleviation, and environmental protection. Proceedings of the 4th International Symposium on Hybrid Rice, Hanoi, Vietnam, May Los Baños (Philippines): International Rice Research Institute. 407 p. Opportunities for and challenges to improving hybrid rice seed yield and seed purity 95

101 Hybrid rice for mechanized agriculture M. Walton Hybrid rice breeding in the United States has been done primarily by RiceTec Inc., which has established a collaboration agreement with the Chinese National Hybrid Rice Research Center at Changsha, Hunan. The latter provides RiceTec with exclusive access to CMS and EGMS line germplasm. Restorer lines are based upon South China indica sources that are traced in large part to materials developed by the International Rice Research Institute. U.S. cultivars that are derived primarily from tropical japonica germplasm are used extensively as pollen parents for RiceTec s two-line rice hybrids. In 2000, RiceTec released XL6 as its first rice hybrid ever commercialized in the U.S. This hybrid yielded about 10 t ha 1 (about 20% higher than the best U.S. varieties). However, it suffered from high lodging and poor milling factors, preventing it from achieving a significant market share. In 2002, two new hybrids, XL7 and XL8, were released; these have less lodging and better milling quality. XL7 is also several days earlier than any variety currently grown in the market and offers producers the option of producing a ratoon crop or planting after wheat. XL8 competes with two leading long-grain varieties, Cocodrie and Wells, which account for 80% of the southern U.S. long-grain production. Challenges to the mechanization of hybrid rice in the U.S. are discussed in this paper. RiceTec, Inc. was established in 1990 for the purpose of developing and commercializing hybrid rice seed in the United States. The company is based in Alvin, Texas, and it focuses on developing hybrids for the southern U.S. rice production area that is composed of parts of Texas, Louisiana, Arkansas, Mississippi, and Missouri. More than 40 scientists and technicians are engaged in breeding, testing, and germplasm development activities at three permanent locations in North America: Alvin, Texas; Newport, Arkansas; and Lajas, Puerto Rico. Approximately 15 Texasbased scientists and technicians conduct research and provide laboratory support in molecular genetics, cell biology, and grain quality. RiceTec uses state-of-the-art molecular marker, tissue culture, and grain quality laboratories to support breeding, product development, and seed production activities. 97

102 Approximately 80% of the breeding effort focuses on developing hybrids for the U.S. long- and medium-grain markets, with the balance devoted to developing specialty rice varieties, including aromatic and arborio types that are marketed through RiceTec s Consumer Business Unit. In addition to the permanent facilities in Texas, Arkansas, and Puerto Rico, RiceTec has developed a winter nursery operation on the island of Kauai in the state of Hawaii and conducts a hybrid testing program in Brazil, Argentina, and Uruguay with the support of RiceTec Ltda., a sister company that is commercializing rice hybrids in South America. RiceTec germplasm RiceTec has incorporated a broad germplasm base into its breeding programs. In 1993, the company established an agreement with the Chinese National Hybrid Rice Research Center (CNHRRC), formerly the Hunan Hybrid Rice Research Center, which provided RiceTec with exclusive access to A-line germplasm developed at CNHRRC for use in North and South America. The agreement was extended to include S-line germplasm in Restorer lines are based upon South China indica sources that are traced in large part to materials developed by the International Rice Research Institute. U.S. cultivars, which are derived primarily from tropical japonica germplasm, are used extensively on the male side of RiceTec s two-line breeding effort. Hybrid rice in the United States RiceTec released its first product, XL6, for commercial sale in XL6 was the first rice hybrid ever commercialized in the United States, and represented a significant milestone in the history of hybrid rice and RiceTec. This hybrid had very high yield, just less than 10 t ha 1, which was about 20% higher than the yield of the best U.S. varieties and almost 50% better than the average U.S. rice yield. However, XL6 suffered from high lodging and poor milling, factors that prevented it from achieving a significant market share. In 2002, RiceTec released two new hybrids for sale, XL7 and XL8. Neither hybrid has the yield potential of XL6, but both are significantly improved for lodging and milling. XL7 is a very early hybrid, several days earlier than any variety currently grown in the market, and it offers producers the option of producing a ratoon crop or being planted after wheat. XL8 competes directly with the two market-leading southern long-grain varieties, Cocodrie and Wells, which collectively account for about 80% of the southern U.S. long-grain production. Challenges to hybrid rice in mechanized agriculture The farming systems of the United States and the temperate rice-producing areas of South America are based on large-scale mechanized approaches to farming. To com- 98 Walton

103 Fig. 1. Rice hybrids developed by RiceTec. pete successfully against varieties developed for use in capital-intensive farming systems, it has been necessary to make several changes to the hybrid rice germplasm that was originally developed for small-scale, intensively managed cultural systems. The RiceTec breeding program has focused heavily on two traits that limit acceptance of any rice hybrid or variety, lodging and milling yield. The propensity for hybrids to lodge has been a major factor limiting acceptance by U.S. farmers. Lodging reduces grain quality and increases the amount of time required to harvest a field, which in turn results in a higher cost of production. Milling yield is at least as important as lodging for hybrid acceptance. Although total grain yield is very important, cultivars that do not have acceptable milling yields, defined in the United States as 70% total milling and 55% whole milling, are discounted by the millers who purchase the grain from farmers. The loss of revenue from milling discounts causes farmers to look unfavorably on a cultivar, regardless of the total grain yield. The first hybrid commercialized by RiceTec in the United States, XL6, had extremely high yields but also suffered from severe lodging and poor milling. RiceTec s newest hybrids, XL7 and XL8, both released commercially in 2002, largely overcame both the lodging and milling problems of XL6 (Fig. 1). Both new hybrids are similar to the best U.S. varieties with respect to lodging and milling; however, RiceTec breeders continue to focus a great deal of attention on both traits. A third major breeding target has been seed yield of the female parent. U.S. and South American farmers typically plant varietal rice at very high seeding rates, ranging from 90 to 130 kg ha 1, and seed costs are very low. RiceTec breeders have focused a great deal of effort on developing male sterile lines with high seed yield potential and male lines with high pollen shed potential to provide RiceTec s Seed Business Unit with hybrids that could be produced economically. Hybrid rice for mechanized agriculture 99

104 Fig. 2. Bay width of male and female parents in U.S. hybrid rice seed production field. Breeding for seed yield-related traits is also required to permit hybrid production on the scale that occurs in the United States. Seed production in the U.S. occurs in fields ranging from 10 to 1,000 ha vis-à-vis Asian production on fields from 1 to 5 ha in size. Female bay widths in the U.S. can be up to 10 m (Fig. 2), whereas in China the bay widths are 1.0 to 1.5 m. The mechanized approach to agriculture reduces the amount of labor required for large-scale farming, but also reduces the intensity of management that is possible. Consequently, for hybrids to be successful in the U.S., RiceTec has had to develop male and female parent lines that have high outcrossing potential and that can be successfully produced using the production practices common to this country. In addition to breeding for high seed yield, RiceTec agronomists have shown growers that it is possible to plant rice at much lower seeding rates than are typically used. RiceTec hybrids are now planted at approximately 35 kg ha 1 in the U.S. and 70 kg ha 1 in South America. Convincing farmers to reduce seeding rates that dramatically has been a major focus of the Seed Business Units in both hemispheres, and the success of RiceTec in doing so is evidence of RiceTec s commitment to, and close contact with, rice farmers. Market challenges Acceptance of hybrid rice in the United States requires hybrids adapted to mechanized agriculture and that produce grain that meets the very specific grain quality parameters of the U.S. rice market. U.S. rice is divided into three market classes based on grain size. Each market class has specific requirements for grain length and width, amylose content, and gelatinization temperature (Table 1). The indica 100 Walton

105 Table 1. Physical and chemical properties of U.S. rice. Item Milled grain Gelatinization temperature Market class Length Width Length-width ( C) (mm) (mm) ratio Long : Medium : Short : germplasm that makes up approximately 75% of RiceTec s germplasm does not match most of the U.S. market criteria, and RiceTec breeders use a variety of germplasm sources and selection methods to modify the base germplasm to meet U.S. market specifications. U.S. long-grain rice, the primary target for RiceTec s breeding program, is also known for very low levels of chalk. Hybrids from indica germplasm tend to have high levels of chalk, which may not be visible in the parent lines but is expressed in hybrid combinations. RiceTec breeders are making progress for this trait, but continue to look for new and improved selection methods. Future challenges RiceTec has been breeding hybrid rice for 12 years and, in that short period of time, has released three hybrids to U.S. farmers, developed and disseminated new cultural practices, and developed the expertise and knowledge necessary to produce hybrid seed on a large scale. In 2003, RiceTec will release a herbicide-tolerant version of XL8 with resistance to the imidazolamine class of herbicides. U.S. farmers are eager to plant the new hybrid as it provides them with new weed control options in a very high-yielding hybrid. However, RiceTec and its breeders will continue to face challenges that must be overcome if hybrid rice is to become a significant factor in the U.S. rice market. The public breeding programs continue to develop new varieties that have higher yield than current varieties (Fig. 3), and these yield gains put renewed pressure on RiceTec breeders to find hybrid combinations with even higher yield potential than the current hybrids. RiceTec breeders have made tremendous progress in breeding for milling yield, starch properties, and grain appearance, but further progress will be necessary if hybrids hope to gain a significant share of the U.S. market. To make this progress, RiceTec breeders will need to find a proper balance between breeding parents that have similar grain quality and appearance traits and that demonstrate high levels of yield heterosis when combined to make hybrids. Finally, RiceTec breeders must continue their efforts to breed for seed production potential. Although RiceTec sells its products to farmers at a very high premium Hybrid rice for mechanized agriculture 101

106 Yield (lbs acre 1 ) 10, RTH Hybrids = x 556,968 9,000 8,500 Hybrids Varieties XL7 XL8 Linear (varieties) Linear (hybrids) 8,000 7,500 7,000 Varieties = x 326,640 6, Fig. 3. Yield gain of hybrids and varieties. to varieties, for RiceTec to thrive as a business it must reduce the cost of seed production and reduce the risk that comes from producing seed on large areas. Notes Author s address: RiceTec, Inc., P.O. Box 1305, Alvin, Texas, USA. Citation: Virmani SS, Mao CX, Hardy B, editors Hybrid rice for food security, poverty alleviation, and environmental protection. Proceedings of the 4th International Symposium on Hybrid Rice, Hanoi, Vietnam, May Los Baños (Philippines): International Rice Research Institute. 407 p. 102 Walton

107 Biotechnological tools in hybrid breeding and seed production

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109 Improving hybrid rice through anther culture and transgenic approaches S. Balachandran, G. Chandel, M.F. Alam, J. Tu, S.S. Virmani, K. Datta, and S.K. Datta Among the available genetic means to break the yield barrier in rice, agreement is widespread that rice hybrids have the potential to increase yield by t ha 1 in farmers fields under irrigated conditions. However, rice hybrids are affected by as many pests and diseases as common rice varieties. For the large-scale adoption of this technology, hybrids need to be resistant to the major pests and diseases prevailing in the target areas. Chemical control of yellow stem borer has been either ineffective or expensive, besides leading to environmental pollution. As an effective plant protection strategy, genetic engineering approaches now enable the production of insecticidal protein within the rice plant itself, which offers environment-friendly protection against insect pests. The incorporation of resistance genes in CMS or maintainer or restorer lines is expected to make hybrids resistant to the target disease or pest. While it is difficult to produce transgenic CMS plants every time, the transformed maintainer line with resistance genes will result in a resistant CMS line by backcrossing. The transgenic restorer line, in contrast, can be directly used for hybrid production. Recently, we have successfully transformed two transgenic maintainer lines (IR68899B and IR68897B) and two restorer lines (MH-63 and BR R) with the truncated chimeric Bt gene, cryiab (driven by 35S and PEPC promoters), and/or the hybrid Bt gene cryiab/cryiac (driven by the actin I promoter). These lines showed a wide range of expression (low to high) of Bt proteins, which were stably inherited. For BR R (T 0 plants), protein expression was 0.61 mg g 1 of fresh leaf vis-à-vis 20 ng mg 1 soluble protein found in the Bt MH-63 line. A selected homozygous MH-63 Bt was hybridized with CMS line Zhenshan 97A to produce the first-ever hybrid Bt rice (Shanyou 63). The hybird rice was field-evaluated for the first time in Wuhan, China, in The transgenic CMS restorer plant and its hybrid exhibited excellent protection against extremely high, repeated infestations of yellow stem borer and natural outbreaks of leaffolder. The transgenic hybrid not only showed high protection against both insects but also recorded 28.9% more yield than the non-bt hybrid. Similarly, Xa21 has been incorporated in maintainer (B) and restorer (R) lines. The combinations of Xa21 and Bt will further enhance 105

110 the yield of hybrid rice and ensure a pesticide-free environment and improved rice breeding. Further work on developing transgenic IR58025B with plant protection is in progress. Our recent research on nutrition (iron, protein, and provitamin-a) improvement and tolerance for abiotic stresses will probably diversify and improve the hybrid rice-breeding program. The prospects for accelerating growth in rice production by conventional means as in the past are less encouraging as there is little scope for expansion of area under rice, especially in the irrigated areas where the high-yielding varieties that contributed to increasing rice production for the past 25 years have tended to plateau and total factor productivity has begun to decline. Among the available genetic means to break the yield barrier in rice, there is a widespread agreement that rice hybrids have the potential to increase yields by t ha 1 in farmers fields under irrigated conditions. Taking advantage of the Chinese success in hybrid rice technology, India has developed a strong hybrid rice program during the last decade, which has been followed more recently by Vietnam, Indonesia, Malaysia, and the Philippines (Virmani 2001). The major problems in hybrid rice breeding are the limited number of parental lines with specific desirable traits, a lower frequency of maintainers and restorers among elite breeding lines, the narrow genetic base, lack of resistance to biotic stresses, and poor grain quality of some parental lines. Rice hybrids are affected by many pests and diseases such as blast, bacterial blight, sheath blight, yellow stem borer, brown planthopper, whitebacked planthopper, leaffolder, and gall midge. For the largescale adoption of this technology, hybrids need to be resistant to the major pests and diseases prevailing in the target areas. To breed improved heterotic rice hybrids, we need to adopt new strategies to enhance the frequency of availability of maintainers and restorers and ensure a constant supply of genetically diverse parental lines. Major advances in biotechnology offer us a variety of new tools such as anther culture, protoplast culture, molecular markers, and DNA transformation for use in crop improvement programs (Datta 2000). The advent of recombinant DNA technology or genetic engineering in plants has dramatically increased the number of options and methods available for improving male sterility systems in crop plants. Possible applications of a few of these approaches for hybrid parental line improvement are discussed. Application of anther culture in hybrid rice breeding After the first complete set of three-line hybrid rice was formed in the early 1970s, the purity of parental lines used in developing hybrid rice has declined markedly because of gene drift and mutation, artificial and biological confounding, and exposure to unfavorable natural stresses. Such a decrease during the long period of propagation and production results in reduced yield potential and grain quality and increased vulnerability to more diseases and pests. It is therefore necessary to take 106 Balachandran et al

111 strict and effective measures to purify these parental lines. The conventional technique of purification is very elaborate and selection is made on phenotype, but this cannot guarantee that selected materials will be genetically homozygotic and stable. Because anther culture can make genes highly homozygotic, it is a more effective method for purification. Anther culture can be used extensively for hybrid rice breeding in the following ways (Zhu et al 1998): 1. Exploiting the anther culture system for haploid protoplast culture, which is essential for genetic engineering. Plants regenerated from microspore-derived cell suspensions would allow the prompt recovery of homozygous diploid transgenic plants. 2. Understanding the genetic mechanism controlling some important characters such as cytoplasmic male sterility (CMS)-fertility restoration, photo-/ temperature-sensitive genetic male sterility (PGMS/TGMS), and wide compatibility (WC) using anther culture derived doubled haploids (DH). Anther culture can effectively eliminate gene interaction existing in the heterozygous genotypes derived from conventional breeding procedures. DH lines also avoid interference from the environment on genetic expression. 3. Developing intermediate lines from the pollen-derived progenies of intervarietal and intersubspecific crosses that can be used as parental lines of heterotic rice hybrids to develop diverse CMS lines. 4. Application of the genetic engineering approach to develop diverse and improved CMS lines possessing novel genes transferred from the transformed maintainer lines (Alam et al 1999). 5. Advancing transgenic lines through anther culture to recover homozygous transgenic doubled haploids in a shorter time. Some recent developments in the use of anther culture for hybrid rice breeding are mentioned below. Purifying parental lines of hybrid rice using anther culture Anther culture was successfully used for purifying CMS line V20A by producing different types with varying stages of pollen abortion and a small proportion of fertile pollen recovered in the normal state of mononucleate abortion (Xu et al 1983). Its hybrid rice Shan You 6 yielded 19.1% higher than the control (Ge et al 1985). Bai et al (1991) reported that hybrids derived from anther culture purified restorer line Minghui 63 were significantly improved in purity, seed-setting rate, yield potential, and resistance. Through anther culture of the male sterile lines of II-32A, You la, and Xie Qing Zhao A and their respective maintainer lines, some normal sterile or fertile pollen diploids were obtained. These purified CMS lines were screened for use through testcrosses, sterility tests, and heterosis trials (Zhang et al 1994a, Chen et al 1994). In the 1990 dry season at IRRI, out of 95 anther culture lines regenerated from CMS line IR54752A, 10 lines were identified possessing 100% male sterility. Two-line hybrid rice derived from P(T)GMS lines is another approach for exploiting heterosis in rice. As most P(T)GMS lines currently used are not stable enough, three P(T)GMS lines (ZaXi30S, 1286S, and 1356S) were purified via anther culture. These purified Improving hybrid rice through anther culture and transgenic approaches 107

112 lines were not only stable in sterility performance but were also comparable with the donors in major characters. Using anther culture in breeding parental lines Significant achievements have been obtained in breeding CMS restorer lines via anther culture. Zhu et al (1993) developed CMS restorer line 2374 with strong combining ability by improving a pollen strain derived from Shan You 2 by the conventional method. Zhu et al (1996, unpublished) also bred a CMS restorer line (1044) via anther culture of the F 1 hybrid, Ce Wang et al (1994) adopted the cross irradiation anther culture procedure to create new restorer lines for CMS. Out of the several anther culture lines developed via anther culture of young panicles of the F 1 (Minghui 63/ZiGui), restorer line Chuan Hui 802 was selected for strong restorability. Using this R line, the hybrid II You 802 (II 32A/Chuan Hui 802) was developed and grown on 66,000 ha in China. Zhou (1996, unpublished) inoculated the anthers of an F 1 hybrid derived from two highly disease-resistant parents and successfully developed a disease-resistant restorer line, DT Ai. A hybrid derived from DT Ai and Wei 20A had excellent disease resistance and high yield potential. Breeding widely compatible restorers (WCRs) for indica CMS is now an available option for the exploitation of intersubspecific heterosis. Although some WCRs in highly heterotic hybrids have been developed through conventional techniques, this method requires a long process to stabilize and subsequently test the WC and CMS restoring ability. By using the anther culture method, however, we can develop WCR lines expeditiously and simultaneously screen them for WC and restoring ability. Li et al (1992) reported the use of anther culture in intersubspecific heterosis breeding for the first time. Zhu et al (1993) created a good-quality WCR named HRl004 through anther culture of the F 1 hybrid (Cpslol7 Skybonnet) in 3 years or five cropping seasons from parent selection to the identification of a WCR. The WC and restoring ability were identified by crossing, respectively, with the four test varieties, IR36, Nanjing 11, Balilla, and Akihikari, and with CMS lines Xie Qing Zhao A, Chang Fei 22A, and You IA. The fertile pollen rate and seed-setting rate of the F 1 hybrids were above 75%, showing that these materials were widely compatible. The seed-setting of F 1 hybrids with CMS was more than 70%, suggesting that these had strong restoring ability. Zhang et al (1994b) reported that 33 of 120 pollen strains from single hybridization combinations of WCV and CMS restorer lines and 15 of 35 composite hybridization combinations could restore the fertility of CMS. Further crossing with the WC test varieties singled out a WCR named H Yin et al (1994) obtained 20 WCRs and 3 intersubspecific hybrids with strong heterosis from pollen-derived progenies of WCV and Cpslo17 crossed with CMS restorer lines. Developing homozygous transgenic lines rapidly via anther culture The stability of the transgene expression in the subsequent generations depends on the homozygosity at the transgenic locus, thus making the transgenic lines fixed. This could be achieved by advancement of selfing generations at least up to T 2 (Tu et al 108 Balachandran et al

113 1998), which requires a minimum of 2 or more years depending on the photosensitive reaction of the genotype(s). However, doubled-haploid breeding through anther culture could be successfully employed to reduce this time period for attaining homozygosity, particularly when the transgenes are integrated in more than one locus in the chromosome. Recently, homozygous transgenic lines of variety Swarna with enhanced sheath blight resistance were obtained within a year from the start of transformation compared with months for advancement by normal selfing generations (Baisakh et al 2001). Genetic engineering in hybrid rice development Engineered male sterility The exploitation of heterosis hinges on the availability of a good male sterility system. In the tropics, over the past decades, many cytoplasmic male sterility (CMS) sources have been successfully developed and used for hybrid seed production in rice. Considerable progress has been made in understanding the organization and expression of plant genes. Efficient methods for transforming many plant species have also been developed. It is now possible to introduce virtually any genetic sequence into a plant genome and modulate its expression with a certain degree of precision. With these tools, several methods have been developed for disrupting normal pollen development (for male sterility) and for restoring normal pollen development (fertility restoration) in the hybrid (Narayanan 1998). 1. Disruption of pollen development by transgene expression One of the earliest and successful attempts to induce male sterility by genetic engineering involved the transfer and tissue-specific expression of a toxin gene that disrupted normal pollen development. The toxin gene from a fungal source, barnase (an RNase), driven by a tapetum-specific promoter (TA 29), was made to express itself specifically in the tapetal tissue of developing tobacco anthers (Mariani et al 1990). Fertility restoration in these male sterile transgenic plants could be achieved by crossing with another transgenic tobacco line that expressed the specific RNase inhibitor, barstar (Mariani et al 1992). Using the same system, male sterility has been successfully engineered in rapeseed, Brassica napus (Denis et al 1993). So far, three pollen-specific genes have been isolated and characterized: maize (ZmI3), tomato (IAT52, Twell et al 1989), and rice (PSI, Zou et al, 1994). The PSI-GUS chimeric gene was introduced into tobacco and expressed in specific tissues that resulted in male sterile plants (Zou et al 1994). Ling et al (1998) were able to develop partially transgenic male sterile rice plants by introducing the PSI-barnase gene. 2. Inhibition of male gametogenesis (MG) genes Male gametogenesis is a complex development process that is controlled by the expression of many genes. Genes whose function seems to be specifically associated with male gametogenesis (MG genes) have been reported in a few crops. Aarts et al (1993) isolated a gene from Arabidopsis, using transposon mutagenesis, the disrup- Improving hybrid rice through anther culture and transgenic approaches 109

114 tion of which leads to male sterility; this locus was designated MS2. With the availability of efficient molecular gene-hunting tools such as gene traps (Sundaresan et al 1995), it is now relatively easy to identify loci involved specifically in male gemetogenesis. Several molecular methods inhibit the expression of a locus. One of the powerful and widely tested methods is the antisense RNA technique, which involves the expression of the sequence of the gene to be inhibited in antisense (complementary to the mrna sequence) orientation through transgenes. A plant can thus be made male sterile by inhibiting any of the MG genes through this technique (Narayanan 1998). 3. Developing CMS lines via protoplast fusion Protoplast fusion is considered as one of the important approaches for achieving cytoplasmic diversification and developing alloplasmic lines from distant species of Oryza and related genera. Protoplast culture has made it possible to transfer CMS into elite breeding lines in one generation instead of the 5 7 repeated backcrosses required in conventional procedures (Brar et al 1998). Some notable examples of successful CMS transfer by protoplast fusion are tobacco, Brassica, Citrus sp., and, rice. Somatic hybrids have been produced through protoplast fusion between cultivated rice and four wild species (Hayashi et al 1988). In cybridization, the nuclear genome of one parent is combined with the organelles of a second parent. In effect, organelles are transferred from one parent to the other in a single step. Protoplast fusion provides a unique opportunity to produce cybrids and recombine cytoplasmically inherited traits. Protoplasts of the donor CMS line are exposed to high doses of irradiation and fused with the iodoacetamide-treated protoplasts of the recipient line. Irradiation inactivates the nucleus and chemical treatment with iodoacetamide prevents cell division. As a result, metabolically complementary cells are capable of developing into plantlets after fusion treatment. The donor-recipient method has been used successfully to transfer CMS sources into fertile lines of rice. This method does not require the use of a selectable cytoplasmic marker of the donor. Yang et al (1988, 1989) produced cybrid plants in rice by electrofusing gammairradiated protoplasts of A-58 CMS and iodoacetamide-treated protoplasts of the fertile cultivar Fujiminori. Cybrids had the peroxidase isozyme of the fertile parent (Fujiminori) but had four plasmid-like DNAs (B1, B2, B3, and B4) from the sterile A-58 CMS parent in their mitochondrial genomes. Cybrids produced through protoplast fusion using the donor-recipient method had the mitochondrial genome of the CMS line and the nuclear genome of the fertile variety (Akagi et al l989). Kyozuka et al (1989) also used the donor-recipient method and transferred CMS of Chinsurah Boro II, an indica rice, into japonica variety Nipponbare. In another study, Akagi and Fujimura (1992) developed an efficient and highly reproducible system for transferring CMS into an array of breeding lines. Using this method, indica CMS has been introduced into 35 japonica cultivars. 110 Balachandran et al

115 Improving parental lines against diseases and insect pests via genetic engineering As a complement to crop protection strategies, the genetic engineering approach has enabled us to develop transgenic rice resistant to biotic stresses. Recently, transgenic rice with Xa21, Bt, and chitinase genes, conferring resistance to bacterial leaf blight, yellow stem borer, and sheath blight, respectively, has become available for evaluation. The advantages of using Bt toxin-producing transgenic rice plants over conventional Bt spray application are so significant that, during the past 10 years, several Bt plants with different versions of truncated and/or synthetic Bt genes have been reported. The average yield losses caused by stem borers (a lepidopteran insect) to the rice crop are often estimated at 10 30% and occasionally as much as 60 95% (Pathak and Khan 1994). Chemical control of yellow stem borer has been either ineffective or expensive, besides leading to environmental pollution. Many indica rice varieties with different Bt genes have been developed and evaluated against yellow stem borer. As the hybrids need to possess multiple disease and insect resistance, a strategy for improving the parental lines of hybrids using the transgenic approach has been followed at IRRI. Transgenic rice plants harboring the cloned Xa21 gene displayed a high level of resistance against the blight pathogen, Xanthomonas oryzae. Several elite indica rice varieties transformed with the Xa21 gene have shown high levels of resistance in the greenhouse and also in field conditions. Tu et al (1998, 2000b) reported the production and evaluation of transgenic indica rice in fields with the Xa21 gene. Hybrid rice breeding involves the three-line system in which a cytoplasmic male sterile line (CMS) used as a female parent is crossed with a fertility restoration (R) line to produce a hybrid. The third line, a maintainer (B) line, is used for maintaining/producing male sterile plants. A hybrid is resistant to a disease/insect when one of the above parents is resistant because of a dominant gene(s) (Virmani 2001). Incorporation of a resistance gene(s) in a CMS or maintainer or restorer line is expected to make the hybrid resistant to the target disease or pest. While it is difficult to produce a transgenic CMS plant every time, one-time genetic transformation of a maintainer line with a resistance gene(s) will automatically result in a resistant CMS line by backcrossing (Alam et al 1999). The transgenic restorer line, in contrast, can be directly used for hybrid production (Tu et al 2000a). A detailed scheme for producing and using transgenic CMS, maintainer (B), and restorer (R) lines resistant to target diseases and insects in a hybrid rice breeding program is illustrated in Figures 1 and 2. Recently, we have successfully transformed two transgenic maintainer lines (IR68899B, IR68897B) and two restorer lines (MH63 and BR R) with the truncated chimeric Bt gene, cryiab (driven by 35S and PEPC), and/or the hybrid Bt gene cryiab/ac driven by the actin 1 promoter (Table 1). These lines showed a wide range of expression (low to high) of Bt proteins and the protein content was stably inherited (Datta et al 1998, Alam et al 1999, Tu et al 2000a). Although the expression of the Bt endotoxin protein was low (20 ng mg 1 soluble protein), a half dose of this endotoxin expressed in the MH63-derived hybrid was sufficient to control lepidopteran Improving hybrid rice through anther culture and transgenic approaches 111

116 Elite maintainer (B) line (susceptible) Transform with disease/insect resistance genes Primary (T 0 ) transgenic maintainer (B) (resistant?) Confirm transgene integration by PCR/Southern Repeated selfing Confirm transgene expression by bioassay/western/northern Homozygous transgenic B lines Fig. 1. Schematic diagram of converting a susceptible maintainer into a resistant transgenic maintainer (B) line. insects (Tu et al 2000a). In the case of BR827-35R (T 0 plants), the protein expression level was 0.61 mg g 1 of fresh leaf. A selected homozygous MH63 Bt was hybridized with CMS line Zhenshan 97A to produce the first-ever hybrid Bt rice (Shanyou 63). The hybrid Bt rice was field-evaluated for the first time in Wuhan, China, during The transgenic CMS restorer line and its hybrid exhibited excellent protection against extremely high, repeated infestations of yellow stem borer and natural outbreaks of leaffolder (Fig. 3). The transgenic hybrid not only showed high protection against both insects but also recorded 28.9% more yield than the non-bt hybrid. These were the first reported field trials of a commercial hybrid Bt rice in which the yield was higher than that of non-bt commercial hybrid rice (Tu et al 2000a). Thus, 112 Balachandran et al

117 Elite restorer (R) (susceptible) Transform with disease/insect resistance genes Primary (T 0 ) transgenic restorer (resistant?) Confirm transgene integration by PCR/Southern Repeated selfing Confirm transgene expression by bioassay/western/northern Homozygous transgenic R lines Fig. 2. Schematic diagram of converting a susceptible restorer into a resistant transgenic restorer (R) line. the successful integration of various Bt genes in hybrid rice provides a good resource for the management of rice pests in Asian agriculture (Table 2). Now that the combination of biotechnological innovations and traditional plant breeding efforts proved successful in a field environment, we are working on future prospects for developing and using more transgenic parental lines in hybrid rice breeding. Conclusions The yield advantage, resistance to biotic and abiotic stresses, adaptability, grain quality, and other traits in rice hybrids are largely determined by the type of parental lines Improving hybrid rice through anther culture and transgenic approaches 113

118 Table 1. Current status of Bt rice hybrids developed at the International Rice Research Institute, Philippines. Molecular analysis Bioassay in Field evaluation glasshouse Variety Bt gene used Promoter Generation/ Southern Western YSB deadhearts status YSB a larval (%) mortality (%) Bt Minghui 63 cryiab/cryiac Actin 1 Homozygous Positive (1.8 kb) Positive 100% (60 kda fusion protein) Bt Shanyou 63 CryIAb/cryIAc Actin 1 Homozygous Positive (1.8 kb) Positive 100% (60 kda fusion protein) IR68899B CryIAb CaMV 35S T 2 segregating Positive (1.8 kb) Positive 100% Not done (60 kda fusion protein) IR68897B CryIAb/cryIAc Actin 1 T 1 segregating Positive (1.8 kb) Not done 100% Not done BR827-35R CryIAb/cryIAc Actin 1 T 1 segregating Positive (1.8 kb) Not done <60% Not done a YSB = yellow stem borer. 114 Balachandran et al

119 A B Fig. 3. Field evaluation of Bt hybrid rice at Wuhan, China. (A) Pest reaction of transgenic Bt hybrids (right) and Minghui 63 control (left) plants against heavy manual infestation of yellow stem borer. (B) Pest reaction of Bt hybrid (left) and non-bt hybrid Shanyou 63 (right) against natural infestation of yellow stem borer. Table 2. Potential benefit of improving hybrid rice by genetic engineering. Country Rice area in 2001 Transgenic approach Benefits (in million ha) Total rice Hybrid rice China million (50%) Insect resistance Low or no pesticide use, economic benefit to farmers India ,000 (0.44%) Plant protection Economic benefit to farmers Vietnam ,000 (6.39%) Quality, plant protection, To farmers and and nutrition improvement environment Bangladesh ,000 (0.13%) Plant protection To farmers and environment Philippines ,000 (0.24%) Plant protection To farmers and environment used. With the advent of transgenic technology, it has now become possible to insert a variety of genes into the plant genome from different sources. Further, the genetic diversity of parental lines can now be better determined by using molecular marker technology, which should aid in selecting parents suitable for making hybrids. Molecular markers can also help in tagging heterotic gene blocks, accumulating favorable genes, and making heterotic combinations. The future hybrids are expected to have wider adaptability to different environments, better yield capability, increased insect and disease resistance, and value-added grain with high nutritional content such as iron, vitamin A, and lysine. For the future success of hybrid technology, the Improving hybrid rice through anther culture and transgenic approaches 115

120 integration of transgenic and molecular breeding approaches would play a vital role in parental line improvement. References Aarts MGM, Dirkse WG, Stiekema WJ, Periera A Transposon-tagging of male sterility gene in Arabidopsis. Nature 363: Akagi H, Fujimura T Cytoplasmic male sterility transfer into japonica variety with cybrid method. In: Proceedings of the Second International Symposium on Hybrid Rice, International Rice Research Institute, Manila, Philippines. Akagi H, Sakamoto M, Negishi T, Fujimura T Construction of rice cybrid plants. Mol. Gen. Genet. 215: Alam MF, Datta K, Abrigo E, Oliva N, Tu J, Virmani SS, Datta SK Transgenic insectresistant maintainer line (IR68899B) for improvement of hybrid rice. Plant Cell Rep. 18: Bai, HS, Wang BH, Ge MF Study on purification of Mingui 63. Crop Res. 5(1): Baisakh N, Datta K, Oliva N, Ona I, Rao GJN, Mew TW, Datta SK Rapid development of homozygous transgenic rice using anther culture harboring rice chitinase gene for enhanced sheath blight resistance. Plant Biotechnol. 18(2): Brar DS, Zhu YG, Ahmed MI, Jachuk PJ, Virmani SS Diversifying the CMS system to improve the sustainability of hybrid rice technology. In: Virmani SS, Siddiq EA, Muralidharan K, editors. Advances in hybrid rice technology. Proceedings of the 3rd International Symposium on Hybrid Rice, November 1996, Hyderabad, India. Manila (Philippines): International Rice Research Institute. p Chen IS, Zhang IH, Ge MF, Wan BH, Bai HS Studies on fertility stability in the anther culture-derived progeny of Xieqingzao A. J. Crops 2:1-4. Datta K, Vasquez A, Tu J, Torrizo L, Alam MF, Oliva N, Abrigo E, Khush GS, Datta SK Constitutive and tissue-specific differential expression of cryia(b) gene in transgenic rice plants conferring enhanced resistance to insect pests. Theor. Appl. Genet. 97: Datta SK Potential benefit of genetic engineering in plant breeding: rice, a case study. Agric. Chem. Biotechnol. 43(4): Denis M, Delourme R, Gourret J-P, Mariani C, Renerd M Expression of engineered nuclear male sterility in Brassica napus. Plant Physiol. 101: Ge MF, Bai HS, Wang BH, Chen IS, Zhang JH Biological effects and field application of the 3-lines of hybrid rice purified by anther culture. In: Wang WQ, Zhu XQ, editors. Agricultural biotechnology in China. Beijing: Science & Technology Division of Agricultural Ministry. p Ge MF, Tan CL, Bai HS, Yang QP Anther culture and its application to purification and rejuvenation of a male-sterile line from abortive wild type rice. J Jiangsu Agric. Sci. 1(1): Hayashi Y, Kyozuka J, Shimamoto Hybrids of rice (Oryza sativa L.) with wild Oryza species obtained by cell fusion. Mol. Gen. Genet. 214:6-10. Kyozuka J, Kaneda T, Shimamoto K Production of a cytoplasmic male sterile rice (Oryza sativa L.) by cell fusion. Bio/Technology 7: Li P, Hu YY, Huan GS, Li RD, Zhou KD Utilization of rice anther culture in breeding intersubspecific hybrids. J. Sichuan Agric. Univ. 10(3): Balachandran et al

121 Ling DH, Tao Li-zen, Ma ZR, Zhang SP, Datta SK Engineered male sterile transgenic plants of rice (Oryza sativa L.) with ps1-barnase gene transformation by particle bombardment. Acta Genet. Sin. 25(5): Mariani C, De Beukeleer M, Truettner J, Leemans J, Goldberg RB Induction of male sterility in plants by chimeric ribonuclease gene. Nature 347: Mariani C, Gossela V, De Beukeleer M, De Block M, Goldberg RB, De Greef W, Leemans J A chimeric ribonuclease-inhibitor gene to male sterile plants. Nature 357: Narayanan KK Developing and using novel sources of male sterility. In: Virmani SS, Siddiq EA, Muralidharan K, editors. Advances in hybrid rice technology. Proceedings of the 3rd International Symposium on Hybrid Rice, Novemeber 1996, Hyderabad, India. Manila (Philippines): International Rice Research Institute. p Pathak MD, Khan ZR Insect pests of rice. Los Baños (Philippines): International Rice Research Institute. p Sundaresan V, Springer P, Volpe T, Howard S, Dean C, Jones JDG Patterns of gene action in plant development revealed by the enhancer trap and gene trap transposable elements. Genes Dev. 9: Tu J, Ona I, Zhang Q, Mew TW, Khush GS, Datta SK Transgenic rice variety IR72 with xa-21 is resistant to bacterial blight. Theor. Appl. Genet. 97: Tu J, Zhang G, Datta K, Xu C, He Y, Zhang Q, Khush GS, Datta SK. 2000a. Field performance of transgenic elite commercial hybrid rice expressing Bacillus thuringiensis δ-endotoxin. Nature Biotechnol. 18: Tu J, Datta K, Zhang Q, Datta SK. 2000b. Field performance of transgenic indica rice (Oryza sativa L.), IR72, with Xa-21. Theor. Appl. Genet. 101: Twell D, Wing R, Yamaguchi J, McCormick S Isolation and expression of an anther specific gene from tomato. Mol. Gen. Genet. 217: Virmani SS Opportunities and challenges of developing and using hybrid rice technology in the tropics. In: Peng S, Hardy B, editors. Rice research for food security and poverty alleviation. Proceedings of the International Rice Research Conference, 31 March-3 April Los Baños (Philippines): International Rice Research Institute. p Wang JY, Zhang AX, Xiang YW, Zhang AZ, Zhou XM Effects of integrated breeding techniques on the selection of restorer lines in hybrid rice. Southwest China J. Agric. Sci. 7(2): Yang ZO, Shikanai T, Yamada Y Plant regeneration from cytoplasmic hybrids of rice (Oryza sativa L.). Theor. Appl. Genet. 77: Yin JH, Zhu DY, Pan XG, Ding XH, Lie YQ, Mao LH Screening japonica wide compatibility restorer rice from pollen strains. Acta Agric. Jiangxi 6(2): Zhang AZ, Xiang YW, Zhang ZX, Wan LY, Zhou XM. 1994b. Use of anther culture method on selection and breeding of restorer line crossing between indica and japonica for rice. Acta Agron. Sin. 20(6): Zhang JH, Chen JS, Liang GH, Tang YH. 1994a. The purification of the CMS lines II-2A and You IA I rice through anther culture. J. Jiangsu Agric. Coll. 15(1): Zhu DY, Pan XG, Chen CY, Jie YQ, Ding XH, Yin JH Using androgenesis in indica rice breeding. Intl. Rice Res. Newsl. 18(1): Improving hybrid rice through anther culture and transgenic approaches 117

122 Zhu DY, Sun ZX, Pan XG, Ding XH, Shen XH, Wan Y, Pan H, Yin JH, Alejar MS, Torrizo L, Datta SK Use of anther culture in hybrid rice breeding. In: Virmani SS, Siddiq EA, Muralidharan K, editors. Advances in hybrid rice technology. Proceedings of the 3rd International Symposium on Hybrid Rice, November 1996, Hyderabad, India. Manila (Philippines): International Rice Research Institute. p Zou JT, Zhan XY, Wu HM, Wang H, Cheung AY Characterization of a rice pollenspecific gene and its expression. Am. J. Bot. 81(5): Notes Authors address: Plant Breeding, Genetics, and Biochemistry Division, International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines. Citation: Virmani SS, Mao CX, Hardy B, editors Hybrid rice for food security, poverty alleviation, and environmental protection. Proceedings of the 4th International Symposium on Hybrid Rice, Hanoi, Vietnam, May Los Baños (Philippines): International Rice Research Institute. 407 p. 118 Balachandran et al

123 Advances in understanding the genetic basis of heterosis in rice Qifa Zhang and Zhikang Li Findings revealed by the two most recent sets of studies designed to characterize the genetic basis of heterosis in rice are summarized in this paper. The first set of studies used a population of recombinant inbred lines (RILs) from a cross between Lemont and Teqing, the backcross populations of RILs with the two parents and two testcross populations. The second set of studies used an immortalized F 2 population produced by intercrossing RILs from a cross between Zhenshan 97 and Minghui 63. Heterosis measurements were used as data input for identifying a single-locus QTL and epistasis governing heterosis. Results from the two studies were in agreement that epistasis played an important role as the genetic basis of heterosis, but the types of epistatic gene actions identified in the two studies were different. The first set of studies suggested the importance of dominant types of interaction in the genetic basis of heterosis. While the second set of studies indicated the prevalence of additive-by-additive interactions, it was also shown that heterotic effects at the single-locus level in combination with the small advantageous effects of double heterozygotes at the two-locus level could adequately explain the genetic basis of heterosis in Shanyou 63. The implications of these findings were discussed. The large-scale adoption of hybrid rice in China, and more recently in India and Vietnam and other Asian countries, has contributed strongly to the improved food availability in these countries (Yuan 1998, Virmani 1998). It is estimated that hybrids can outyield conventional cultivars by 30 40% in production fields (Yuan 1992). More than 100% mid-parent heterosis has been frequently observed in experimental plots (Zhang et al 1994, 1995, 1996, Zhao et al 1999). In addition, hybrids often appear to be more adaptive and more resistant to diseases and they perform more stably than conventional cultivars. The success of hybrid rice breeding, together with a relatively small genome size, saturated molecular linkage maps (Causse et al 1994, Harushima et al 1998), and rapid advances in genome sequencing (Feng et al 2002, Goff et al 2002, Sasaki et 119

124 al 2002, Yu et al 2002), has provided a rare opportunity for dissecting the genetic basis of heterosis. Several studies have been conducted in rice with the goal of characterizing the genetic basis of heterosis, making use of high-density molecular marker linkage maps. For example, Xiao et al (1995) conducted a genetic study of quantitative traits in an intersubspecific cross and suggested that dominance was the genetic basis of heterosis in rice. However, the data presented in that paper showed that the level of heterosis was low in the cross they used compared with many data sets obtained from experimental plots, which may affect the inference of the genetic components governing heterosis. On the other hand, Yu et al (1997) analyzed the genetic components underlying yield and its component traits using an F 2:3 population derived from a highly heterotic rice cross, and detected a large number of digenic interactions involving loci distributed throughout the rice genome. They suggested that epistasis played an important role as the genetic basis of heterosis. However, both of the abovementioned analyses of heterosis were based on the performance measurement of the trait rather than heterosis, and the genetic basis of heterosis was inferred from genetic components estimated for the trait performance. Although heterosis and trait performance are closely related, they are nonetheless distinct in many respects, both statistically and biologically. Thus, to have a real picture of the genetic components underlying heterosis, it is necessary to use the measurements of heterosis as the data input in the analyses. In the last two years, two sets of experiments have been reported that aimed to characterize the genetic basis of heterosis using heterosis measurements as the input data. The first set of experiments was conducted by backcrossing a population of recombinant inbred lines (RILs) with the two parents and also crossing the RILs with two testers, resulting in heterosis measurements for five related populations (Li et al 2001, Luo et al 2001). The second set of experiments was conducted by producing an immortalized F 2 population, from which the measurements for heterosis were obtained (Hua et al 2002, 2003). This paper intends to summarize the findings and the features revealed in the analyses of these two sets of experiments. Genetic basis of heterosis in a population derived from an indica/japonica hybrid In the first set of experiments, a cross was made between Teqing, a high-yielding indica cultivar from China, and Lemont, a japonica cultivar from the United States with wide compatibility (Ikehashi and Araki 1984), from which 254 RILs were obtained by single-seed descent (Li et al 2001, Luo et al 2001). The RILs were crossed to the two parental lines, resulting in two backcross hybrid populations, 177 hybrids from the crosses of RILs with Teqing (TQBCF 1 ) and 172 hybrids from the crosses of RILs with Lemont (LTBCF 1 ). In addition, 192 and 187 hybrids were obtained by testcrossing the RILs with Zhong 413 (a restorer line with wide compatibility developed in China) and IR64 (a cultivar widely used in South and Southeast Asian countries), respectively. The RILs, the two BC 1 F 1 populations (LTBCF 1 and TQBCF 1 ), the two testcross populations (Z413TCF 1 and IR64TCF 1 ), the parents (Lemont and Teqing), and 120 Qifa Zhang and Zhikang Li

125 their F 1 were evaluated in two separate field experiments at two locations in 1996, Zhejiang Agricultural University (ZAU) and the China National Rice Research Institute (CNRRI), both in Zhejiang Province (Table 1). Measurements were taken for grain yield and other yield-component traits, including panicles per plant, grains per panicle, and grain weight. Heterosis for yield and for all three yield-component traits was observed in the BC and TC hybrid populations (Table 1). On the basis of mid-parent heterosis, H MP (= F 1 MP), the IR64TCF 1 population showed the highest heterosis in yield, the LTBCF 1 population the second, the TQBCF 1 the third, and the Z413TCF 1 the lowest. However, the relative levels of heterosis of the component traits differed from one cross to another. Within each of the F 1 populations, individual hybrids varied considerably in their performance and heterosis. Most BC or TC hybrids showed highly significant positive heterosis. However, hybrids showing significant negative heterosis for these traits were also observed, but were much less frequent in all four F 1 populations. Data for 179 restriction fragment length polymorphism (RFLP) markers and four morphological markers were obtained for all the RILs and parental lines. A linkage map was constructed using Mapmaker 3.0 (Lincoln et al 1992), which spanned 1,918.7 cm and covered 12 rice chromosomes. The trait performance and mid-parent heterosis of individual BC and TC F 1 hybrids were used to identify QTLs (quantitative trait loci) contributing to heterosis. A mixed linear model was used for interval mapping of both main-effect and digenic epistatic QTLs, for which a computer software (QTLMAPPER version 1.0) was developed, allowing simultaneous interval mapping of both main-effect and digenic epistatic QTLs in an RIL, doubled-haploid, or BC population (Wang et al 1999). The genetic expectations of the parameters estimated in the above model differ according to the nature of the mapping population and the input data. For the RIL population, the main effects a i and a j are the additive effects of the two putative QTLs (Q i and Q j ) and aa ij is the additive epistatic effect between Q i and Q j (Wang et al 1999). For the BCF 1 populations, however, a i and a j are the combined effects of additive and dominance effects when estimated from the F 1 mean values, and the QTL dominance effects when estimated from the mid-parent heterosis values. Similarly, the estimated epistatic effect using H MP measurements is the dominance by dominance interaction effect (dd ij ) between epistatic QTLs, while the effects from the mean F 1 values contained both additive and nonadditive epistatic components. Large numbers of main-effect and epistatic QTLs were resolved for performance and heterosis of the four traits in the various populations. For illustration, we use the results of grains per panicle to demonstrate the main points. Four main-effect QTLs affecting this trait (one in ZAU and three in CNRRI) were identified in the RILs (Table 2). Eleven QTLs affecting F 1 mean values and/or heterosis were detected in the BC or testcross F 1 populations. Of the 11 QTLs, two (between C225c and G2132a on chromosome 8 and between RG1094f and C16 on chromosome 10) showed additive effects as they were detectable only by the F 1 mean values, one (between G103b and RZ698 on chromosome 9) detected in the LTBCF 1 s appeared Advances in understanding the genetic basis of heterosis in rice 121

126 Table 1. Summary statistics of performance and mid-parent heterosis (H MP ) of the Lemont/Teqing RILs, the two backcross F 1, and two testcross F 1 populations (RILs two testers, Z413 and IR64). Item Yield (t ha 1 ) Panicles plant 1 Grains panicle 1 1,000-grain weight (g) Mean SD Range Mean SD Range Mean SD Range Mean SD Range Zhejiang Agricultural University Lemont Teqing F 1 (LT TQ) H MP LTBCF (LTBC) H MP TQBCF (TQBC) H MP RILs China National Rice Research Institute Lemont Teqing F 1 (LT TQ) H MP Z IR Z413TCF (Z413TC) H MP IR64TCF (IR64TC) H MP RILs Qifa Zhang and Zhikang Li

127 Table 2. Main-effect QTLs associated with grains panicle 1 in the Lemont/Teqing RILs, and heterosis in their BCF 1 and TCF 1 populations in the ZAU and CNRRI experiments. Population Location Chromosome Marker interval RILs F 1 Heterosis LOD Effect R 2 LOD Effect R 2 LOD Effect R 2 RILs ZAU 1 RZ14-C944b RILs CNRRI 3 G249-RG RILs CNRRI 6 RG653-RZ RILs CNRRI 9 G103b-RZ LTBCF 1 ZAU 9 G103b-RZ TQBCF 1 ZAU 6 C235a-G294d TQBCF 1 ZAU 9 RG451-RZ TQBCF 1 ZAU 10 RG1094f-C Z413F 1 CNRRI 3 G249-RG Z413F 1 CNRRI 4 Ph-G IR64F 1 CNRRI 3 C515-RG348a IR64F 1 CNRRI 6 G1314b-HHU IR64F 1 CNRRI 8 C225c-G2132a IR64F 1 CNRRI 9 CDO395-CDO IR64F 1 CNRRI 12 RG901-G Advances in understanding the genetic basis of heterosis in rice 123

128 to be dominant, and the remaining eight appeared to be overdominant since the QTL effects estimated from heterosis values were equal to or greater than their effects estimated from F 1 mean values. Nineteen epistatic QTL pairs affecting grains per panicle were identified from the F 1 mean and/or heterosis of the BC and TC populations, including six in the LTBCF 1 s, five in the TQBCF 1 s, five in the Z413TCF 1 s, and three in the IR64TCF 1 s, respectively (Table 3). A majority of the epistatic QTLs represent dominance by dominance interactions. Two conclusions were reached from these analyses. First, most QTLs associated with heterosis appeared to be involved in epistasis. Second, most QTLs contributing to heterosis appeared to be overdominant. These observations implicate epistasis and overdominance as the major genetic basis of heterosis, within the limit of this set of experimental materials. Genetic basis of heterosis in a population derived from the best commercial hybrid, Shanyou 63 The second set of experiments was conducted by Hua et al (2002, 2003). In this study, a new experimental design was employed that produced an immortalized F 2 population by using 240 F 9 RILs, derived by single-seed descent from a cross between Zhenshan 97 and Minghui 63, the parents of the most widely cultivated rice hybrid in China. In this design, crosses were made between the RILs chosen by random permutations of the 240 RILs. In each round of permutation, the 240 RILs were randomly divided into two groups, and the lines in the two groups were paired at random without replacement to provide parents for 120 crosses. This procedure was repeated three times, resulting in a population of 360 crosses. This population resembles an F 2 population in that the compositions and frequencies of single- and multilocus genotypes are the same as those in an F 2 population. Field trials of the immortalized F 2 population were conducted in the rice-growing seasons of 1998 and 1999 on the experimental farm of Huazhong Agricultural University in Wuhan, China, in which the hybrids and the corresponding parental RILs were planted in the same plots. Traits examined were yield per plant, tillers per plant, grains per panicle, and grain weight. Mid-parent heterosis was measured for all the traits and used as the data input for analyzing the genetic basis of heterosis. Molecular marker linkage maps consisting of 231 polymorphic loci, including 174 RFLP and 57 simple sequence repeat (SSR) loci, were constructed for both the RIL and immortalized F 2 populations. In detecting heterotic loci (HL) using a single-locus model, it was assumed that the two homozygotes of each locus did not contribute to heterosis, and a locus was considered to be an HL only when showing significant difference in heterosis between the heterozygote and the mean of the two homozygotes. This idea for detecting HL was incorporated in a composite interval mapping model (Zeng 1993, 1994). The entire genome was searched at the probability level for digenic interactions for each trait with two-way analyses of variance (ANOVA) using all pos- 124 Qifa Zhang and Zhikang Li

129 Table 3. Digenic epistatic QTLs affecting F 1 hybrid performance and heterosis of grains panicle 1 detected in the Lemont /Teqing BCF 1 RILs Lemont (LT), RILs Teqing (TQ), RILs Z413 (Z413), and RILs IR64 (IR64) populations. Population QTL i QTL j F 1 Heterosis Character Marker interval Character Marker interval LOD a i a j aa ij R 2 (%) LOD a i a j aa ij R 2 (%) LT 1 C112-RG RZ53-RZ *** *** 17.5 LT 1 R210-RZ382 2 RG83-G *** 8.1 LT 3 RG450-RG100 8 G187-G56a *** ** 13.2 LT 5 RG556-gl1 8 C424b-RZ ** 1.00 *** 10.2 LT 6 RZ762-C76 8 G187-G56a ** *** 16.5 LT 9 RZ777-CDO82 10 C16-RG * 1.07 *** ** 0.81 ** 6.6 TQ 1 RG462-CDO118 6 G200a-RZ *** *** 6.0 TQ 1 CDO455-RZ776a 1 RG472-RG ** 4.9 TQ 4 HHU39a-RG RG16-RZ797b * 0.58 *** * 0.46 ** 4.5 TQ 4 G177-RZ590b 6 RG424-G1314b *** ** 0.64 *** 9.0 TQ 4 RG214-Ph 11 RZ781-C *** *** 10.8 TQ 3 C746-CDO337 6 G1314b-HHU ** 4.7 TQ 5 RG556-gl1 11 RG1109-RZ537b ** 4.8 Z413 1 RZ14-C944b 6 G1468b-RG ** ** 5.1 Z413 1 CDO118-CDO455 5 Y1049-R569a *** *** 12.2 Z413 1 C131-RG472 4 RG1094e-Y1065Lc *** *** 7.7 Z413 2 RG139-C624x 8 C825a-CSU ** 0.44 *** ** 0.42 ** 7.5 Z413 4 Ph-G379 8 G187-G56a ** ** 5.4 IR64 3 G249-RG RG1109-RZ537b ** ** 0.20 * 0.35 ** 6.4 IR64 4 G177-RZ590b 11 RG16-RZ797b * 0.33 *** ** 0.36 *** 6.6 IR64 8 C825a-CSU RZ797b-RG1094d ** 0.20 * ** 0.32 ** 5.2 Advances in understanding the genetic basis of heterosis in rice 125

130 Table 4. Mid-parent heterosis of yield and yield-component traits in Shanyou 63 and the immortalized F 2 population. Trait Shanyou 63 Immortalized F 2 Heterosis (%) Mean (%) Range (%) 1998 Yield (t ha 1 ) Tillers plant Grains panicle ,000-grain weight (g) Yield (t ha 1 ) Tillers plant Grains panicle ,000-grain weight (g) sible two-locus combinations of marker genotypes. Randomization tests were conducted to identify interactions that are more likely to be really significant. Each significant interaction was partitioned into four components, each specified by a single degree of freedom: additive (first locus) additive (second locus) (AA), additive dominance (AD), dominance additive (DA), and dominance dominance (DD). Statistical significance for each term was assessed using an orthogonal contrast test provided by the statistical package STATISTICA (StatSoft 1997). The hybrid Shanyou 63 showed very strong heterosis in yield and grains per panicle (Table 4). The amounts of heterosis varied widely for all the traits in the immortalized F 2 population, from highly negative to highly positive. A total of 33 HL were identified in the two years for heterosis of the four traits (Table 5). Only two of the HL were detected in both years; the remaining 31 HL were detected only in one year. To further characterize the genetic effects of the loci showing significant heterotic effects, the additive and dominance effects were calculated using the performance data (before subtracting the parental means) at the exact genomic locations of the HL (Table 5) using composite interval mapping, even though QTLs were not detected at most of the HL (Hua et al 2002). It can be seen that the genetic effects of the trait performance evaluated at the HL varied from partial dominance and full dominance to overdominance, with several of the HL showing large overdominance. The two-locus combinations that showed significant interactions as detected using two-way ANOVA at P = are listed in Table 6. Also listed are the significant interactions identified by randomization tests, from which it can be seen that several interactions simultaneously survived the randomization tests in both years for each of the traits. The numbers of three different types of interaction terms (AA, AD/DA, and DD) partitioned using the orthogonal contrast tests for the two-locus combinations that were confirmed by the randomization tests are given in Table 7. In 126 Qifa Zhang and Zhikang Li

131 Table 5. Putative heterotic loci (HL) identified for yield and yield-component traits in the immortalized F 2 population. Trait HL Flanking markers LOD h a A b D c d/a d 1998 Yield yd1 C567-C yd3 RG393-G yd6 RG653-G yd8 G2132-R Tillers tp6b RM225-C plant 1 tp6c RZ588-P tp9 RM215-R1952b tp12 C909B-RM Grains gp1 RM237-C panicle 1 gp4 G235-R gp8 RG333-C gp10 C153A-RM gp11 RM20a-C Grain gw3 RZ403-C weight gw4 G102-RM gw6b Y4073L-C751A gw6c C962-RZ gw11a RM209-RM gw11b RG2-RM gw12 C966-G1128a Yield yd5 R830-R yd7 C1023-R yd9a C1232-R yd9b RM219-RZ yd12 C909B-RM Tillers tp6a Wx-RM plant 1 tp11 G257-RM Grains gp6 RZ667-RG panicle 1 gp7 C1023-R gp12 C732-RM20b Grain gw1 C86-RG weight gw3 RZ403-C gw6a Y4073L-C751A gw6b R2869-C gw10 C153A-RM a Heterotic effect of the HL defined as the difference in heterosis between the heterozygote and the means of the two homozygotes. b,c Additive and dominance effects calculated using the performance data at the exact genomic location of the HL. d Ratio of dominance to the absolute value of the additive effect. Advances in understanding the genetic basis of heterosis in rice 127

132 Table 6. Numbers of significant interactions for heterosis of yield and yieldcomponent traits identified by searching all possible two-locus combinations and confirmed by randomization tests. Trait Whole-genome searching a Randomization test b Common Common Yield Tillers plant Grains panicle Grain weight a Significant at P = identified by the whole-genome search. b Interactions that were identified by the randomization test in one year and significant at P = identified by the whole-genome search in the other year are also listed. Table 7. Summary of significant interactions for the heterosis of yield and yield-component traits based on the randomization test. Trait Interaction Common Yield Positive pairs AA AD (DA) DD Tillers plant 1 Positive pairs AA AD (DA) DD Grains panicle 1 Positive pairs AA AD (DA) DD Grain weight Positive pairs AA AD (DA) DD all four traits, AA occurred at predominantly high frequencies, followed by AD/DA, with DD being the least frequent. In two-locus combinations showing significant AA, the parental two-locus homozygotes (11/11 or 22/22) in many cases showed marginal advantages vis-à-vis the means of the two parental genotypes (11/11 and 22/22). However, the complementary two-locus homozygotes (11/22 or 22/11) frequently appeared to be the best genotypes by showing large deviations from the means of the two parental genotypes as well as the Minghui 63 genotypes (data not shown, but can be found in Hua et al 2003). In two-locus combinations showing significant DD, all the double heterozygotes (12/12) appeared to be advantageous compared with the means of the two 128 Qifa Zhang and Zhikang Li

133 parental genotypes (Table 8). However, the best two-locus genotypes were frequently those that were homozygous at one locus and heterozygous at the other locus (11/12, 12/11, 22/12, or 12/22). The heterotic values were also examined for two-locus combinations showing AD/DA interactions (data not shown). The general trend is similar: the parental two-locus homozygotes (11/11 or 22/22) had marginal advantages in some cases, the complementary two-locus homozygotes (11/22 or 22/11) frequently showed large effects on heterosis, and single heterozygotes (11/12, 12/11, 22/12, and 12/22) can also be the best two-locus genotypes in a few cases. It should be noted that in no case did the double heterozygote show the highest level of heterosis. These features were also well illustrated when analyzed using the performance data (trait measurements before subtracting the parental means) (Hua et al 2002). To interpret the genetic basis of heterosis based on the analyses, the authors made a distinction between the heterosis observed in the immortalized F 2 population and the heterosis expressed in the F 1 hybrid. They also chose grains per panicle as the trait for detailed analysis because this trait is less complex than yield and the results may intuitively be more comprehensible, and because this trait shows a high level of heterosis and high heritability that have been repeatedly observed in previous studies (Zhang et al 1994, 1996). The analyses of the immortalized F 2 population showed that all kinds of genetic effects, including heterotic effects (because of partial-dominance, full dominance, and overdominance) at the single-locus level, and all three types of interactions (AA, AD/DA, and DD) at the two-locus level, were involved in the genetic basis of heterosis. Thus, at the population level, all kinds of genetic effects can be contributors to the genetic basis of heterosis. Thus, the effects of dominance, overdominance, and epistasis of various forms are not mutually exclusive in the genetic basis of heterosis. All of these components have roles to play depending on the genetic architecture of the population. For the hybrid Shanyou 63, however, only genetic components associated with heterozygotes are relevant, given the conditions that all the analyses were based on the loci that were polymorphic between the two parents, Minghui 63 and Zhenshan 97. Hence, only single-locus heterotic effects (because of partial-dominance, full dominance, and overdominance) and DD interactions were pertinent to the interpretation of heterosis in Shanyou 63. Assuming complete independence of the singlelocus heterotic effects and digenic interactions involving DD detected in the analyses, which might be violated because of linkage, the genetic basis of heterosis for grains per panicle in the F 1 hybrid may tentatively be sketched as follows. Summing up the heterotic effects over the HL, as can be seen from Table 5, would produce a total of grains per panicle. Summation of the deviations of double heterozygotes from the means of the two parental genotypes over the two-locus combinations showing DD yielded a total of grains per panicle (Table 8). Thus, together, the effects detected at the single-locus and two-locus levels could account for (86%) of the seeds that were measured as the amount of heterosis in the F 1 (Table 4). This is an excellent approximation considering that the analysis failed to reveal many of the single-locus heterotic effects and DD because of the statistical Advances in understanding the genetic basis of heterosis in rice 129

134 Table 8. Comparative advantage of the double heterozygote in the amount of heterosis in each of the two-locus combinations showing significant DD for grains panicle 1 in Locus 1 a Locus 2 a Var % by Double heterozygote Best Best DD homozygote b genotype b Heterosis Over Over best Over best midparent c homozygote genotype RM243 (1) RM234 (7) e d 11/22 12/11 RG634 (2) C347 (8) d 11/22 22/12 RM31 (5) RM201 (9) /22 11/22 P (6) R496 (12) d f 22/11 22/12 P (6) RM12 (12) f f 22/11 22/12 a The numbers in parentheses indicate the chromosomal locations of the markers. b Genotype of the first locus/second locus; 11, homozygous for the Minghui 63 allele; 22, homozygous for Zhenshan 97 allele; 12, heterozygote. c Mean of the two parental genotypes. d,e,f Significant difference from zero at 0.05, 0.01, probability levels, respectively. 130 Qifa Zhang and Zhikang Li

135 thresholds imposed, and that the heritability for this trait, although high, is far from unity. These results strongly suggest that heterotic effects (because of partial-dominance, full dominance, and overdominance) at the single-locus level, in combination with the small advantageous effects of double heterozygotes (because of DD) at the two-locus level, can adequately account for the genetic basis of heterosis in Shanyou 63, the most widely cultivated rice hybrid in China. Conclusions These two experiments made use of populations from two types of crosses: an indica japonica cross and a cross representing the best commercial indica hybrid, and both crosses are highly heterotic. The results from the two experiments are in agreement such that both showed that epistasis played an important role as the genetic basis of heterosis, which is also consistent with the results of a previous study (Yu et al 1997). However, the conclusions of the two experiments differ in the types of epistatic gene actions. The first set of experiments suggested that dominant types of digenic interactions are important in the genetic basis of heterosis, whereas the second set of experiments showed that additive by additive interactions occur most frequently in the immortalized F 2 population. In addition, it was also shown that heterotic effects (because of partial-dominance, full dominance, and overdominance) at the single-locus level, in combination with the small advantageous effects of double heterozygotes (because of DD) at the two-locus level, can adequately explain the genetic basis of heterosis in Shanyou 63. Several possibilities exist to account for the discrepancies in the results of the two experiments, including differences in the genetic materials, the experimental designs, and the statistical genetic models as well as assumptions involved in the analyses. Resolving these differences may help reconcile the long-debated questions surrounding the genetic basis of heterosis. References Causse MA, Fulton TM, Cho YG, Ahn SN, Chunwongse J, Wu K, Xiao J, Yu ZH, Ronald PC, Harrington SE, Second G, McCouch SR, Tanksley SD Saturated molecular map of the rice genome based on an interspecific backcross population. Genetics 138: Feng Q, Zhang Y, Hao P, Wang S, Fu G, Huang Y, Li Y, Zhu J, Liu Y, Hu X, Jia P, Zhang Y, Zhao Q, Ying K, Yu S, Tang Y, Weng Q, Zhang L, Lu Y, Mu J, Lu Y, Zhang LS, Yu Z, Fan D, Liu X, Lu T, Li C, Wu Y, Sun T, Lei H, Li T, Hu H, Guan J, Wu M, Zhang R, Zhou B, Chen Z, Chen L, Jin Z, Wang R, Yin H, Cai Z, Ren S, Lü G, Gu W, Zhu J, Tu W, Jia J, Zhang Y, Chen J, Kang H, Chen X, Shao C, Sun Y, Hu Q, Zhang X, Zhang W, Wang L, Ding C, Sheng H, Gu J, Chen S, Ni L, Zhu F, Chen W, Lan L, Lai Y, Cheng Z, Gu M, Jiang J, Li J, Hong G, Xue Y, Han B Sequence and analysis of rice chromosome 4. Nature 420: Advances in understanding the genetic basis of heterosis in rice 131

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138 Notes Authors addresses: Qifa Zhang, National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan , China, phone: , fax: , Zhikang Li, Plant Breeding, Genetics, and Biochemistry Division, International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines. Acknowledgements: Qifa Zhang acknowledges financial support from the National Program on the Development of Basic Research and Zhikang Li acknowledges financial support from the Rockefeller Foundation. Citation: Virmani SS, Mao CX, Hardy B, editors Hybrid rice for food security, poverty alleviation, and environmental protection. Proceedings of the 4th International Symposium on Hybrid Rice, Hanoi, Vietnam, May Los Baños (Philippines): International Rice Research Institute. 407 p. 134 Qifa Zhang and Zhikang Li

139 Molecular approaches for fixing the heterozygosity of hybrid rice Xuezhi Bi, J. de Palma, R. Oane, G.S. Khush, and J. Bennett The heterotic yield advantage of hybrid rice is not available to poor farmers because costly F 1 seed must be purchased every season. If the heterozygosity of the hybrid could be fixed, through some novel form of sexual or asexual seed formation, production costs would fall and farmers would be able to reproduce hybrid seed in their own fields. Here we consider two approaches: (1) a sexual pathway similar to that leading to permanent hybridity in a few plant genera such as Oenothera and Isotoma and (2) an asexual pathway based on the much more common phenomenon of apomixis. We have focused our experiments on the second pathway. Apomixis is known among relatives of maize, wheat, and millet but is unproven for relatives of rice. In collaboration with CSIRO Plant Industry, IRRI is pursuing the goal of a synthetic form of apomixis that would be compatible with hybrid rice production ( one-line hybrid rice ). CSIRO has already accomplished fertilization-independent (FI) endosperm formation in Arabidopsis thaliana through mutagenesis and is now attempting to reproduce this achievement in rice through RNA interference. If this objective can be attained in conjunction with another major objective FI embryo formation in the nucellus we would be well on the way to achieving a form of FI apomixis for hybrid rice. However, when fertilization fails for genetic or environmental reasons, the ovary normally fails to expand. If this were to happen in FI apomicts, it would greatly restrict the size of the endosperm. We have now succeeded in bypassing this important checkpoint in more than 50 independent transformants by using OsASP1::gus constructs. OsAsp1 is a putative aspartyl proteinase that is the rice orthologue of the barley nucellin gene. The OsASP1::gus transformants show two phenotypes: (1) total male sterility through poor pollen development and failure of dehiscence and (2) the formation of full-size pericarp and testa devoid of embryo and endosperm. They are, however, completely female fertile and have been propagated as hemizygotes. This discovery opens the way to FI formation of full-size endosperm. We report also on progress toward achieving FI embryogenesis in rice through ectopic expression of transcription factor genes such as OsLEC1. 135

140 Heterotic yield advantage is about 15 20% for indica/indica hybrid rice compared with the best indica inbreds (Virmani 1994, Li and Yuan 1999). Preliminary data suggest that heterotic values of up to 40% may be possible for indica/tropical japonica hybrids (Khush et al 1998). The heterotic yield advantage of hybrid rice is not available to poor farmers because costly F 1 seed must be purchased every season. When farmers try to reproduce hybrid seed, both heterozygosity and yield advantage decline as a result of recombination events accompanying meiosis (Virmani 1994). Most hybrid rice is currently produced by the original three-line method, with a small percentage produced by the newer two-line method (Yuan 1998). If the heterozygous state could be genetically fixed through some form of apomictic seed production that avoided meiosis, costs would decline and farmers would be able to reproduce hybrid seed in their own fields. Yuan (1998) has used the term one-line hybrids to describe this approach. In collaboration with the Commonwealth Scientific and Industrial Research Organization in Australia, IRRI is pursuing the goal of developing a synthetic form of apomixis that would be compatible with hybrid rice production. Here we begin by discussing in general terms the possible pathways to fixing heterozygosity, and then focus on the particular apomictic pathway that is being followed currently by CSIRO and IRRI. CSIRO s Division of Plant Industry has already accomplished fertilization-independent endosperm formation in Arabidopsis thaliana through mutagenesis (Chaudhury et al 1997) and is now attempting to reproduce this achievement in rice through a transgenic approach. We report two complementary transgenic studies on rice at IRRI: fertilization-independent formation of the pericarp and seed coat and progress toward fertilization-independent embryogenesis. Fixing heterozygosity First, we discuss the meiotic and ameiotic pathways to fixing heterozygosity in hybrid rice. Heterozygosity is greatest in an F 1 hybrid between two distantly related parental lines and declines sharply in the F 2 and later generations (Fig. 1). The decline is due principally to the events of meiosis. During premeiotic interphase, the chromosomes replicate, increasing the DNA content of the nucleus from 2C to 4C. As the centromeres of the replicated chromosomes do not separate, the two sister chromatids of each chromosome remain connected. Nonsister chromatids of homologous chromosome pairs undergo synapsis and homologous recombination. Following their attachment as a group to the spindle apparatus at the first meiotic division, homologous chromosomes segregate randomly into the two daughter cells with the sister chromatids of each still linked together. At the second meiotic division, the sister chromatids separate into the tetrad of daughter spores, each of which contains one complete haploid (1C) set of chromosomes derived randomly from the maternal and paternal sets. This basic process occurs in both ovary and anthers, except that only one female spore survives to form the embryo sac containing the egg, whereas all four male spores survive to form pollen with two sperm cells. Fertilization of the egg by a sperm restores the 2C state, whereas fertilization of the binucleate central 136 Xuezhi Bi et al

141 Homozygous regions Maternal chromosome Paternal chromosome No meiotic recombination, exclusively paternal chromosomes in pollen, exclusively maternal chromosomes in egg Meiotic Hypothetical F 2 Fixed maximal heterozygosity Normal F 2 Reduced heterozygosity Meiotic Meiotic recombination and random segregation F 1 hybrid Maximal heterozygosity Centromeres on spindle Ameiotic Without random segregation and with minimal mitotic recombination Apomictic F 2 Fixed maximal heterozygosity Fig. 1. Schematic representation of F 1 hybrid rice and three types of derived F 2 generations normal, apomictic, and hypothetical. Nonsister chromatids of two pairs of homologous chromosomes are depicted at the start of the first meiotic division, adjacent on the spindle prior to the onset of homologous recombination. For simplicity, the sister chromatids produced in the premeiotic S phase are not represented. Lightly and darkly shaded chromosomal regions are of maternal and paternal origin, respectively. cell (1C + 1C) by the other sperm creates the triploid (3C) state characteristic of endosperm. The loss of heterozygosity arises from three processes: (1) the recombination that occurs at synapsis, (2) the segregation of the homologues at meiosis I, and (3) the fertilization of the egg by a sperm that differs from it in the specific outcomes of the first two processes. We have been thinking about what would be needed to genetically fix heterozygosity in rice. In principle, two contrasting pathways might fix heterozygosity. The first possibility is a sexual pathway, in which meiosis occurred without the processes leading to a loss of heterozygosity. The second is an asexual pathway in which meiosis was bypassed completely. We shall now discuss the possible features of both pathways (Fig. 1). The first is now largely hypothetical, although populations of Oenothera, Isotoma, and several less studied plant genera achieve stable hybridity by a route that has several features that would be desired in the sexual pathway. The second is less hypothetical because it seeks to reproduce features seen widely among certain types of polyploid apomictic plants. We discuss the first pathway briefly and then focus the remainder of this article on a novel approach to achieving apomixis for diploid hybrid rice. Molecular approaches for fixing the heterozygosity of hybrid rice 137

142 Hypothetical pathway to fixed heterozygosity To fix the heterozygosity of hybrid rice while retaining meiosis, it would be necessary to block three processes: (1) recombination between nonsister homologous chromatids at synapsis, (2) random segregation of homologous chromosomes at the first meiotic division, and (3) fertilization between an egg and a sperm that differed randomly in their content of maternal and paternal alleles. How would such a result be achieved? Three changes would appear to be necessary (Fig. 1): during meiotic prophase, synapsis would have to occur without recombination, during meiosis I, the maternal and paternal set of chromosomes would have to segregate to opposite poles, and there would have to be a balanced gametic lethality that ensured that only the maternal set of chromosomes would be transmitted through the egg, while only the paternal set of chromosomes would be transmitted through the sperm. It is interesting that these novel features seem to be the basis for the permanent hybridity seen in a few plant genera such as Oenothera (Cleland 1962) and Isotoma (James 1965). The phenomenon has been studied most intensively in certain races of Oenothera. During meiosis I in these races, the 14 chromosomes do not form into seven pairs of bivalents as in normal plants, but join up end-to-end to form an undulating circle whose plane lies at right angles to the axis of the spindle (Cleland 1962). The maternal and paternal chromosomes alternate around the circle and segregate as maternal and paternal sets into the daughter cells. During meiosis II, the sister chromatids within the maternal or paternal set separate to form two haploid chromosome sets. It is not clear whether the exclusive production of heterozygous offspring is due to a balanced gametic lethality or to a zygotic lethality of homozygous offspring. The circle of 14 chromosomes forms in the cells of a hybrid plant whenever the maternal and paternal chromosomes show a very specific sort of segmental interchange heterozygosity (Cleland 1962). It has been established that, in one parent, as much as a whole arm of each chromosome has been replaced by an arm of a nonhomologous chromosome. However, the replacement is not reciprocal because that would generate only 4-member circles. Rather, the interchanges are such that each maternal chromosome synapses with two different paternal chromosomes and vice-versa. There are obviously many ways of achieving this situation, and Cleland and his colleagues detected examples of most of them among populations of Oenothera. In some races of Oenothera, smaller rings are seen, with the chromosomes that are excluded from the ring forming normal bivalents. The Oenothera system of chromosome rings is not ideal for maintaining heterozygosity. For one thing, it is quite mutagenic, and aneuploids are often detected. For another, the chromosomal interchanges required to achieve complete ring formation (24 chromosomes per ring in rice) would severely limit the flexibility of hybrid formation. The balanced lethal system would also require a lot of attention. Nevertheless, it may be a system worth considering as a model for a meiotic pathway to 138 Xuezhi Bi et al

143 fixed heterozygosity. We now discuss an ameiotic pathway to fixed heterozygosity, namely, apomixis. Apomictic pathway to fixed heterozygosity Apomixis is found in more than 300 species of plants, including close relatives of maize, wheat, sorghum, and millet (Asker and Jerling 1992), but it appears to be absent from rice and its relatives (Brar et al 1995, Bennett et al 2001). The three major types of apomixis are diplospory, apospory, and adventitious embryony (Koltunow 1993). In diplospory, the most common form of apomixis among grasses, a modified sexual embryo sac produces an embryo without meiosis. Apospory and adventitious embryony produce an ameiotic embryo in competition with the sexual embryo. Research on mapping apomixis genes in diplosporous and aposporous grasses has progressed considerably but has not yet reached the point where the identity or even the number of apomixis genes is clear (Savidan 2000). It is therefore not yet possible to begin a project on achieving apomixis in rice based on a molecular understanding of events in natural apomicts. It is also unlikely that naturally occurring apomixis could be used directly in hybrid rice production. Apomixis is normally associated with polyploids and is either obligate or facultative; for hybrid rice, apomixis would have to be compatible with diploidy and would have to be switched off and on as the breeder requires. These considerations suggest that some form of synthetic apomixis would have to be developed. Synthetic apomixis A synthetic approach to achieving apomixis in sexual plants was proposed by Peacock (1992), who introduced the concept of the embryo sac induction (ESI) gene. According to this concept, the ESI gene, normally turned on in the megaspore after meiosis, might possibly be turned on by mutation without meiosis. If so, an embryo might be formed with the same genetic constitution as the maternal tissue. The most convenient plant in which to search for the ESI gene would be Arabidopsis because of its well-developed genetic, molecular, and genomic resources. A genetic screen was designed to detect mutants of Arabidopsis producing fertilization-independent seed (FIS) (Peacock et al 1995, Koltunow et al 1995). The first three fis mutants were reported by Chaudhury et al (1997). To capitalize on the fis mutants and related developments, CSIRO and IRRI began a three-phase, 15-year project to achieve apomixis in hybrid rice (Bennett et al 1998). Phase 1 was to be devoted to the development of molecular tools required to achieve apomixis in rice and involved research on Arabidopsis, rice, and the model apomict Hieracium (Koltunow et al 2000). Phase 2 was to focus on the application of these tools to achieve a basic form of apomixis in rice. Phase 3 was seen as the time to integrate this form of apomixis into hybrid rice breeding programs. The year 2002 marked the end of phase 1. Molecular approaches for fixing the heterozygosity of hybrid rice 139

144 Fertilization-independent endosperm production Analysis of the three fis mutants revealed that autonomous endosperm development took place with minimal embryo formation (Chaudhury et al 1997). Female meiosis and embryo sac formation occurred normally, and then fusion of the two haploid nuclei of the central cell led spontaneously to the formation of diploid endosperm without the need for fertilization. The corresponding genes FIS1, FIS2, and FIS3 were found to belong to the polycomb group of transcription factors (Luo et al 1999, 2000, Chaudhury et al 2001). FIS1 was found to be identical to MEA (Grossniklaus et al 1998) and FIS3 to FIE (Ohad et al 1999). FIS2 has been found to be homologous to two other Arabidopsis genes, EMF2 and VRN2, and to a Drosophila gene, Su(z)12 (Chaudhury et al 2001). We assume that natural apomicts, instead of invoking some alternative pathway of autonomous endosperm development, use the FIS-like genes as part of their apomictic reproduction mechanism. If so, inactivation of genes orthologous to FIS1, FIS2, and FIS3 could be used to trigger autonomous endosperm development in any target plant. CSIRO Plant Industry is therefore isolating orthologous genes from rice to develop interfering RNA (RNAi) constructs that will inactivate these genes in vivo (Wesley et al 2001) to stimulate the autonomous development of endosperm in rice. Synthetic apomixis: fertilization-dependent or -independent? The potential availability of fertilization-independent (FI) endosperm formation raises a key question about synthetic apomixis: Can it actually be engineered to be entirely fertilization-independent? After all, we plan to generate apomictic embryos from cells of the nucellus without fertilization. FI apomixis could be desirable for environmental reasons (Bennett et al 2001). One potential problem lies in the hybrid maternal tissue that also constitutes the seed: the palea, lemma, lodicules, pericarp, and testa. The first three tissues develop and function before fertilization and therefore pose no problem. By contrast, the ovary tissues that will eventually expand after fertilization to form the pericarp and testa almost invariably fail to expand in the absence of fertilization. If fertilization fails for genetic or environmental reasons, a checkpoint prevents further expansion of these tissues within the already fully expanded palea and lemma. The operation of this checkpoint is clearly evident in almost all forms of cytoplasmic male sterility in rice; the only known exception is the formation of pseudograins in rice plants in which the boro II cytoplasm operates in a Wu 10 nuclear background (Chaudhury et al 1982). Fortunately, as we report below, we now have available a means of bypassing this checkpoint in rice. Fertilization-independent formation of pericarp and testa Our induction of full development of the pericarp and testa without fertilization was a by-product of our search for a promoter to direct nucellus-specific expression of transcription factors that control embryonic genes. The nucellus is the large mass of 140 Xuezhi Bi et al

145 cells surrounding the embryo sac within the ovule. About 14 days before fertilization, a nucellar cell just under the dome of the ovule develops into the megaspore mother cell. This diploid cell will undergo meiosis to form a tetrad of haploid cells, only one of which will survive. Known as the megaspore, the survivor undergoes three mitoses to form the 8-nucleate embryo sac. At about the time of fertilization, the nucellus begins to undergo programmed cell death to supply the young zygotic embryo and the young endosperm with nutrients. Several nucellus-specific genes have been identified in barley. We focused on isolating the rice orthologue of one of these genes. It encoded a putative aspartyl proteinase that was named nucellin by Chen and Foolad (1997). The barley nucellin gene encodes a polypeptide of 410 amino acids that is expressed exclusively in the nucellus, starting about 1 day before fertilization and most abundantly at 3 4 days after fertilization. Chen and Foolad (1997) speculated that nucellin plays a role in the programmed cell death of the nucellus. Data on the barley nucellin gene aided the isolation of the rice orthologue. We examined its expression pattern with reverse transcriptase-pcr and RNA in situ hybridization and found that the rice nucellin gene is expressed most actively in young embryos and considerably less in the nucellus, the anthers, pollen, and other tissues of the flower. We used the barley nucellin gene in RT-PCR and RNA in situ hybridization in barley flowers and observed the same pattern of expression. We concluded that the expression analysis of Chen and Foolad (1997) was not valid and decided to call this putative aspartic proteinase OsAsp1 rather than rice nucellin. We transformed two rice cultivars (Nipponbare and Murasaki) with three different constructs in which the gus reporter gene was under the control of an extended rice OsASP1 promoter (promoter plus first exon with or without the first intron). All 60 independent OsASP1::gus transformants grew normally through vegetative and reproductive development until the late stages of pollen maturation, at which point starch accumulation failed in most pollen, pollen shedding failed completely, and the plants were sterile as judged by the failure to form embryo and endosperm. However, before anthesis, ovary development appeared normal. When the transgenic plants were fertilized with pollen from nontransgenic plants, seed set was normal and the seeds were viable. The distribution of the gus gene in the progeny was 3:1 (gus + : gus ), even greater than the expected 1:1 ratio. These results indicated that the OsASP1::gus transformants were male sterile but female fertile. We are propagating the transformants as hemizygotes. The most remarkable feature of the OsASP1::gus transformants was the enlargement of the ovary in spite of male sterility (Fig. 2). Other features were also unique, especially the failure of the stigma, anthers, and filaments to degenerate after anthesis. Examination of fixed thin sections shows that both pericarp and testa are formed, but the enlarged ovaries are devoid of embryo and endosperm (Fig. 3). This phenomenon may be very useful in ensuring full seed enlargement in apomicts capable of FI embryogenesis and FI endosperm formation. Our working hypothesis is Molecular approaches for fixing the heterozygosity of hybrid rice 141

146 A B C D E Fig. 2. Rice plants transformed with OsASP1::gus constructs are male-sterile but bypass a checkpoint limiting ovary expansion when fertilization fails. (A) Control plants at anthesis. (B) Control plants 5 days after anthesis. (C) Transgenic plants 5 days after anthesis. (D,E) Transgenic plants 10 days after anthesis. (E) Transgenic plants stained for GUS activity. that the OsASP1::gus construct is unusually effective in silencing or co-suppressing endogenous OsASP1 expression (Wesley et al 2001). The molecular basis of this phenomenon requires more attention and the propagation of the hemizygote will facilitate such studies. This behavior is quite different from that observed in most known rice lines showing cytoplasmic male sterility (CMS). FI formation of pericarp and testa has been seen with only one CMS line, an unusual japonica CMS line studied by Chaudhury et al (1982). It contains the boro II cytoplasm in the Wu 10 nuclear background. As with OsASP1::gus transformants, the empty pericarp and seed coat are fully expanded and filled with clear liquid. It will be interesting to examine expression of the endog- 142 Xuezhi Bi et al

147 A En B Fig. 3. Basal region of longitudinal sections of ovary of rice cultivar Nipponbare stained with acridine orange. (A) Nontransgenic control. (B) Plant transformed with an OsASP1::gus construct. Em = embryo, En = endosperm, Pe = pericarp, Te = testa, bar = 100 µm. enous OsASP1 gene in this line. Our transformants and the boro II/Wu 10 line may provide important clues concerning the nature of the checkpoint that blocks ovary enlargement in the absence of fertilization and its relationship, if any, with male sterility. Fertilization-independent embryogenesis: ectopic expression of embryonic genes Our approach to achieving FI embryogenesis in the rice nucellus is based in part on the success that has already been achieved with ectopic embryogenesis in Arabidopsis. Molecular approaches for fixing the heterozygosity of hybrid rice 143

148 We began by isolating rice orthologues of Arabidopsis LEC1 and PKL genes that are reported to have a capacity to induce ectopic embryogenesis when overexpressed (AtLEC1, Lotan et al 1998) or inactivated (AtPKL, Ogas et al 1999). We have isolated two LEC1 homologues and one PKL homologue from rice (Bennett et al 2001). The two LEC1 homologues (OsLEC1A and OsLEC1B) are very closely related to AtLEC1 in the conserved HAP3 DNA-binding and subunit interaction domains but are even more closely related to a LEC1-like gene recently detected in unannotated Arabidopsis DNA sequence. OsLEC1B, like AtLEC1, is expressed in immature embryos and in cotyledon and radicle meristems. It is also expressed to high levels in somatic embryos. By contrast, OsLEC1A is expressed in immature embryos but not in somatic embryos. When rice was transformed with an Actin1::OsLEC1A construct, transcripts of OsLEC1A were present in leaves of all transformants but were absent from leaves of control plants (Table 1). In contrast with leaves of controls, leaves of all transformants contained several seed-specific transcripts (encoding OsBZ8 transcription factor and glutelin-b1 and α-globulin seed storage proteins). Seed-specific transcription factors OsLEC1B and OsVP1 and a late embryogenesis abundant (LEA) protein (OsEm1) were not expressed in leaves. On the other hand, embryo-preferred transcripts encoding 16 kda and 18 kda oleosins were detected in one of the six transgenic plants. These results suggest that OsLEC1 is capable of inducing a range of seed-specific proteins in leaves. This range can be modified in some genetic backgrounds to induce embryo-preferred genes, but some genes remain uninducible by this transcription factor. We must now determine whether Actin1::OsLEC1B and Actin1::antisensePKL induce a different spectrum of seed-specific genes in transgenic rice. We have also identified the rice orthologue of the BABYBOOM (BBM) gene that causes ectopic embryogenesis when overexpressed in Brassica (Boutilier et al 2002). We had originally thought that the next step would be to express these genes under the control of a nucellus-specific promoter. The rice orthologue of the barley nucellin gene (Chen and Foolad 1997) appeared to offer such a promoter until we showed (see previous section) that both the barley gene and its rice orthologue are much more abundantly expressed in the immature embryo than in the nucellus. We propose now to use gene expression in the megaspore mother cell at the time of meiosis as the springboard for controlling gene expression in surrounding cells (see below). Future objectives The CSIRO-IRRI collaboration on synthetic apomixis entered its second 5-year phase in July 2003, with the award of a new grant by the Australian Centre for International Agricultural Research, which also funded the first phase. IRRI s activities in relation to synthetic apomixis will focus on four objectives: (1) understanding the mode of action of OsAsp1::gus constructs and refining the mechanism by which FI formation of pericarp and testa occurs; (2) combining this phenomenon with FI endosperm 144 Xuezhi Bi et al

149 Table 1. Examination of transgenic plants containing Act1::OsLEC1A construct for ectopic expression of selected rice genes in leaves. Transcripts were detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Acc = GenBank accession. Tissue > Leaf Spikelet Cultivar > Murasaki Nipponbare IR64 Gene Negative Transgenic plant number Negative Positive control control control Acc. no. Name AY OsLEC1A AY OsLEC1B + U42208 OsBZ D16640 OsVP1 + U22102 OsEm + AF REB X63990 Globulin X54314 GlutB U45322 Globulin-like + + U43930 Ole U43931 Ole D16096 GAPDH Molecular approaches for fixing the heterozygosity of hybrid rice 145

150 Phase 1 ( ) FI endosperm in Arabidopsis Identification of OsFIS genes FI pericarp and testa in rice Identification of OsLEC1 and OsDMC1 Phase 2 ( ) FI pericarp and testa Role of OsAsp1 Mechanism of passing fertilization checkpoint FI diploid endosperm (full-sized) Achieved by crossing? Replace transgenes by mutations? FI diploid endosperm RNAi to suppress OsFIS genes Fate of pericarp and testa? FI embryogenesis Rice homologues of embryogenesis genes of Arabidopsis Ectopic expression in nucellus under control of megaspore mother cell Apomictic hybrid rice Viability and fertility? Grain quality and stress tolerance? Yield potential? Phase 3 ( ) Ploidy of endosperm Biosafety One-line hybrid rice Fig. 4. Three-phase research project aiming to produce synthetic apomixis for one-line hybrid rice production. This project is a collaboration between the Division of Plant Industry of the Commonwealth Scientific and Industrial Research Organization (CSIRO) of Australia and the International Rice Research Institute in the Philippines. FI = fertilization-independent. formation as achieved by CSIRO colleagues to increase the size of FI endosperms; (3) understanding how to induce FI embryogenesis in the rice nucellus based on results emerging from work on other plants, especially Arabidopsis; and (4) combining these phenotypes to produce FI apomictic seeds. These objectives are summarized in Figure 4. Understanding FI-independent pericarp and testa expansion The mode of action of OsAsp1::gus constructs is fascinating and raises many questions. What is the nature of the checkpoint on pericarp and testa expansion? What 146 Xuezhi Bi et al

151 role does a putative aspartate proteinase OsAsp1 play in this checkpoint? How does expression of the OsAsp1::gus transgene lead to the observed phenotypes of male sterility and FI expansion of maternal (i.e., hybrid plant) components of the seed? Are there any additional, unwanted side-effects of the construct that would have to be eliminated by refinement of this approach? Equally fascinating but probably not relevant to achieving apomixis in hybrid rice are questions about the importance of the checkpoint in the evolution of cereals and in the ability of cereals to optimize grain number under stress. Full-size apomictic endosperm If down-regulation of OsFIS genes by RNAi leads to FI endosperm formation without bypassing the checkpoint on FI ovary expansion, the size of the endosperm will be limited to the size of the ovary at anthesis. It will be important to combine the phenotype produced by RNAi of OsFIS with that produced by OsAsp1::gus. We have recently found that the use of pollen from nontransgenic plants to cross-pollinate male-sterile T 0 plants carrying the OsAsp1::gus constructs produces viable seeds that carry the transgene. If OsFIS-RNAi plants are male-fertile, it should be possible to use pollen from OsFIS-RNAi plants to cross-pollinate OsAsp1::gus plants and thereby combine the two phenotypes. If enlarged endosperm forms in a FI manner, a major step forward to our goal of apomictic hybrid rice will have been achieved. An intriguing point is that we might be able to reach the same point in the future simply with mutants that knock out these two types of genes. The mutants would be discovered by a reverse genetics approach based on knowledge of the identities and sequences of the genes. The double transformants or the double mutants, however, would probably lack a functional embryo. Repairing this defect would be the major task of Phase 2. Apomictic embryogenesis FI embryogenesis lies at the core of achieving synthetic apomixis. Fortunately, rapid progress is being made in understanding natural and ectopic embryogenesis in Arabidopsis and to a lesser extent in other plants. We shall examine the embryogenic capacity of rice homologues of Arabidopsis genes that regulate embryogenesis. These genes include not only LEC1 and PKL but also LEC2 (Stone et al 2001), BBM (Boutilier et al 2002), WUSCHEL (Zuo et al 2002), and functionally related genes. We shall use microarray analysis to determine the fraction of embryonic genes that are expressed ectopically in various easily analyzed tissues overexpressing these transcription factors. We shall also isolate molecular markers for embryogenesis. The markers will include several already available genes (OsLEC1A, OsLEC1B, OsASP1, VP1, OsEm, and oleosin) as well as others that will be isolated specifically for this purpose. Zinc-finger proteins are one of the largest groups of transcription factors and have already been exploited as stage-specific markers for anther development in petunia (Kobayashi et al 1998). The DNA-binding motif QALGGH is frequently present twice or more in this class of protein, making it relatively simple to amplify Molecular approaches for fixing the heterozygosity of hybrid rice 147

152 and clone the corresponding cdnas (Kubo et al 1998). These cdna clones will be used to make gene-specific RT-PCR primers and in situ hybridization probes to assay the ectopic expression of the genes in response to embryo-inducing genes. However, the key is to induce FI embryogenesis in the nucellus of hybrid rice. At one stage, we planned to use microarray analysis, RT-PCR, and in situ hybridization to isolate nucellus-specific promoters for rice. Microarray analysis (Kawasaki et al 2001) would identify cdnas expressed in the ovary at the correct time (5 8 days before anthesis), while RT-PCR would identify the subset of cdnas expressed in the ovary but in no other tissue of the plant, including the anthers. In situ hybridization (Kathiresan et al 2002) would then identify a smaller subset of cdnas expressed exclusively in the nucellus at the correct time. From these cdnas, nucellus-specific promoters would be isolated and spliced with rice transcription factors to induce embryonic gene expression in the nucellus as an important step to achieving nucellar adventitious embryony. Now, we favor a different approach based on using the megaspore mother cell to control gene expression in neighboring cells through cell-cell or cell-to-cell interactions (Fig. 4). The approach may be based on the meiosis-specificity of a truncated promoter of the OsDMC1A gene (A. Kathiresan, J. Bennett, manuscript in preparation). This gene was cloned by Kathiresan et al (2002) and shows higher preference for expression during meiosis than its recent duplicate, OsDMC1B. Questions for the future The final step of Phase 2 of the project will be to combine FI embryogenesis and FI formation of full-sized endosperm to produce viable, fertile apomictic hybrid rice seeds and to examine them for genetic stability, grain quality, stress tolerance, and heterotic yield potential. Two questions will be uppermost in our minds: (1) Is heterozygosity entirely responsible for heterosis, or are epigenetic (parent-of-origin) effects also important? (2) Is a diploid endosperm compatible with normal yield, or would it be necessary to restore the natural triploid endosperm? (Bennett et al 2001). These questions and others concerning the integration of synthetic apomixis into hybrid rice breeding programs (as one-line hybrids) will be considered fully in the third 5-year phase beginning in July References Asker SE, Jerling L Apomixis in plants. Boca Raton, Fla. (USA): CRC. Bennett J, Ladha JK, Schmit V, Sheehy J New frontier projects: beyond the pipeline. In: Dowling NG, Greenfield SM, Fischer KS, editors. Sustainability of rice in the global food system. Los Baños (Philippines): International Rice Research Institute. p Bennett J, Bi XZ, Kathiresan A, Khush GS Molecular tools for achieving synthetic apomixis in hybrid rice. In: Khush GS, Brar DS, Hardy B, editors. Rice genetics IV. New Delhi (India): Science Publishers, and Los Baños (Philippines): International Rice Research Institute. p Boutilier K, Offringa R, Sharma VK, Kieft H, Ouellet T, Zhang L, Hattori J, Liu CM, van 148 Xuezhi Bi et al

153 Lammeren AA, Miki BL, Custers JB, van Lookeren Campagne MM Ectopic expression of BABY BOOM triggers a conversion from vegetative to embryonic growth. Plant Cell 14: Brar DS, Elloran RM, Lanuang MC, Tolentino VS, Khush GS Screening wild species of rice for apomixis. Philip. J. Crop Sci. 20:6. Chaudhury AM, Koltunow A, Payne T, Luo M, Tucker MR, Dennis ES, Peacock WJ Control of early seed development. Annu. Rev. Cell Dev. Biol. 17: Chaudhury AM, Ming L, Miller C, Craig S, Dennis ES, Peacock WJ Fertilizationindependent seed development in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 94: Chaudhury RC, Virmani SS, Khush GS, Juliano BO A pseudograin on a cytoplasmic male-sterile rice line. Int. Rice Res. Newsl. 7(6):3-4. Chen F, Foolad MR Molecular organization of a gene in barley which encodes a protein similar to aspartic protease and its specific expression in nucellar cells during degeneration. Plant Mol. Biol. 35: Cleland RE The cytogenetics of Oenothera. Adv. Genet. 11: Grossniklaus U, Vielle-Calzada J, Hoeppner MA, Gagliano WB Maternal control of embryogenesis by MEDEA, a polycomb group gene in Arabidopsis. Science 280: James SH Complex hybridity in Isotoma petraea. I. The occurrence of interchange heterozygosity, autogamy and a balanced lethal system. Heredity 20: Kathiresan A, Khush GS, Bennett J Two rice DMC1 genes are differentially expressed during meiosis and haploid and diploid mitosis. Sexual Plant Reprod. 14: Kawasaki S, Borchert C, Deyholos M, Wang H, Brazille S, Kawai K, Galbraith D, Bohnert HJ Gene expression profiles during the initial phase of salt stress in rice. Plant Cell 13: Khush, GS, Aquino RC, Virmani SS, Bharaj TS Using tropical japonica germplasm to enhance heterosis in rice. In: Virmani SS, Siddiq EA, Muralidharan K, editors. Advances in hybrid rice technology. Manila (Philippines): International Rice Research Institute. p Kobayashi A, Sakamoto A, Kubo K, Rybka Z, Kanno Y, Takatsuji H Seven zinc-finger transcription factors are expressed sequentially during the development of anthers in petunia. Plant J. 13: Koltunow AM Apomixis: embryo sac and embryos formed without meiosis or fertilization in ovules. Plant Cell 5: Koltunow AM, Bicknell RA, Chaudhury AM Apomixis: molecular strategies for the generation of genetically identical seeds without fertilization. Plant Physiol. 108: Koltunow AM, Johnson SD, Bicknell RA Apomixis is not developmentally conserved in related genetically characterised Hieracium plants of varying ploidy. Sexual Plant Reprod. 12: Kubo K, Sakamoto A, Kobayashi A, Rybka Z, Kanno Y, Nakagawa H, Takatsuji H Cys2/His2 zinc-finger protein family of petunia: evolution and general mechanism of target-sequence recognition. Nucleic Acids Res. 26: Molecular approaches for fixing the heterozygosity of hybrid rice 149

154 Li JM, Yuan LP Hybrid rice: genetics, breeding, and seed production. Plant Breed. Rev. 17: Lotan T, Ohto M, Yee KM, West MA, Lo R, Kwong RW, Yamagishi K, Fischer RL, Goldberg RB, Harada JJ Arabidopsis LEAFY COTYLEDON1 is sufficient to induce embryo development in vegetative cells. Cell 93: Luo P, Bilodeau P, Koltunow A, Dennis ES, Peacock WJ, Chaudhury AM Genes controlling fertilization-independent seed development in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 98: Luo M, Bilodeau P, Dennis ES, Peacock WJ, Chaudhury A Interaction and parent-oforigin effects for FIS2, MEA, and FIE in the endosperm and embryo of developing Arabidopsis seeds. Proc. Natl. Acad. Sci. USA 97: Ogas J, Kaufmann S, Henderson J, Somerville C PICKLE is a CHD3 chromatinremodeling factor that regulates the transition from embryonic to vegetative development in Arabidopsis. Proc. Natl. Acad. Sci. USA 96: Ohad N, Yadegari R, Margossian L, Hannon M, Michaeli D, Harada JJ, Goldberg RB, Fischer RL Mutations in FIE, a WD polycomb group gene, allow endosperm development without fertilization. Plant Cell 11: Peacock WJ Genetic engineering and mutagenesis for apomixis in rice. Apomixis Newsl. 4:3-7. Peacock J, Ming L, Craig S, Dennis E, Chaudhury A A mutagenesis programme for apomixis genes in Arabidopsis. In: Induced mutations and molecular techniques for crop improvement. Vienna (Austria): International Atomic Energy Agency. p Savidan Y Apomixis: genetics and breeding. Plant Breed. Rev. 18: Stone SL, Kwong LW, Yee KM, Pelletier J, Lepiniec L, Fischer RL, Goldberg RB, Harada JJ LEAFY COTYLEDON2 encodes a B3 domain transcription factor that induces embryo development. Proc. Natl. Acad. Sci. USA 98: Virmani SS Heterosis and hybrid rice breeding. New York, N.Y. (USA): Springer- Verlag. Wesley SV, Helliwell CA, Smith NA, Wang M, Rouse DT, Liu Q, Gooding PS, Singh SP, Abbott D, Stoutjesdijk PA, Robinson SP, Gleave AP, Green AG, Waterhouse PM Construct design for efficient, effective and high-throughput gene silencing in plants. Plant J. 27: Yuan LP Hybrid development and use: innovative approach and challenges. Int. Rice Comm. Newsl. 47:7-15. Zuo J, Niu QW, Frugis G, Chua NH The WUSCHEL gene promotes vegetative-toembryonic transition in Arabidopsis. Plant J. 30: Notes Authors addresses: Xuezhi Bi, J. de Palma, R. Oane, G.S. Khush, and J. Bennett, Plant Breeding, Genetics, and Biochemistry Division, International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines. j.bennett@cgiar.org. Xuezhi Bi, current address: Department of Biological Sciences, National University of Singapore, Blk S2, 14 Science Drive 4, Singapore Acknowledgments: We thank the Australian Centre for International Agricultural Research for financial support of this project; Arumugam Kathiresan, Abdul Chaudhury, and Anna Koltunow for discussions; and Evelyn Liwanag, Gina Borja, and Noel Malabanan for technical assistance. Citation: Virmani SS, Mao CX, Hardy B, editors Hybrid rice for food security, poverty Bi alleviation, et and environmental protection. Proceedings of the 4th International 150 Xuezhi Symposium on Hybrid Rice, Hanoi, Vietnam, May Los Baños (Philippines): International Rice Research Institute. 407 p.

155 Management of hybrid rice

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157 Physiological bases of heterosis and crop management strategies for hybrid rice in the tropics S. Peng, J. Yang, R.C. Laza, A.L. Sanico, R.M. Visperas, and T.T. Son Commercial production of three-line hybrid rice has expanded in some tropical Asian countries. Heterotic level, crop management, hybrid seed production, and grain quality are the major limiting factors for the further expansion of hybrid rice in this region. Traits responsible for heterosis for grain yield are different under different growing conditions. Under favorable growing conditions, the higher yield of hybrid rice is attributed to greater biomass production. Under suboptimum growing conditions such as low radiation, a higher harvest index contributes to the higher yield of hybrid rice. Greater leaf area production rather than tiller production is responsible for the higher biomass accumulation and grain yield of hybrid rice compared with the best conventional check variety in the tropics. A high tillering rate causes the early vigor of hybrid rice grown in temperate China. Hybrid rice technology is more profitable in rice-growing areas with medium and high yields ( 5 t ha 1 ). Hybrid rice is suitable for both the dry and wet seasons. Both heterosis and yield differences are greater at a high N rate than at a low N rate. Hybrid rice needs a different N management strategy from conventional rice to maximize yield and minimize disease damage. Leaf area and tiller and biomass production of A lines do not limit the seed yield of hybrid rice. In fact, crop growth could be too high to cause a reduction in the outcrossing rate. Widening transplanting spacing up to cm and transplanting split tillers are feasible ways to reduce the seed requirement to as low as 2.6 kg ha 1 without reducing grain yield. The commercial production of three-line hybrid rice has expanded in some tropical Asian countries such as India, Vietnam, and the Philippines. However, the area planted to hybrid rice is still quite small in those countries. The major limitations to the largescale adoption of hybrid rice technology in the tropics are the inadequate level of standard heterosis for grain yield, poor agronomic management of hybrid rice, low yield of hybrid seed production, high seed cost, and poor grain quality. Under favorable growing conditions, heterosis for grain yield is usually attributed to higher biomass production (Song et al 1990, Yamauchi 1994). It is often reported that hybrid 153

158 rice has better remobilization of carbohydrate from straw to grain than conventional rice (Song et al 1990). Under what conditions has this advantage of remobilization been reflected in yield traits such as harvest index (HI)? The early vigor of hybrid rice developed in temperate areas has been mainly attributed to its higher tillering rate (Agata 1990, Peng et al 1998, Ying et al 1998). Is this also true for hybrid rice in the tropics? Standard heterosis in breeders replicated yield trials is usually high, with an average of 20% to 25%. Information on the level of standard heterosis in agronomic trials and in farmers fields is limited. The level of standard heterosis under different yields, growing seasons, and N rates is necessary information for establishing a deployment strategy for hybrid rice technology. In this paper, we have summarized three data sets from breeders replicated yield trials, published data of agronomic trials on hybrid rice in journals, and agronomic trials conducted by IRRI s crop physiology laboratory in the Philippines to compare the level of standard heterosis and yield advantage of hybrid rice under different growing conditions. Crop management of hybrid rice developed in the tropics was not studied as intensively as that of hybrid rice in China. Some studies were conducted without using conventional rice as a check variety. Others used only a few levels of treatment and often concluded that hybrid and conventional rice responded similarly to the crop management treatment because genotype crop management interactions were not statistically significant. These results cannot prove whether hybrid rice needs a crop management strategy different from that used in conventional rice. To determine the optimal crop management strategy for hybrid rice, one should compare the standard heterosis and yield advantage of hybrid rice at each treatment level. In this paper, experiments with different rates and timing of N application are included to elucidate whether hybrid rice needs a different crop management strategy from that of conventional rice to maximize grain yield and minimize disease damage. The yield of commercial hybrid seed production in the tropics is lower than that in China (Virmani 2001). The low outcrossing rate is mainly responsible for the low hybrid seed yield. Crop management strategies such as planting date, planting density, and gibberellic acid (GA 3 ) application have proven to be effective in increasing outcrossing rate and seed yield. It is unknown whether crop growth and sink size (spikelets m 2 ) in the female parent (A line) are sufficient to support high yield in hybrid seed production in the tropics. One of the effective ways to cut seed cost is to reduce the seed requirement during crop establishment. Widening transplanting spacing and transplanting split tillers can reduce the seed requirement substantially. Will those practices reduce grain yield, especially at high yield? Two field experiments were conducted at the IRRI experiment farm to study this question. Traits responsible for heterosis for grain yield While the physiological bases of heterosis for grain yield are unclear, the morphological and agronomic traits responsible for heterosis for grain yield are relatively known. Studies often show that the yield advantage of hybrids is attributed to higher biomass production compared with the best conventional varieties (Song et al 1990, 154 Peng et al

159 Table 1. Grain yield (oven-dry weight), total biomass at physiological maturity (PM), and harvest index of rice genotypes from different groups. IRRI farm, 1998 wet season (WS) and 1999 dry season (DS). Genotype Number Grain yield Biomass at Harvest of entries (g m 2 ) PM (g m 2 ) index (%) 1998 WS F 1 hybrid , Indica inbred , NPT indica , Av SE DS F 1 hybrid , Indica inbred , NPT , NPT indica , Av SE Yamauchi 1994). Others reported that hybrid rice has a higher HI than conventional varieties (Ponnuthurai et al 1984, Blanco et al 1990). The high HI was attributed to the high ratio of reserves translocated from the culm and sheath to the spikelets during the ripening period (Song et al 1990). We compared grain yield and the contribution of biomass partitioning and translocation to grain yield among four genotypic groups: F 1 hybrids, indica inbreds, new plant type (NPT) lines, and NPT indica lines in the 1998 wet season (WS) and 1999 dry season (DS) in the Philippines. The average yield of F 1 hybrid rice was 17% and 4% higher than that of indica inbreds in the 1998 WS and 1999 DS, respectively (Table 1). Grain yield was highly and positively correlated to HI (r 2 = ) and the relationship between grain yield and biomass production was relatively weak. Therefore, the high grain yield of F 1 hybrids was attributed to high HI and not to biomass at physiological maturity. The high HI of F 1 hybrids was due to the great amount of biomass that was accumulated before flowering and translocated to the grains during grain filling (Table 2). Biomass accumulation from flowering to physiological maturity was lower in F 1 hybrids than in other genotypic groups. The mean daily total radiation during the 1999 DS was only 18.6 MJ m 2, about 10% lower than the 10-year average from the 1989 DS to 1998 DS. Maximum grain yield is 10 t ha 1 in the DS in tropical irrigated rice systems under normal climatic conditions (Yoshida 1981). In this study, the highest grain yield expressed at 14% moisture level was 8 t ha 1 in the 1999 DS. In general, the WS is unfavorable for rice production because of reduced total radiation compared with the normal DS. Therefore, both the 1998 WS (with mean daily total radiation of 18.2 MJ m 2 ) and 1999 DS are considered as suboptimum for rice production. Further improvement in rice yield potential under favorable growing conditions might come from increased biomass production rather than increased HI (Peng et al 1999). Un- Physiological bases of heterosis and crop management strategies for hybrid rice in the tropics 155

160 Table 2. Biomass at flowering, biomass production after flowering (W r ), and biomass translocated from straw to grain (T) of rice genotypes from different groups. IRRI farm, 1998 wet season (WS) and 1999 dry season (DS). Genotype Number Biomass W r T of entries at flowering (g m 2 ) (g m 2 ) (g m 2 ) 1998 WS F 1 hybrid Indica inbred NPT indica Av SE DS F 1 hybrid 11 1, Indica inbred 11 1, NPT NPT indica 11 1, Av SE der suboptimum growing conditions, however, maintaining high HI could be more important than increasing biomass production to achieve high actual grain yield. Vigor in initial growth has been considered as the principal trigger for heterosis and increased biomass and yield of hybrid rice in the tropics (Akita 1988, Blanco et al 1990). Physiological and morphological studies have been conducted to determine the underlying mechanism for the early vigor of hybrid rice (Yamauchi and Yoshida 1985, Maruyama et al 1987, Akita et al 1990, Blanco et al 1990, Kabaki 1993). The early vigor of hybrid rice developed in temperate areas has been mainly attributed to its higher tillering rate (Agata 1990, Peng et al 1998, Ying et al 1998). Earlier reports indicated that hybrids had higher tillering ability than their parental lines (Jones 1924, Sen and Mitra 1958). The larger leaf area index resulting from an increased number of tillers was the main factor for achieving heterosis for crop growth rate (CGR) of japonica/indica hybrid rice at the early vegetative stage under temperate conditions (Kabaki 1993). Yamauchi and Yoshida (1985) and Yamauchi (1994) also reported that the vigorous growth of the hybrids compared with their parental lines was due to their high leaf area growth rate (LAGR) caused by high tillering. For hybrid rice in the tropics, however, it is not clear what factors contribute to the greater increase in dry matter of rice hybrids during vegetative growth compared with that of conventional varieties. We analyzed data from seven field experiments conducted from 1994 to 1997 in both the DS and WS at the IRRI farm. Each experiment had one to four pairs of comparisons between the best hybrids and the best conventional varieties. The tillering rate of hybrids from transplanting to midtillering was lower than or equal to that of conventional varieties (Fig. 1A). Hybrid rice had the same or greater LAGR than conventional varieties during the same period (Fig. 1B). The crop growth rate from transplanting to midtillering was closely correlated with LAGR 156 Peng et al

161 Tillering rate (m 2 d 1 ) Hybrid Conventional Leaf area growth rate (dm 2 m 2 d 1 ) Crop growth rate (g m 2 d 1 ) DS 1995 DS 1996 DS 1994 WS 1995 WS 1997 DS Year and season Fig. 1. Tillering rate, leaf area growth rate, and crop growth rate of hybrid and conventional varieties from transplanting to midtillering in the dry (DS) and wet (WS) seasons in different years at the IRRI farm. Bars represent average standard error of mean of four replications. Physiological bases of heterosis and crop management strategies for hybrid rice in the tropics 157

162 (r 2 = 0.96) but not with tillering rate (r 2 = 0.35). The difference in LAGR and CGR between hybrids and conventional varieties was not associated with tillering. Therefore, the higher LAGR, independent from tillering, contributed to the greater CGR of recently developed hybrid rice in the tropics. The specific leaf weight of conventional varieties was generally higher than that of hybrids, implying that the hybrids had thinner leaf blades than the conventional varieties. A large leaf blade is often associated with a thin leaf blade. The higher LAGR of the hybrids than that of the conventional varieties could be attributed to their thinner leaf blades. Standard heterosis under different yield level The level of standard heterosis under different yield levels is useful information for establishing a deployment strategy for hybrid rice technology. We summarized data of breeders replicated yield trials (RYT) conducted in multilocations of India, the Philippines, Vietnam, Pakistan, and Malaysia in the DS and WS from 1986 to 1999 (Virmani et al 1996, IRRI 1995, 1997, 1998, 1999, 2000). Standard heterosis was calculated as the yield difference between the hybrid and conventional check variety divided by the yield of the conventional check variety, expressed as a percentage. Average yield was calculated from the grain yield of the hybrid and conventional check variety and used to represent the yield level of that particular site and season. This data set consisted of 75 experiments conducted in 14 years and 155 data points (Fig. 2). It covered a wide range of yield from 2.2 to 11.6 t ha 1. Standard heterosis ranged from 14% to 67%, with a mean of 23.4%. Average standard heterosis at low (<5 t ha 1 ), medium (5 7 t ha 1 ), and high (>7 t ha 1 ) yield levels was 24.6%, 24.9%, and 17.9%, respectively. Although standard heterosis decreased at high yield, the average yield difference between hybrid and conventional rice was not different from that at medium yield level. At high yield level, average yield was 7.9 t ha 1 and hybrid rice produced 1.3 t ha 1 more than conventional rice. At medium yield level, average yield was 6.0 t ha 1 and hybrid rice produced 1.3 t ha 1 more than conventional rice. At low yield level, average yield was 4.3 t ha 1 and hybrid rice produced 0.9 t ha 1 more than conventional rice. The results suggest that hybrid rice technology could be more profitable in the rice-growing areas with medium and high yield levels. Average standard heterosis was higher in the WS than in the DS (26.1% vs 19.6%). Since average grain yield was also higher in the DS than in the WS (6.7 vs 5.3 t ha 1 ), the average yield difference between hybrid and conventional rice was the same in both seasons (i.e., 1.2 t ha 1 ). Therefore, hybrid rice technology is suitable for both the DS and WS. The same analysis was conducted by summarizing published data of agronomic trials on hybrid rice (Fig. 3). This data set consisted of eight experiments conducted in the DS and WS from 1993 to 1999 in India and 25 data points. It covered yield from 2.3 to 8.6 t ha 1 with an N rate ranging from 0 to 180 kg ha 1. At each N level, the best hybrid was compared with the best conventional check variety. Standard heterosis ranged from 15% to 48%, with a mean of 16.4%. At low yield level (<5 t ha 1 ), average standard heterosis was 18.1% and average yield was 3.9 t ha 1. At 158 Peng et al

163 Standard heterosis (%) n = 155 Mean = 23.4% Average grain yield (t ha 1 ) Fig. 2. Standard heterosis under different yield levels. Data were from breeders replicated yield trials conducted in multilocations in India, the Philippines, Vietnam, Pakistan, and Malaysia in the dry and wet seasons from 1986 to 1999 (Virmani et al 1996, IRRI, 1995, 1997, 1998, 1999, 2000). 11 medium and high yield levels ( 5 t ha 1 ), average standard heterosis was 15.7% and average yield was 6.2 t ha 1. Hybrid rice produced 0.6 t ha 1 more at low yield level and 0.8 t ha 1 more at medium and high yield levels than conventional rice. This supports the conclusion that hybrid rice technology could be more profitable in the rice-growing areas with medium and high yield levels. When the N rate was greater than 120 kg ha 1, average yield, standard heterosis, and the yield difference between hybrid and conventional rice was 6.5 t ha 1, 25.3%, and 1.4 t ha 1, respectively. At the low N rate ( 120 kg ha 1 ), average yield, standard heterosis, and the yield difference between hybrid and conventional rice was 5.2 t ha 1, 13.0%, and 0.5 t ha 1, respectively. Therefore, both heterosis and yield difference were greater at a high N rate than at a low N rate. The third data set was from agronomic trials on hybrid rice conducted by IRRI s crop physiology laboratory in the DS and WS from 1994 to 2001 at IRRI and at Philippine Rice Research Institute (PhilRice) farms, Philippines (Fig. 4). This data set consisted of 28 experiments and 44 data points. It covered yield from 4.4 to 11.2 t ha 1 with an N rate ranging from 0 to 235 kg ha 1. At each N level, the best hybrid was compared with the best conventional check variety. Standard heterosis ranged Physiological bases of heterosis and crop management strategies for hybrid rice in the tropics 159

164 Standard heterosis (%) n = 25 Mean = 16.4% Average grain yield (t ha 1 ) Fig. 3. Standard heterosis under different yield levels. Data were from published data of agronomic trials on hybrid rice conducted in the dry and wet seasons from 1993 to 1999 in India. from 15% to 40%, with a mean of 5.1%, which was significantly lower than the average standard heterosis in the previous two data sets. In this data set, variation in yield was mainly caused by season (DS vs WS) and by N rate. Average standard heterosis was higher in the WS than in the DS (7.4% vs 4.2%). Since average grain yield was also higher in the DS than in the WS (8.3 vs 6.0 t ha 1 ), the average yield difference between hybrid and conventional rice was insignificant between the two seasons (i.e., 0.39 t ha 1 in the WS and 0.34 t ha 1 in the DS). This supports the conclusion that hybrid rice technology is suitable for both the DS and WS. When the N rate was greater than 120 kg ha 1, average yield, standard heterosis, and the yield difference between hybrid and conventional rice was 8.9 t ha 1, 6.8%, and 0.5 t ha 1, respectively. At a low N rate ( 120 kg ha 1 ), average yield, standard heterosis, and the yield difference between hybrid and conventional rice was 6.2 t ha 1, 2.9%, and 0.1 t ha 1, respectively. The results indicated again that both heterosis and yield difference were greater at a high N rate than at a low N rate. Therefore, hybrid rice technology could be more profitable under high N input. However, this statement does not mean that hybrid rice requires more N than conventional rice under the same yield level. 160 Peng et al

165 Standard heterosis (%) n = 44 Mean = 5.1% Average grain yield (t ha 1 ) Fig. 4. Standard heterosis under different yield levels. Data were from agronomic trials on hybrid rice conducted by IRRI s crop physiology laboratory in the dry and wet seasons from 1994 to 2001 at IRRI and Philippine Rice Research Institute (PhilRice) farms, Philippines. Crop management for increasing the grain yield of hybrid rice Compared with hybrid rice breeding, little research has been conducted on crop management of hybrid rice in the tropics. The International Rice Research Notes (IRRN) published only 10 research papers on crop management of hybrid rice cultivation from 1990 to 2001, with the first published paper on this subject in 1996 (Table 3). All those 10 papers were submitted by Indian scientists. Nitrogen management and crop establishment were the major topics in those papers (Table 4). A conventional check variety was included in seven of the 10 papers. Among the seven papers with a conventional check variety, six papers reported positive standard heterosis for grain yield. However, none of the seven papers showed a significant genotype crop management interaction. In other words, hybrids and conventional check varieties responded similarly to the crop management treatments. This does not necessarily mean that hybrid rice should be managed as conventional rice is. Most of those papers were published in IRRN from 1996 to 1999 with a focus on N management and crop establishment. This trend was also true in journals such as Oryza, the Indian Journal of Agricultural Science, and Indian Journal of Agronomy. Physiological bases of heterosis and crop management strategies for hybrid rice in the tropics 161

166 Table 3. Number of research papers published in the International Rice Research Notes (IRRN) on crop management of hybrid rice cultivation and seed production from 1990 to Year Crop Crop management management on hybrid rice on hybrid seed cultivation production Total 10 2 Table 4. Treatments of 10 research papers published in the International Rice Research Notes (IRRN) on crop management of hybrid rice cultivation from 1990 to Treatment Number of research papers Rate of nitrogen application 6 Timing of nitrogen application 4 Nitrogen source 1 Seedling number per hill 3 Seeding rate in seedbed 2 Seedling age at transplanting 1 Transplanting spacing 2 Transplanting date 1 Does hybrid rice need a different crop management strategy from conventional rice in the tropics to maximize grain yield and N-use efficiency and minimize disease damage? Peng et al (1998) reported that skipping basal or midtillering N application was more detrimental to hybrid rice than to conventional rice in terms of grain yield. The beneficial effect of N application at the flowering stage for improving grain filling was greater for hybrid rice than for conventional rice. Furthermore, standard heterosis was highest under optimal N management. The field experiment conducted at the IRRI farm in the 2000 DS further supported this statement (Table 5). We determined the effects of the timing and rate of N application on standard heterosis of IR68284H. A split application of 100 kg N ha 1 at basal and midtillering increased the grain yield of IR68284H by 10% compared with the application of 100 kg ha 1 at 162 Peng et al

167 Table 5. Grain yield of IR72 and IR68284H and their standard heterosis as affected by timing and rate of N application. IRRI farm, 2000 dry season. Timing of N application N rate Grain yield (t ha 1 ) Standard (kg ha 1 ) heterosis (%) BS a MT PI FL IR72 IR68284H ± 0.4 b 7.5 ± ± ± ± ± a BS = basal, MT = midtillering, PI = panicle initiation, FL = flowering. b Mean ± SE. only basal. However, IR72 did not respond to this split application. Reducing the total N rate from 200 to 125 kg ha 1 caused a yield reduction of 13% in IR68284H but only 5% in IR72. Standard heterosis was significantly higher with the better timing and optimal rate of N application. Real-time N management using the chlorophyll meter (SPAD) was tested for IR68284H with IR72 as the control at the IRRI farm in the 2001 DS (Table 6). Three N treatments were (1) zero-n, (2) fixed-split application of 60 kg ha 1 at basal, 40 at midtillering, 60 at panicle initiation, and 45 at flowering, and (3) SPAD-based N management. In the SPAD-based N treatment, N was applied when the SPAD reading was below 35 in IR72 and 34 in IR68284H from 21 days after transplanting (DAT) to flowering. A lower SPAD threshold was used for the hybrid rice than for the conventional rice because hybrid rice usually has a lower leaf N content and specific leaf weight than conventional rice. The SPAD reading was taken weekly by measuring the five topmost fully expanded leaves from each plot. The SPAD value was calculated as the mean of the five leaves and four replications. The amount of N application was 45 kg ha 1 if the SPAD value was below the threshold around the panicle initiation stage and 30 kg ha 1 in the rest. In IR72, N was applied at 28 and 51 DAT with 30 and 45 kg ha 1, respectively (Fig. 5). In IR68284H, N was applied at 23, 44, and 64 DAT with 30, 45, and 30 kg ha 1, respectively. The SPAD-based N management resulted in a significantly lower total N rate but higher agronomic N- use efficiency compared with the fixed-split N treatment. The SPAD-based N management reduced grain yield of IR72 significantly compared with the fixed split-n treatment. However, grain yield was not significantly different between the two treatments in IR68284H. It is obvious that the hybrid responded to the real-time N management differently from the conventional rice. An experiment was conducted in the 2001 WS to determine the effect of timing and rate of N application and transplanting spacing on bacterial leaf streak damage in hybrid rice (Table 7). IR68284H was used with IR72 as the conventional check variety. The total N rate ranged from 60 to 90 kg ha 1 with different proportions of N applied before the maximum tillering stage. Transplanting spacings were cm and cm. The degree of bacterial leaf streak infestation was scored at 58 DAT using a rating scale of 0 to 9, wherein zero means no occurrence and 9 represents severe damage. Significantly higher bacterial leaf streak infestation was observed in Physiological bases of heterosis and crop management strategies for hybrid rice in the tropics 163

168 SPAD value 45 IR72 IR68284H Days after transplanting Fig. 5. Chlorophyll meter (SPAD) readings of SPAD-based N management treatment in IR72 and IR68284H, IRRI farm, 2001 dry season. Nitrogen was applied when the SPAD reading was below 35 in IR72 and 34 in IR68284H from 21 days after transplanting to flowering. Numbers in the figure indicate the amount of N in kg ha 1 applied each time. Bars represent standard error of mean of four replications. Table 6. Grain yield and agronomic N-use efficiency (ANUE) of IR72 and IR68284H under fixed-split and chlorophyll meter (SPAD)-based N management. IRRI farm, 2001 dry season. Genotype N treatment N rate Grain yield ANUE (kg ha 1 ) (t ha 1 ) (kg kg 1 ) IR72 Control ± 0.12 a Fixed-split ± SPAD-based b ± IR68284H Control ± 0.10 Fixed-split ± SPAD-based c ± a Mean ± SE. b SPAD threshold was 35, 30 kg N ha 1 was applied at 28 days after transplanting (DAT) and 45 kg N ha 1 at 51 DAT. c SPAD threshold was 34. At 23, 44, and 64 DAT, N was applied at 30, 45, and 30 kg ha 1, respectively. 164 Peng et al

169 Table 7. Effect of N and transplanting spacing on bacterial leaf streak in IR72 and IR68284H. IRRI farm, 2001 wet season. Timing of N application N rate IR72 IR68284H (kg ha 1 ) BS a MT MaT PI b Mean Mean ± 0.0 c 0.3 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Mean a BS = basal, MT = midtillering, MaT = maximum tillering, and PI = panicle initiation. b Transplanting spacing at cm and cm. c Mean ± SE. Rating scale was 0 to 9, wherein zero means no occurrence and 9 represents severe damage by bacterial leaf streak. Physiological bases of heterosis and crop management strategies for hybrid rice in the tropics 165

170 Table 8. Comparison in crop growth at flowering between A lines of hybrid seed production fields in the 2001 dry season with IR68284H, which produced 10.7 t ha 1 in the 1997 dry season at the IRRI farm. Entry Panicles Plant height LAI a TDW b CGR c m 2 (cm) (g m 2 ) (g m 2 d 1 ) IR58025A , IR73328A , IR72081A , IR69624A , IR68888A , Mean , IR68284H , a Leaf area index at flowering stage. b Total dry weight at flowering stage. c Crop growth rate from transplanting to flowering stage. IR68284H than in IR72. The transplanting spacing had no effect on the incidence of bacterial leaf streak. Reducing the N rate at the basal and midtillering stage resulted in less disease incidence, especially in IR68284H. Therefore, a crop management strategy should be developed specifically for hybrid rice to fully express its yield potential. Crop management for increasing hybrid seed yield and reducing seed requirement The yield of hybrid seed production, next to the level of standard heterosis, is the most important constraint to the expansion of hybrid rice technology. The yield of commercial hybrid seed production is usually less than 1 t ha 1 in the tropics, mainly because of the low outcrossing rate. One may also ask if CGR and spikelet number per unit area of female parent (A line) limit the yield of hybrid seed production. A growth analysis was conducted at the flowering stage on five A lines in five hybrid seed production fields in the 2001 DS at the IRRI farm (Table 8). Panicle number m 2, leaf area index, and total dry matter of the five A lines at the flowering stage were actually greater than those of a rice crop that produced a grain yield of 10.7 t ha 1. This suggests that biomass production and sink size in the female parent were sufficient to support high seed yield. In fact, growth in leaf area and dry matter of the A line should be reduced moderately to facilitate outcrossing by minimizing the physical barrier of pollen distribution. This can be done by optimizing the N rate in the hybrid seed production field. In addition to increasing hybrid seed yield, reducing the seed requirement during crop establishment while maintaining hybrid rice yield is another way to minimize the cost of hybrid seeds. At the IRRI farm during the 1997 DS, we planted 28- d-old seedlings of IR64615H, IR68877H, IR68284H, and IR72 (check variety) at one seedling hill 1 using four different spacings: 10 30, 20 20, 15 30, and cm. Nitrogen (180 kg ha 1 ) was applied as four equal splits. Another experiment was conducted in the 1997 WS using three different spacings (20 20 cm, Peng et al

171 Grain yield (t ha 1 ) cm cm cm cm 1997 DS IR72 IR64615H IR68877H IR68284H cm cm cm 1997 WS IR72 IR68877H IR68284H Variety Fig. 6. Grain yield of hybrid rice and IR72 (conventional check variety) under different transplanting spacings (cm) at the IRRI farm, 1997 dry and wet seasons. Bars represent standard error of mean of four replications. cm, cm) as main plots, varieties (IR68877H, IR68284H, and IR72 as the check variety) as subplots, and seedling split (1 seedling hill 1 as a control and 1 tiller hill 1 as the seedling split treatment) as sub-subplots. In this experiment, we maximized the use of seedlings by splitting the tiller-bearing plants into single tillers and transplanting 1 tiller hill 1. The crop received a total of 75 kg N ha 1. The hybrid varieties produced significantly greater yield than IR72 across all treatments and seasons. The use of different transplanting spacings and seedling splitting treatments gave no significant differences in yield within a variety (Fig. 6 and Table 9). The Physiological bases of heterosis and crop management strategies for hybrid rice in the tropics 167

172 Table 9. Effect of seedling split on grain yield (t ha 1 ) of three varieties grown under three transplanting spacings. IRRI farm, 1997 wet season. Variety Treatment Transplanting spacing (cm cm) Mean IR72 Control a Split b IR68877H Control Split IR68284H Control Split a Transplanted with 1 seedling hill 1. b Transplanted with split tiller at 1 tiller hill 1. Mother tiller was also considered as 1 tiller. Table 10. Seed requirements of different crop establishment methods. Crop establishment method Seed requirement (kg ha 1 ) Direct wet seeding 100 Transplanting at cm and 5 seedlings hil 1 39 a Transplanting at cm and 1 seedling hill Transplanting at cm and 1 tiller hill b a Assuming 1,000-grain weight = 25 g (fresh weight) and 80% of seeds produce seedlings. b Assuming each seedling bears 1 tiller. results suggest that it is possible to widen plant spacing up to cm without decreasing yield, even at high yield levels. With this spacing, it is estimated that only 5.2 kg seed ha 1 is needed and this can be reduced even further by 50% (2.6 kg ha 1 ) if split tillers are used. This seed requirement is far lower than that of other types of crop establishment (Table 10). References Agata W Mechanism of high yield achievement in Chinese F 1 rice compared with cultivated rice varieties. Jpn. J. Crop Sci. 59(Extra issue 2): Akita S Physiological bases of heterosis in rice. In: Hybrid rice. Proceedings of the International Symposium on Hybrid Rice. Los Baños (Philippines): International Rice Research Institute. p Akita S, Blanco LC, Katayama K Physiological mechanism of heterosis in seedling growth of indica F 1 rice hybrids. Jpn. J. Crop Sci. 59(3): Blanco LC, Casal C, Akita S, Virmani SS Biomass, grain yield, and harvest index of F 1 rice hybrids and inbreds. Int. Rice Res. Newsl. 15:9-10. IRRI (International Rice Research Institute) Program report for Los Baños (Phil- 168 Peng et al

173 ippines): International Rice Research Institute. 311 p. IRRI (International Rice Research Institute) Program report for Los Baños (Philippines): International Rice Research Institute. 176 p. IRRI (International Rice Research Institute) Program report for Los Baños (Philippines): International Rice Research Institute. 175 p. IRRI (International Rice Research Institute) Program report for Los Baños (Philippines): International Rice Research Institute. 188 p. IRRI (International Rice Research Institute) Program report for Los Baños (Philippines): International Rice Research Institute. 175 p. Jones JW Hybrid vigor in rice. Agron. J. 18: Kabaki N Growth and yield of japonica-indica hybrid rice. JARQ 27: Maruyama S, Miyazato K, Nose A Studies on matter production of F 1 hybrid in rice. Jpn. J. Crop Sci. 51(Extra issue 2): Peng S, Cassman KG, Virmani SS, Sheehy J, Khush GS Yield potential trends of tropical rice since the release of IR8 and the challenge of increasing rice yield potential. Crop Sci. 39: Peng S, Yang J, Garcia FV, Laza RC, Visperas RM, Sanico AL, Chavez AQ, Virmani SS Physiology-based crop management for yield maximization of hybrid rice. In: Virmani SS, Siddiq EA, Muralidharan K, editors. Advances in hybrid rice technology. Proceedings of the Third International Symposium on Hybrid Rice, November 1996, Hyderabad, India. Los Baños (Philippines): International Rice Research Institute. p Ponnuthurai S, Virmani SS, Vergara BS Comparative studies on the growth and grain yield of some F 1 rice (Oryza sativa L.) hybrids. Philipp. J. Crop Sci. 9(3): Sen PK, Mitra GN Inheritance of photoperiodic reaction in rice. Nature 182: Song X, Agata W, Kawamitsu Y Studies on dry matter and grain production of F 1 hybrid rice in China. I. Characteristics of dry matter production. Jpn. J. Crop Sci. 59(1): Virmani SS Opportunities and challenges of developing and using hybrid rice technology in the tropics. In: Peng S, Hardy B, editors. Rice research for food security and poverty alleviation. Proceedings of the International Rice Research Conference, 31 March-3 April 2000, Los Baños, Philippines. Los Baños (Philippines): International Rice Research Institute. p Virmani SS, Khush GS, Pingali PL Hybrid rice for tropics: potential, research priorities and policy issues. In: Ilyas Ahmed M, Viraktamath BC, Ramesha MS, Vijaya Kumar CHM, editors. Hybrid rice technology. Hyderabad (India): Directorate of Rice Research. p Yamauchi M Physiological bases of higher yield potential in F 1 hybrids. In: Virmani SS, editor. Hybrid rice technology: new developments and future prospects. Los Baños (Philippines): International Rice Research Institute. p Yamauchi M, Yoshida S Heterosis in net photosynthetic rate, leaf area, tillering, and some physiological characters of 35 F 1 rice hybrids. J. Exp. Bot. 36(163): Ying J, Peng S, He QR, Yang H, Yang CD, Visperas RM, Cassman KG Comparison of high-yield rice in tropical and subtropical environments. I. Determinants of grain and dry matter yields. Field Crops Res. 57(1): Yoshida S Fundamentals of rice crop science. Los Baños (Philippines): International Rice Research Institute. 269 p. Physiological bases of heterosis and crop management strategies for hybrid rice in the tropics 169

174 Notes Authors addresses: S. Peng, R.C. Laza, A.L. Sanico, R.M. Visperas, International Rice Research Institute, Philippines; J. Yang, Yangzhou University, China; T.T. Son, National Institute for Soils and Fertilizers, Vietnam. Citation: Virmani SS, Mao CX, Hardy B, editors Hybrid rice for food security, poverty alleviation, and environmental protection. Proceedings of the 4th International Symposium on Hybrid Rice, Hanoi, Vietnam, May Los Baños (Philippines): International Rice Research Institute. 407 p. 170 Peng et al

175 Technological dissemination strategies

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177 Public, private, and NGO-sector partnership for developing and promoting hybrid rice technology M. Ilyas-Ahmed, Ish Kumar, B.C. Viraktamath, J.S. Sindhu, and Y. Yogeswara Rao There is no greater incentive for public, private, and NGO-sector partnership in agricultural research and development (R&D) than to together meet the enormous challenge posed by global food security, which will require that our limited global resources be used effectively to develop sustainable systems that also conserve natural resources. Comparative advantages of each sector are to be identified and harnessed for achieving the common goal of global food security. In agricultural systems around the world, the roles of the public and private sector and the relationship between them is changing. Recently, there has been a growing awareness, in both sectors, of significant benefits that can be derived from partnership. Investment by the public sector in agricultural R&D has been declining, whereas investment by the private sector has been increasing in both developed and developing countries. Concern is growing that the current level of investment, by both the public and private sector pooled together, may also be inadequate to ensure food security in the coming decade, thus highlighting the urgent need for partnership. In this article, several ways to establish effective partnership are outlined. The current status of hybrid rice in tropical Asian countries has been considered and the strength and level of involvement of the public, private, and NGO sectors have been described. The comparative advantages of these sectors in the development and promotion of hybrid rice are enumerated. As a case study, the current status of partnership among these sectors in India is given. Finally, a model for establishing effective partnership for various activities among different sectors for developing and promoting hybrid rice in tropical Asian countries is proposed. The public sector, which has a comparative advantage in technology generation, can complement the expertise of the private sector in large-scale seed production and distribution and both sectors, along with NGOs, can jointly undertake technology transfer to develop and promote hybrid rice. IRRI can play an effective role in developing and implementing a detailed action plan for partnerships for the member countries under the IRRI/ADB project on hybrid rice. 173

178 The world s current population is 6 billion, which is expected to double by 2050 (James 1996). Ninety-seven percent of this population increase will occur in the developing countries of Asia, Africa, and Latin America (Swaminathan 1995). Coincidentally, these are the regions where rice is the staple food. Even today, 30% of the population in the developing world lives in abject poverty, barely surviving on US$1 or less per day for food, shelter, and other essential needs. The challenge for the future is global food security, which will require at least doubling and preferably tripling food production by This increased production has to come from the existing cultivated area or less of the agricultural land, with less water and with other deteriorating and declining resources. The enormity of the challenge of food security is best illustrated by the fact that, during the next 50 years, the global population will consume twice as much food as has been consumed since agriculture began 10,000 years ago (James 1996). To meet this global challenge of food security effectively, it is imperative that all possible resources be used judiciously and efficiently. The need for partnership Partnership can be defined as two or more organizations with complementary areas of expertise committing resources and working together to achieve a mutually beneficial outcome that would have been difficult for each one of them to achieve alone. There is no greater incentive for public, private, and NGO-sector partnership in agricultural research than the enormous challenge posed by global food security, which will require that limited global resources be used in the most effective way to develop sustainable systems that also conserve natural resources. To meet this challenge requires new partnerships in agricultural research and development between the public and private sector that optimize the comparative advantages of each in pursuit of mutual objectives. Forging these new public-private partnerships would permit the most effective use of the limited global resources for the development of sustainable agricultural systems. In the past, policymakers in developing countries did not recognize the private sector as an important resource for carrying out national programs. Recently, there has been a marked and progressive change in which the private sector is now generally acknowledged to be a key player in development. This view is endorsed by the international development and finance community, which recognizes the private sector as an increasingly important national and international resource (James and Persley 1990). The government should not view for-profit private-sector activities as detrimental to the public good because these private-sector activities are often the most effective way to achieve national goals set by governments. There is, and will continue to be, a critical and essential role for governments in developing countries to examine policy issues in agriculture and to implement technical programs that optimize social welfare for the public good. In agricultural systems around the world, the roles of the public and private sector, and the relationship between them, are chang- 174 Ilyas-Ahmed et al

179 ing. This is due to the expanding research and development capability of the private sector, the reevaluation of the role of the state in providing research services, the introduction of intellectual property regimes, and a more liberal trade and economic environment. In this new setting, it is important to examine the patterns of interaction between these two sectors, focusing on adjustments that need to be made to achieve the goals efficiently. The private sector is an efficient mechanism to deliver seed-based technologies to clients. There are many opportunities in this partnership to enhance the impact of agricultural research for increasing production and productivity. There are opportunities to share skills, knowledge, and costs. There is a complementarity of the agenda. During the 1990s, there has been a growing awareness, in both the public and private sector, of the significant benefits that can be derived from partnership and collaboration. A major challenge for both sectors is to find ways to collaborate in sharing and transferring appropriate new and superior technologies. Private-sector investments in agricultural research and development (R&D) are conservatively estimated to be about US$11.0 billion in developed countries and $2.0 billion in developing countries vis-à-vis $8.5 and $8.8 billion in developed and developing countries, respectively, by the public sector. Thus, both sectors are together investing about $30 billion in agricultural R&D. But this $30 billion investment is inadequate to meet current global agricultural R&D needs. Considerable efficiencies will accrue, however, if the same $30 billion is invested in a coordinated manner by the public and private sector as partners to face the major challenge of global food security. Emerging issues in partnership Partnership is about creating more value together than can be created alone. Partnerships need to be developed carefully. New relationships cannot emerge overnight. They need to be founded and nurtured on trust and transparency. There is a need to make changes to accommodate the preferences and working practices of new partners. Issues of intellectual property rights, confidentiality, and public interest have to be considered, discussed, and negotiated (Dar 2001). Both sectors are entering into these new sets of relationships with caution, but also with hope. The public sector is having to reevaluate the boundaries of its publicgood mandate. It is asking questions such as, Does public-good research have to be publicly funded and publicly executed? Who owns research products jointly developed by the public and private sector? How can public ownership be retained? The private sector also has many valid concerns, such as, Can it recoup its investment in research? Will other companies also have access to the products jointly developed with the public sector? The two sectors also have different professional and administrative traditions. Can the public sector adapt to the urgency of the private sector? These issues need to be considered in depth and a mutually agreeable model for partnership needs to be developed. Public, private, and NGO-sector partnership for developing and promoting hybrid rice technology 175

180 The private sector s primary interest concerns profit and therefore its approach is based on developing and producing good-quality products that its clients want. The public sector, on the other hand, has a more diffuse, although no less important, purpose. This very clear articulation of the tangible purpose for the private sector has two consequences. First, administrative systems, time frames, and professional incentives are set up around the profit motive. Second, successful companies structure themselves, and their relationships and alliances, around ensuring that they make a profit. In contrast to the private sector, public-sector research institutions have a less tangible purpose public goods. As a result, performance of the system is harder to monitor in output terms and, as a consequence, an administrative system has evolved in the public sector that focuses on input monitoring. The procedures of checks and counterchecks that these administrative norms put in place are usually referred to as bureaucracy. Such procedural norms cause time delays, which are ill-suited to the commercial urgency of the private sector (Hall et al 2001). The issue in establishing an effective partnership is how to reconcile these different approaches in outlook and implementation. Some of the major issues to be resolved in public- and private-sector partnership are, Should taxpayers money be used for R&D that will ultimately benefit a company and its shareholders? Multinational companies signing deals with publicly funded research organizations will surely dictate the direction of research possibly to the detriment of small-scale farmers unable to purchase a commercial company s products. On the other hand, it can be argued with equal justification that, if taxpayers have contributed large amounts of money over the years to agricultural research, what is wrong with using commercial companies to transfer the technology to farmers? What are the practical difficulties of combining slower, more bureaucratic procedures of a publicly funded government body that has a very different agenda and political constraint with a commercial company s interests and focused and dynamic approach? What safeguards can be put in place to ensure that all partners, including farmers, get a fair deal? Investment trends in agricultural R&D by the public and private sector Public-sector investment A study conducted by the International Food Policy Research Institute (Brown and Haddad 1994) revealed that the proportion of Official Development Assistance (ODA) for agricultural R&D declined from 20% in 1980 to 14% in This study also showed that real external assistance to agriculture for developing countries declined from $12 billion in 1980 to $10 billion in In the past, ODA and official investment assistance have been important for obtaining additional financial support from national programs for agricultural research. There is evidence now that this external support is declining and at the same time developing countries are providing less support to agricultural R&D. External support to national agricultural research and 176 Ilyas-Ahmed et al

181 Table 1. Investments in agricultural R&D (expressed as a percentage of national agricultural GDP). Region or Number of Most recent country countries year Developing regions Sub-Saharan Africa a (excluding South Africa) South Africa a Asia and the Pacific (excluding China) China b Latin America and the Caribbean West Asia and North Africa Developed countries United States c Australia d a 1991 estimate. b c d extension systems (NARES) is reported to be 35% for sub-saharan Africa, 26% for Asia and the Pacific, and 7% for Latin America and the Caribbean. This declining support of public-sector funds from ODA to aid agricultural research in developing countries further highlights the importance of increased participation by the private sector in partnership with the public sector. Private-sector investments from the North for all sectors in developing countries of the South are estimated to be more than $170 billion annually, almost three times the public-sector ODA funding for all sectors, which is estimated to be around $60 billion (Serageldin and Sfir-Younis 1996). Table 1 shows the public investment in agricultural R&D for four periods. The data reveal that developing-country investments averaged approximately 0.5% of national GDP, whereas the corresponding figures for developed countries averaged around 2.0%. After the 1980s, the proportion of investment by developing countries either leveled off or was declining in some developing countries. Concern is growing that the current level of investment will not be adequate for delivering the technology contribution necessary to increase food productivity enough to ensure food security in the future. In such a scenario, one option that must be explored is a better use of currently available global resources, including the integration of public- and private-sector research resources, to achieve mutual objectives more effectively and efficiently, nationally and internationally. Private-sector investment There are no comprehensive and uniformly generated global estimates of privatesector investment in agricultural R&D in developed and developing countries. How- Public, private, and NGO-sector partnership for developing and promoting hybrid rice technology 177

182 ever, in general, investment by the private sector in agricultural R&D has increased over the last 3 4 decades. In the United States during the 1960s, private-sector investment was 5% less than that of the public sector, whereas, during the early 1990s, it was $3.3 billion, 27% higher than that of the public sector. Table 2 shows the trend in private-sector investment in agricultural R&D in the United States. Private-sector agricultural R&D spending has increased almost 20 times from 1960 to The trend has been more or less similar in other developed countries, where the private sector invests almost 50% of the total expenditure on agricultural R&D. Investment by the private sector in developing countries is much smaller and is concentrated in a few more advanced developing countries such as Argentina, Brazil, India, and Mexico (Pray and Echeverria 1991). The private sector has started investing in a few other developing countries recently. In many developing countries, private-sector investment increased faster than public-sector investment during the 1970s, 80s, and 90s because of the introduction of policies in these countries for increased private-sector participation in agricultural R&D. This trend is likely to continue. Given the nature of the market place and the competition among private-sector corporations, comprehensive data on agricultural R&D are not readily available in the public domain. Though the private sector invests in several industries such as fertilizer, plant protection chemicals, seed and animal health products, machinery and equipment, and biotechnology, etc., for the purpose of this article, it is more relevant to consider the private investment in the seed industry. The value of the global seed trade is estimated to be $45 billion annually, equally shared by three different segments: commercial seed, which is dominated by the private sector; farm-saved seed; and seed from the public sector. The latter is particularly prevalent in developing countries and centrally planned economies. The consumption of agricultural seed, which includes farm-saved seed, is approximately 120 million tons per year. The global consumption of agricultural seed by continent and by crop is shown in Tables 3 and 4, respectively. Asia and the Commonwealth of Independent States are the largest consumers of seed, almost two-thirds of the global market. Consumption has been stagnant during the last decade, except in Asia, where it has increased by 18% since Onethird of the seed used in Asia is rice. Cereals dominate the world seed market, accounting for approximately twothirds of the 120-million-ton market. The private sector dominates the $15 billion annual global commercial seed market. There are approximately 1,500 seed companies worldwide, with 600 based in the United States and 400 in Europe. Fifteen major multinational seed companies account for one-third of the turnover of the $15 billion global commercial seed market. In developing countries, where it is estimated that 80% of the seed is currently supplied by the public sector and farmer-saved seed, private-sector activity in the seed industry is expected to become increasingly strong. Private-sector growth is 178 Ilyas-Ahmed et al

183 Table 2. Trends in private-sector spending on agricultural R&D input-oriented, postharvest, and food processing, 1960 to 1992 (millions of current dollars). Year Input-oriented Postharvest and Total food processing Chemicals Agricultural Veterinary/ Plant Current Real machinery pharmaceuticals breeding , , , , , , ,648.0 Source: Adapted from United States Department of Agriculture (1995). Public, private, and NGO-sector partnership for developing and promoting hybrid rice technology 179

184 Table 3. Total world consumption of agricultural seed, by continent (million tons, including farm-saved seed). Region Commonwealth of Independent States South America Europe North and Central America Asia Africa Oceania Total (world) Source: FAO (Rabobank 1994). Table 4. Total world consumption of agricultural seed, by crop (million tons). Crop Wheat Barley Rice Maize Other grains Root and tuber crops Pulses Oilseeds Total Source: FAO likely to be important in Asia, as well as in Latin America and selected countries in Africa. Current status of hybrid rice in tropical Asia Before considering the various aspects of partnership for developing and promoting hybrid rice, the status of hybrid rice and involvement of the public, private, and NGO sector in various tropical countries in Asia (India, Vietnam, Philippines, Bangladesh, Indonesia, and Sri Lanka) where this technology is now being developed and promoted are considered briefly. Following the remarkable success of hybrid rice in China, the International Rice Research Institute (IRRI) in collaboration with NARES has been making efforts to develop and introduce this innovative technology in some tropical Asian countries for the last one and a half decades. As a result of these persistent efforts, the technology has been developed and introduced in some of these countries. The number of hybrids released and the area covered are given in Tables 5 and Ilyas-Ahmed et al

185 Table 5. Number of hybrids released in tropical Asian countries. Country Public Private Total sector sector India Vietnam Philippines Bangladesh Indonesia Total Table 6. Area under hybrid rice. Country Area (ha) India 200,000 Vietnam 480,000 Philippines 10,000 Bangladesh 30,000 Indonesia 5,000 Total 725,000 Though the number of hybrids developed by the public sector is greater than that of the private sector, a much larger area is covered under a few privately bred hybrids because of the increased efficiency of the private sector in seed production and marketing. The area currently cultivated with hybrids in some of these countries appears in Table 6. In addition to these countries, hybrids have been developed and introduced for commercial cultivation on a small scale recently in the United States, Egypt, and Brazil. In more than 10 other countries, serious R&D efforts for the development of this technology are under way. The involvement of the public, private, and NGO sector in tropical Asian countries is given in Table 7. In most of the countries considered above, the public sector is strong or moderately strong in technology generation and technology transfer, but is weak in largescale seed production. The private sector is strong in seed production in India and has potential to become strong in other countries. The NGO sector is involved in Bangladesh in all three components technology generation, large-scale seed production, and technology transfer. In other countries, the NGO sector needs to be strengthened and involved more actively in technology transfer activities. Comparative advantages of different sectors Each sector (public, private, and NGOs/cooperatives) has certain comparative advantages/strengths of its own, which can be optimally harnessed for the common Public, private, and NGO-sector partnership for developing and promoting hybrid rice technology 181

186 Table 7. Involvement and strength of various sectors in hybrid rice technology. Country Public Private NGOs/ Remarks sector sector cooperatives India Strong Strong Moderately strong Public sector is strong in technology generation. Private sector is strong in large-scale seed production and marketing. Transfer of technology efforts to be intensified. Vietnam Strong Weak Weak Public sector is strong in technology generation and transfer. Public and private sectors are weak in large-scale seed production. Philippines Strong Weak Moderately strong Public sector is strong in technology generation and transfer. Public and private sectors are weak in large-scale seed production. Bangladesh Moderately strong Moderately strong Strong One NGO (BRAC) is involved in all three components technology generation, largescale seed production, and technology transfer. It is getting good support from public sector. Indonesia Moderately strong Moderately strong Weak Partnership between public and private sector needs to be strengthened. NGOs to be involved actively. Sri Lanka Moderately strong Weak Weak All three sectors need to be strengthened and involved more actively in partnership. 182 Ilyas-Ahmed et al

187 goal of the expeditious development and large-scale adoption of hybrid rice in a partnership mode. The comparative advantages of each of these sectors are briefly mentioned below. Public sector Accessibility to a large collection of germplasm Vast and well-trained human resources Well-developed infrastructure Easy accessibility to government/policymakers Effective linkages with national/international public-sector organizations/ institutions Hence, the public sector has a comparative advantage in technology generation. Private sector Result-oriented and focused approach Strong and efficient marketing and distribution network Closer and effective linkages and interaction among research, production, marketing, and extension personnel Fewer or no bureaucratic interferences/delays The private sector has a comparative advantage in large-scale seed production and distribution. It is also effective, on a small scale, in technology generation and transfer. NGOs/cooperatives Closer and intimate contact with farmers at grass-roots level Self-motivated and more dedicated personnel Similar to private sector minus the profit motive. Social good and serving the farming community are the declared motives. Fewer or no bureaucratic interferences/delays. The NGOs/cooperative sector has a comparative advantage in technology transfer. In the few cases where NGOs are very well developed with a vast infrastructure and human resources network, such as in the case of BRAC in Bangladesh, this sector can also undertake, on a limited scale, technology generation and large-scale seed production. Now, the specific task is to critically consider the stage of development and level of involvement of these sectors on a country-to-country basis and work out the modalities for an effective model of partnership for the development and promotion of hybrid rice in the individual countries. Current status of partnership in India As a case study, the current status of partnership in India, where all three sectors are involved, is briefly given below, before describing a possible model for partnership Public, private, and NGO-sector partnership for developing and promoting hybrid rice technology 183

188 among various sectors for developing and promoting hybrid rice in tropical Asian countries. A goal-oriented applied program on the development and use of hybrid rice in India was begun by the Indian Council of Agricultural Research (ICAR) in December IRRI, as a collaborative partner, supplied the needed germplasm and technical support. The project was further strengthened in 1991 through financial support from the UNDP providing for consultancies, fellowship training abroad, and needed equipment and technical support from the FAO. A private R&D foundation, the Mahyco Research Foundation (MRF), and IRRI/ADB project on hybrid rice have extended limited financial support since 1996 and 1998, respectively. The first set of hybrids was released five years after the program began, in A company in the private sector began R&D on hybrid rice in By 1992, another 4 5 companies started working on hybrid rice. Now, 15 private-sector companies are active in hybrid rice R&D and large-scale seed production, but five companies are involved in a big way. Among NGOs/cooperatives, a few Farm Science Centers (Krishi Vigyan Kendras) and cooperatives were involved initially for 2 3 years in large-scale seed production and training of farmers. But now they are not very active. Seventeen hybrids, 13 from the public sector and four from the private sector, have been released so far. In addition, the private sector is marketing 5 6 unreleased but truthfully labeled hybrids. Hybrid rice is estimated to be cultivated annually on 200,000 hectares. More than 95% of the 4,000 tons of hybrid seed produced annually is by the private sector. Private hybrids grown on a large scale are A-6201 and PHB-71. Out of the 13 publicly bred released hybrids, seed of only five hybrids (KRH-2, Sahyadri, NDRH-2, DRRH-1, and CORH-2) is being produced on a very limited scale by the National Seed Corporation, state seed corporations, and state agricultural universities. Though some of the publicly bred hybrids are as good as the private ones, in yield potential and grain quality, they are not cultivated on a large scale because public seed corporations are unable to take up large-scale seed production efficiently. There has been a kind of partnership on a limited scale among public, private, and NGO sectors in India for the development and promotion of hybrid rice. The private sector in India has been provided with all the needed germplasm and training in seed production by the public sector. Private hybrids are evaluated in national trials free of cost. After thorough evaluation in the national network, four privately bred hybrids have been released through the Central Variety Release Committee. Private hybrids are promoted in the national compact block frontline demonstrations conducted across the country. National screening facilities for resistance to pests and diseases and for grain quality characteristics are extended to the private sector. It is also involved actively in all planning and review meetings. A private R&D foundation, the Mahyco Research Foundation (MRF), has partially supported the national hybrid rice program through a grant of $750,000 from 1996 to In addition, it is helping the national program by multiplying and supplying CMS lines and restorer lines to all the interested public and private institutions and conducting strategic research on molecular tagging of restorer gene(s), 184 Ilyas-Ahmed et al

189 deploying the eui gene in CMS lines, and doing molecular testing of the genetic purity of parental lines. NGOs such as farm science centers and a few cooperatives in West Bengal State are helping in technology transfer activities such as training of farmers and farm women and in conducting compact block frontline demonstrations. One farm science center at Gaddipally in Andhra Pradesh State had taken up hybrid rice seed production on 100 ha annually for 2 years. But this activity had to be discontinued because of the problems encountered in genetic purity of the seed produced. Involvement of NGOs has been on a much smaller scale and in a very limited way. As is evident from all of this, though there has been some kind of unplanned partnership among the various sectors in India, there is a need to critically consider the strength of each sector and specifically plan for an effective partnership model for the development and promotion of hybrid rice in the country. Proposed model for partnership The concept of public-private partnerships is often touted, but in reality there are few examples of true partnerships. In view of the need for partnerships and the advantages to be derived by such an arrangement, as described earlier in this article, there is a very strong case for working out in detail modalities for developing partnerships among the public, private, and NGO sectors for the development and promotion of hybrid rice in tropical Asian countries. The stage of development, and level of involvement, of each of these sectors is to be critically considered on a country-tocountry basis, before preparing an action plan for developing partnerships. Prospective activities for partnership and possible ways of collaboration among the different sectors are briefly described below. Partnership for developing hybrid rice technology The public and private sector can collaborate and establish a partnership for developing hybrid rice technology in the following possible ways. The public sector can share the hybrids it developed with interested companies in the private sector and NGOs on a payment and nonexclusive basis One of the constraints to the popularization of publicly bred hybrids has been the lack of a proper seed production agency to take up large-scale seed production. Public-sector seed corporations have unfortunately failed in this task. Even in other crops, wherever hybrid technology has been successful, the bulk of the hybrid seed (70 80%) is produced by the private sector. This will be no different in hybrid rice. So, the breeders seed/foundation seed of parental lines can be produced by the public institution that has developed the hybrid, which can sell it to interested partners in the private sector on a nonexclusive basis. The reason for advocating a nonexclusive basis is that, since the technology has been developed by the public sector using taxpayers money, it should be made available to all interested partners in the private sector. Second, such an approach helps in the wider dissemination of the technology Public, private, and NGO-sector partnership for developing and promoting hybrid rice technology 185

190 because of the efforts of many companies in different regions of a country and avoids the possible disadvantages associated with a monopoly by a single large multinational company. Initially, parental lines are sold for a fixed amount to all interested companies. Already, such a practice is being followed in India for selling the virusresistant tomato varieties developed by a public-sector institution. Second, about 70 80% of the private-sector seed companies do not have their own R&D set-up. All such companies are willing to purchase the parental lines of hybrids released from the public sector. In some cases, even the companies having their own R&D set-up may also purchase the parental lines, produce the hybrid seed on a large scale, and market it either in the same name as given by the public sector or under their own brand name. In the latter case, there should be a stipulation to always mention the name given by the public sector and the name of the public institution on the seed packet. Regarding sharing the germplasm and elite breeding material developed by the public sector, plant breeders are reluctant to share with the private sector till the final product is developed and registered. Hence, it may not be possible at this stage to persuade public-sector plant breeders to willingly share their germplasm and elite breeding material from the F 2 generation onward with the private sector, in the larger interest of developing and promoting the technology for the country. Contractual research in the public sector Since the public sector has a vast, well-established infrastructure with a large number of well-trained human resources, the private sector can avail of that by having contractual research with the public sector on a payment basis. The private sector can have a contract with the public sector for developing parental lines with specific characteristics such as a particular duration, grain type, resistance to diseases/pests, etc., for a period of 3 5 years, instead of duplicating such efforts in their own R&D set-up. Companies without an R&D set-up can benefit more by such an arrangement and partnership between the public and private sector. Evaluation of privately bred hybrids in a national network Another area of partnership between the public and private sector is to have the privately bred hybrids jointly evaluated with public hybrids in the vast national network on a payment basis. Currently in India, privately bred hybrids are jointly evaluated along with public hybrids in the national network on a no-cost basis. Sharing screening facilities Excellent and well-developed public-sector facilities for screening for resistance to major pests and diseases and also for grain quality characteristics can be shared by the private sector on a payment basis. Though some efforts have begun in this direction, they need to be systematized and widely publicized, so that all interested companies in the private sector can avail of these facilities. 186 Ilyas-Ahmed et al

191 Short consultancy visits The private sector has indicated that short consultancy visits by public-sector breeders to its research and seed production farms for 2 3 days duration would be of immense help. The public sector should permit its personnel to undertake such short consultancy visits. The private sector should bear all the expenses involved, along with a nominal fee as institutional charges. Participation of the private sector in field days Another activity for establishing partnership between the public and private sector is to invite private-sector personnel to specially organized field days, at which they can critically observe hybrids being evaluated in national/regional trials and CMS line and restorer line trials. Involvement of the private sector in planning and review meetings Private-sector personnel can be invited and actively involved in the planning as well as review meetings of the national hybrid rice program. Private-sector investment in public-sector breeding research A consortium of private-sector companies can jointly invest in public breeding research, which will be useful to all of them. Alarmed by the declining investment by developed countries in agricultural research in developing countries, a coalition of small and large seed companies in India is helping to fill the breach. Fourteen companies have pledged $109,000 annually to help support applied plant breeding research at ICRISAT for five years. All the material developed through this research will remain as international public goods, freely available to all. Targeted investments by the private sector have a fast impact on poverty reduction and increased productivity. Investment by private-sector research foundations in public-sector research Private-sector research foundations can invest in public-sector breeding research and partially support the ongoing research programs, the products of which are of interest to the private sector. The Mahyco Research Foundation (MRF), a private research foundation, has partially supported the ongoing national hybrid rice program by investing $750,000 from 1996 to Partnership for promoting hybrid rice technology To promote hybrid rice technology, the public sector with its vast and extensive extension network can play a major role. The private sector also does technology transfer on a much smaller scale, but in a very focused and effective way. The one possibility for partnership in technology transfer efforts is to conduct the frontline demonstrations jointly by pooling funds and human resources. Another possibility is that the private sector can provide the funds for that purpose to the public sector on a contractual basis. Publicity through television, video, and print media can be taken Public, private, and NGO-sector partnership for developing and promoting hybrid rice technology 187

192 up jointly and brochures and booklets on the package of practices to be adopted can be published in local languages by pooling funds from the public and private sector. Both sectors are now undertaking technology transfer separately, without the involvement of the other sector, and this is leading to a duplication of efforts and waste of financial resources. A proper model needs to be developed and implemented to avoid duplication of efforts and to use limited resources in technology transfer effectively. The public sector has a comparative advantage in technology generation, but its performance has been rather poor in large-scale seed production. The private sector has demonstrated its efficiency in large-scale seed production and distribution. By establishing partnerships, the public sector can avail of the expertise and efficiency of the private sector in large-scale seed production and distribution to take the technology developed to the ultimate clientele: the farming community. All three sectors public, private, and NGOs can jointly be involved in technology transfer. The International Rice Research Institute, under the second phase of the IRRI/ ADB project on hybrid rice, should take the lead in conducting interactive meetings of all the stakeholders in the member countries to work out the details for establishing effective partnership among the public, private, and NGO sectors on a countryto-country basis. An action plan is required to be prepared and implemented to derive the benefits of partnership among these sectors for the development and promotion of hybrid rice. References Brown L, Haddad L Agricultural growth as a key to poverty alleviation. Brief 7, 2020 vision of the International Food Policy Research Institute (IFPRI), Washington, D.C., USA. Dar WD Perspectives on public-private sector interactions: the way for the future. In: Hall AJ, Yoganand B, Rasheed Sulaiman V, Clark NG, editors. Sharing perspectives on public-private sector interactions. Proceedings of a workshop held 10 April 2001, ICRISAT, Patancheru, India. National Centre for Agricultural Economics and Policy Research (NCAP) and International Crops Research Institute for the Semi-Arid Tropics (ICRISAT). p 5-6. Hall AJ, Rasheed Sulaiman V, Clark NG, Yoganand B Shared perspectives: a synthesis of obstacles and opportunities. In: Hall AJ, Yoganand B, Rasheed Sulaiman V, Clark NG, editors. Sharing perspectives on public-private sector interactions. Proceedings of a workshop held 10 April 2001, ICRISAT, Patancheru, India. National Centre for Agricultural Economics and Policy Research (NCAP) and International Crops Research Institute for the Semi-Arid Tropics (ICRISAT). p James C Agricultural research and development: the need for public-private partnerships. Issues in Agriculture No. 9. Consultative Group on International Agricultural Research, Washington, D.C. James C, Persley GJ Role of private sector. In: Persley GJ, editor. Agricultural biotechnology: opportunities for international development. Wallingford (UK): CAB International. p Ilyas-Ahmed et al

193 Pray CE, Echeverria RG Private-sector agricultural research in less developed countries. In: Pardey PG, Roseblooms J, Anderson JR, editors. Agricultural research policy: international quantitative perspectives. Cambridge (UK): Cambridge University Press. p Serageldin I, Sfir-Younis Effective financing of the environmentally sustainable development. Proceedings of the third annual World Bank Conference on Environmentally Sustainable Development. Washington, D.C. (USA): World Bank. p 11. Swaminathan MS Population, environment and food security. Issues in Agriculture No. 7. Washington, D.C. (USA): Consultative Group on International Agricultural Research. Notes Authors addresses: M. Ilyas-Ahmed and B.C. Viraktamath, Directorate of Rice Research, Rajendranagar, Hyderabad, India; Ish Kumar, Hybrid Rice International Ltd., Tolichowki, Hyderabad, India, now at International Rice Research Institute, Los Baños, Philippines; J.S. Sindhu, Asia Pacific Seed Association, Kasetsart, Bangkok, Thailand; Y. Yogeswara Rao, Hyderabad, India. Citation: Virmani SS, Mao CX, Hardy B, editors Hybrid rice for food security, poverty alleviation, and environmental protection. Proceedings of the 4th International Symposium on Hybrid Rice, Hanoi, Vietnam, May Los Baños (Philippines): International Rice Research Institute. 407 p. Public, private, and NGO-sector partnership for developing and promoting hybrid rice technology 189

194 Table 4. Yield gain of hybrids released for on-farm trials and/or cultivation. Countries Hybrids Inbreds Yield gain Yield (t ha 1 ) (t ha 1 ) (t ha 1 ) gain (%) Bangladesh Sonar Bangla Alok India Philippines Dry season Wet season Vietnam Sri Lanka South Korea Indonesia No field trials conducted Myanmar No field trials conducted Thailand No field trials conducted Sources: Various country progress reports, Husain et al (2001), Directorate of Rice Research, India (2000). Despite the great potential of hybrid rice technology to increase rice production and help reduce food insecurity in Asia, several technical and nontechnical constraints hinder the production, development, and wide-scale adoption of hybrid rice technology. Economic profitability of hybrid rice production among farmers Increased yield and thus production do not automatically translate into higher net profits. Studies were recently conducted in India and Bangladesh to evaluate the initial economic returns of the technology and the results are mixed. Table 5 shows details of costs and returns for different rice varieties (hybrids and high-yielding varieties, HYVs) in Bangladesh (Husain et al 2001) and Table 6 for India (Janaiah and Hossain 2002). In Bangladesh, hybrid varieties Alok and Sonar Bangla are compared with HYVs for the highest grain value, costs, and returns. The highest grain value was obtained for Sonar Bangla. Gross returns were also the highest for Sonar Bangla and the lowest for HYVs. Net returns, however, were the highest for Sonar Bangla and the lowest for Alok. Combined costs and returns for both hybrids show that it has been relatively more profitable to cultivate hybrids than HYVs, but the difference is not statistically significant. In India, the hybrids outyielded the inbreds, but with lower net returns. The results of these early studies identify three factors that hinder the faster and wider adoption of hybrid rice among farmers. First is the inadequate yield advantage of some of the hybrids over the inbreds. Second is the output price, for which 196 Sombilla et al

195 International Task Force on Hybrid Rice S.S. Virmani, Dat Van Tran, and J.S. Sindhu The establishment of an International Task Force on Hybrid Rice (INTAFOHR) was first proposed during the 2nd International Symposium on Hybrid Rice held at IRRI in 1992 to promote the technology outside China. Subsequently, this was endorsed by several countries in various international fora organized by FAO and IRRI. Subsequently, goals, objectives, participation, strategies, and implementation arrangements for INTAFOHR were proposed. A hybrid rice network project was launched in 1998 in the form of an IRRI-ADB project titled Development and Use of Hybrid Rice in Asia. This project aimed to expedite the development and use of hybrid rice technology in tropical Asia. Three international agencies (IRRI, FAO, and APSA) and six member countries (Bangladesh, India, Indonesia, the Philippines, Sri Lanka, and Vietnam), possessing high proportions of irrigated area and a high labor/land ratio, collaborated in the project. In 2000, China also joined as a collaborating partner in this project. This paper describes the roles of the coordinating partners, major achievements, and future directions and strategies of the project. Hybrid rice technology aims to increase rice yield potential beyond the level of semidwarf high-yielding rice varieties by exploiting the phenomenon of heterosis or hybrid vigor. China has successfully developed and used this technology for more than 25 years, which has contributed significantly to its food security, environmental protection, and rural employment generation through the hybrid seed industry. Outside China, IRRI has been actively involved since 1979 in developing this technology, and sharing it freely with national programs that are interested in using it to their advantage. Encouraged by the experience of China and IRRI, about 20 countries outside China have shown interest in developing and using this technology. This involves close coordination of three components technology generation, seed production, and technology transfer, including policy support at the national and international level. To expedite this process outside China, establishment of the International Task Force on Hybrid Rice (INTAFOHR) was first proposed in 1992 during the Second International Symposium on Hybrid Rice held at IRRI. Subsequently, 209

196 this idea was endorsed by various countries in several international fora and INTAFOHR was finally formed in 1995 with an agreement between IRRI and FAO. During the Third International Symposium on Hybrid Rice, a panel discussion was held involving representatives of 16 countries to specify goals, objectives, membership, strategies, and implementation arrangements for INTAFOHR. Following these guidelines, in 1997, a project proposal was submitted by IRRI to the Asian Development Bank (ADB) for funding. This proposal was approved and an International Network on Hybrid Rice was launched in 1998 in the form of an IRRI-ADB project titled Development and Use of Hybrid Rice in Asia. This was a component of the INTAFOHR with a focus on Asia. We describe this network and highlight its achievements and future prospects in the following sections. Membership and objectives In 1998, when the network was launched as an IRRI-ADB project, six countries (Bangladesh, India, Indonesia, the Philippines, Sri Lanka, and Vietnam), keen to develop and use hybrid rice technology in collaboration with IRRI, FAO, and APSA, joined together. China joined the network as a fourth collaborator in Three new countries (Myanmar, Thailand, and the Republic of Korea) also joined the network in 2002, after the successful completion of the first phase of the project. The objectives of the first phase of the network were to (1) strengthen the research capabilities of member countries to develop hybrid rice technology and (2) strengthen the hybrid rice seed production capability of the public, private, and NGObased seed industry in the member countries. Roles of collaborating partners IRRI was given the responsibility of coordinating the project and providing technical backstopping to the member countries through strategic and applied research for technology generation. It was also responsible for free germplasm sharing, training, and consultancy services to public and private research institutions actively involved in producing the technology. FAO took on the responsibility of strengthening collaboration between research and extension work on hybrid rice, sensitizing policymakers in the member countries to support the development and use of the technology, and strengthening hybrid seed industries in public, private, and NGO sectors. Beyond this project, FAO has also been providing its own financial support through technical cooperation projects to some of the member countries to improve their capacity in hybrid rice research, seed production, and technology transfer. This assistance has also been used by the member countries to strengthen their physical research infrastructure on hybrid rice. The member countries also used FAO s assistance for formulating their hybrid rice research and development programs. APSA provided a regular annual forum for the seed industry in Asia to identify and discuss issues influencing the commercialization of hybrid rice technology and 210 Virmani et al

197 to provide feedback on researchable issues to IRRI and national programs. It has also been helping to identify policy intervention areas for the seed industry to promote the technology and assisting in strengthening collaboration among public and private seed industries. China s collaborative role in the project involves human resource development, consultancy services, sensitizing policymakers, and information exchange. Major achievements 1. Establishment of effective regional collaboration Through this network, we have been able to establish effective regional collaboration on hybrid rice in Asia. To allow closer interaction with other networks concerned with nutrient, pest, weed, and water management, this network functions as a workgroup under IRRI s Irrigated Rice Research Consortium. This arrangement is extremely helpful for developing and using the technology. 2. Coordinated international hybrid rice trials Since 1999, we have successfully organized coordinated international hybrid rice trials in member countries. These trials include elite public rice hybrids for evaluation and use in the member countries. Results of the 1999 and 2000 trials (Table 1) indicated that hybrids frequently performed better than the inbred check HYVs in high-yielding environments in which check HYVs yielded higher than 6.5 t ha 1. In low-yielding environments (yields of t ha 1 ), more frequently the yield superiority of hybrids was not manifested. In medium-yielding environments (yield of t ha 1 ), the yield superiority of hybrids over inbreds was inconsistent. Through these trials, member countries also selected the rice hybrids best suited to them for further testing and evaluation. Parental lines for such hybrids were also made available by IRRI to the national agricultural research and extension systems (NARES). 3. Strengthening NARES capacity One achievement of the project with immediate and long-term value was the improvement of human resources and facilities in member countries. More than 150 researchers were trained on hybrid rice breeding and/or seed production in training programs in IRRI, China, and India. Nearly 4,000 seed growers and extension workers were trained locally on seed production and hybrid rice cultivation in more than 100 in-country training programs in the six countries. Improvements in human resources and facilities helped in the release of new hybrids through local breeding efforts and the development of seed production technology in member countries. Encouraged by the progress made in the project, member countries strengthened their national hybrid rice programs with increased investment from national funds as well as donors such as FAO and the United Nations Development Programme, and through bilateral projects with the UK and China. Indonesia s national allocation for hybrid rice research increased four times to Rp. 15 billion, at the same time the country received a technical support project (TCP) from FAO. Vietnam s national International Task Force on Hybrid Rice 211

198 Table 1. Comparison of hybrid and inbred mean yields over different groups of environments and Coordinated International Hybrid Rice Yield Trials, 1999 and Environmental No. of sites Range of yield No. of sites and No. of sites and mean yield (kg ha 1 ) difference in mean positive mean negative kg ha 1 (F 1 I) difference (kg ha 1 difference (kg ha 1 ) ) Low (2,501 4,500) to (+247) ( 329) ( 682) Medium (4,501 6,500) 9 9 1,667 to 886 to , (+443) (+538) ( 660) (508) High (>6,501) to 18 to (+423) (+530) ( 325) ( 18) Overall ,667 to 886 to , (+407) (+535) ( 359) ( 429) a I = inbred. 212 Virmani et al

199 Table 2. FAO support to national hybrid rice programs during Country Project No. Period Approved budget FAO/TCP (US$000) Vietnam VIE/2251 5/92 12/ Vietnam VIE/6614 6/96 12/ Myanmar MYA/6612 3/97 3/ Bangladesh BGD/6613 5/97 4/ Philippines PH/8821 1/98 12/ Egypt EGY/8923 9/99 12/ budget support increased to $70,000 for research and development (R&D) and $589,000 for seed production in 2001 vis-à-vis $13,000 year 1 in 1991 to The Philippine government dramatically increased its investment in hybrid rice R&D during the project period from P 7 million to P 121 million for the Department of Agriculture Program and from P 1 million to P 20 million for PhilRice to support 18 key research projects. In Bangladesh, the PETRRA (Poverty Elimination Through Rice Research Activities) project funded by the UK included a hybrid rice project valued at $600,000 for 2001 to 2004, thus giving a needed boost to the national hybrid rice program. FAO financial support for hybrid rice during the last decade to national programs amounted to $1.76 million (Table 2). 4. Technology generation During , 19 rice hybrids were released in the member countries. Several of these showed a yield advantage of more than 1 t ha 1 over inbred HYVs in on-farm trials (Fig. 1). Many of these hybrids were derived from parental lines provided by IRRI to the national programs. IRRI continued to develop new parental lines, including cytoplasmic male sterile (CMS), maintainer, restorer, and thermosensitive genic male sterile (TGMS) lines under its strategic research program conducted under this project. Hybrid seed yields of up to 2.5 t ha 1 were obtained in member countries. 5. Increased participation of the hybrid seed industry in member countries The seed production and marketing component of hybrid rice technology has encouraged increased participation of seed enterprises (in the public, private, and NGO sectors) for its development and use in member countries. Since 1998, at least 20 new seed enterprises have become involved in hybrid rice research and/or seed production and marketing. In India, private seed companies accounted for 90% of the F 1 seed supply (3,000 t) in 2001, whereas, in Vietnam, the local seed industry supplied about 20% of the hybrid seed requirement (11,000 t), with 80% still imported from China. In the Philippines, organized community-based farmer groups are also involved in this enterprise. In Bangladesh, one NGO (the Bangladesh Rural Advancement Committee), along with some private companies and the public-sector International Task Force on Hybrid Rice 213

200 Yield (t ha 1 ) Sonar Bangla 7.5 Best inbred Hybrid 5.2 Sahyadr Mestizo HYT Bangladesh (sample farms, boro season) India (15 locations, rabi) Philippines (on-farm compact farms at 5 locations, 1998 dry season) Vietnam (1999 summer season) Fig. 1. Yield of some released hybrids compared with high-yielding inbreds in multilocation trials. Table 3. Number of different seed enterprises involved in hybrid rice research and development in Asia. Country Private companies NGOs Public seed Cooperatives, Total enterprises associations Bangladesh India Indonesia Philippines Sri Lanka 2 2 Vietnam Total Bangladesh Agricultural Development Corporation (BADC) are participating in hybrid rice seed production and marketing. The current situation of seed enterprises dealing with hybrid rice in member countries is given in Table Consultancy assistance A total of 33 consultancy missions (Table 4) were carried out in the project to monitor, review, assess, recommend, and extend technical assistance to NARES in their hybrid rice programs. Experts on hybrid rice breeding, seed production, agronomy, and the seed industry came from IRRI (3), FAO (3), China (3), India (4), the Philippines (2), and Vietnam (2). These consultancies also helped in identifying the major 214 Virmani et al

201 Table 4. Hybrid rice-related consultancy missions from 1998 to Country No. of consultancy missions Bangladesh 7 Indonesia 6 India 5 Philippines 5 Sri Lanka 5 Vietnam 5 Total 33 constraints to the development and large-scale adoption of the technology in member countries. 7. Identification of policy intervention areas Four policy workshops, one study tour, and three studies were conducted from 1999 to Policy workshops both network-wide (organized by FAO) and in-country (two in the Philippines and one in Bangladesh) were organized under the project. We also organized a 3-day study tour in India for policymakers of network member countries. Two study missions examined the constraints and policies for network members, one study focused on Vietnam, and a commissioned paper studied intellectual property rights (IPR) in relation to the development and use of hybrid rice technology. The following policy intervention points were identified through various policyrelated activities: Recognize the use of hybrid rice technology as a strategy to increase rice production for food security and employment generation and identify and support a capable R&D agency to play the lead role in the national hybrid rice program and coordinate the activities of various stakeholders from the public, private, and NGO sectors with a mission approach. Establish collaboration with other member countries and international agencies involved in hybrid rice for the development and promotion of the technology. Discuss with member countries in the hybrid rice network concerns about germplasm exchange and establish an appropriate national policy on the free exchange of germplasm for R&D on hybrid rice in view of IPR legislation. At the initial stage of technology use, consider a subsidy for seed production and cultivation to cover the risks. Give attention to the pricing, local manufacture of gibberellic acid (GA 3 ), and/or substitution with alternative chemicals because GA 3 is an essential but expensive imported input in F 1 seed production. International Task Force on Hybrid Rice 215

202 Table 5. Area planted to hybrid rice in Asian countries where its use has increased significantly. Country Hybrid rice area (ha) Vietnam 187, ,000 India 100, ,000 Bangladesh 15,000 Philippines 6,000 Total 287, ,000 Provide seed-quality assurance to farmers based on seed certification and/ or a truthful labeling policy and upgrade and modernize seed certification and quality service laboratories to handle hybrid rice seed certification requirements. Make arrangements by which interested private seed companies would be able to have access to public parental materials to produce and market hybrid seeds. 8. Impact By 2001, the area planted to hybrid rice in four member countries (Table 5) had expanded by 413,300 ha, more than a twofold increase over the 1997 area (287,700 ha), thus raising the total to 701,000 ha. The total amount of seeds planted to the 413,300 ha additional area would amount to 8,266 t (at a seeding rate of 20 kg ha 1 ), valued at $16.5 million (at $2 kg 1 ), indicating the development of a sizeable seed industry. With 50 person-days incremental labor per hectare to produce seed at 1 t ha 1 yield, a total of 413,300 person-days additional labor employment was realized. Given an average of 1 t ha 1 additional yield of hybrids, the 413,300 ha contributed to the production of an additional 413,300 t of paddy valued at $50 million. Therefore, in its early phase of development and use, hybrid rice technology is already contributing significantly toward increasing productivity, income, and rural employment in Asia. The network was rated highly successful by the ADB review team and a second phase of the project titled Sustaining Food Security in Asia through the Development and Dissemination of Hybrid Rice Technology was granted for three years, beginning in 2002, in which three new member countries (Republic of Korea, Myanmar, and Thailand) were added. Future directions and strategies As projected, food security in the developing countries of tropical Asia has a precarious outlook in the light of increasing population, declining productivity, and limited 216 Virmani et al

203 land and water resources. To sustain food security and alleviate poverty in these countries, yield per unit area per unit time should increase and the unit cost of rice production decrease. Thus, affordable rice supplies must be increased to meet the demand from the increasing population. Under a conducive policy environment, the use of hybrid rice technology as a strategy for food security has been illustrated in China. Recent developments, including the achievements of this project, have shown that this technology also has good prospects for contributing to food security and poverty alleviation in tropical rice-growing countries with a high proportion of irrigated rice area and a high land-labor ratio. The major challenge is how to overcome certain constraints (such as poor grain quality, the high cost of seeds, inadequate agronomic management) and create a more favorable environment for the rapid development and dissemination of hybrid rice technology for the large-scale cultivation of hybrid rice in areas with favorable conditions to make a significant impact. A socioeconomic analysis of the technology s impact will need to be done to keep its application on the right track. A complementary but relatively lesser effort would be to assess the prospects of hybrid rice for poverty alleviation among resource-poor irrigated rice farmers and those in irrigated and direct-seeded irrigated rice ecosystems. Toward these ends, a concerted effort is necessary in carrying out hybrid rice technology refinement and generation and establishing an efficient system of seed production and supply of affordable high-quality seed and well-focused technology transfer. This would require further strengthening of the NARES for hybrid rice, with strong national policy and program support to allow more effective coordination of efforts among researchers, technology transfer experts, and the private seed industry. The second phase of the ADB-funded project would help meet these challenges. To broaden the scope of INTAFOHR beyond Asia, different donor support is needed to make this a truly global network. Notes Authors addresses: S.S. Virmani, International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines; Dat Van Tran, Food and Agriculture Organization of the United Nations (FAO), Rome, Italy; J.S. Sindhu, Asia Pacific Seed Association, P.O. Box 1030, Bangkok 10903, Thailand. Acknowledgment: The financial assistance provided by the Asian Development Bank in the form of two projects established since 1998 under this network is gratefully acknowledged. Citation: Virmani SS, Mao CX, Hardy B, editors Hybrid rice for food security, poverty alleviation, and environmental protection. Proceedings of the 4th International Symposium on Hybrid Rice, Hanoi, Vietnam, May Los Baños (Philippines): International Rice Research Institute. 407 p. International Task Force on Hybrid Rice 217

204 Hybrid rice: how to go forward? Dat Van Tran All stakeholders, including decision-makers, rice scientists, extension workers, F 1 seed producers, and rice growers, have recognized the advantages of hybrid rice technology. Rice hybrids not only outyield commercial inbred varieties by 15% to 20% but also generate rural employment opportunities and potential land saving for other uses. These promising benefits have encouraged many governments to focus on developing and disseminating hybrid rice, especially in countries where rice imports have become a burden on the national economy, population continues to grow, and expansion of rice area is not possible. However, progress in large-scale adoption under tropical conditions seems slow, although, in countries such as Vietnam and India, the government has taken aggressive measures to solve technical and nontechnical problems. How should we go forward to expedite farmers adoption of this technology? This paper reviews the factors thought to be responsible for the slow progress and suggests that technological constraints could be the major limitation to technology transfer. Obviously, technological development has not been able to keep pace with the public interest in hybrid rice, in terms of research activities and hybrid seed production. Regardless of substantial improvement in human capacity, national hybrid rice programs would stagnate should progress in research be slow for a long period, if the level of F 1 seed yield remained unchanged, and if the government decreased its interest in the matter. Rice is the staple food for more than 3 billion people in the world. About 90% of the world s rice is produced by small-scale farmers in low-income developing countries and consumed locally. Rice has become an important element of national food security in an increasing number of developing countries. During the Green Revolution, rice production increased substantially mainly because of an increase in yield. In the 1980s, yield growth increased at more than 2% per year. Since 1990, the growth of the world s rice yield has decelerated considerably (Tran 2002). Looking ahead 30 years, rice specialists estimate that feeding the population would require nearly 30% more rice, for a total of 760 million t of paddy rice. In 2000, world rice production 219

205 reached around 600 million t on 154 million ha (FAO 2002). The challenge for rice scientists is to ensure sustainable yield growth with less land, labor, water, and pesticides, while protecting the environment and health of the people. Hybrid rice is an emerging technology, which can increase rice yield by 15 20% over that of commercial varieties thanks to its heterosis. This technology can contribute to increased rice production to meet the world s future demand. The inaugural meeting of the IRRI-ADB Project on Sustaining Food Security in Asia Through the Development of Hybrid Rice Technology, held at IRRI in the Philippines on March 2002, marked considerable progress in six participating countries national hybrid rice development and dissemination program. In 2001, the total area cultivated to hybrid rice reached 700,000 ha. India, Vietnam, and the Philippines have made substantial progress in developing skilled manpower, research, and seed production. The Indonesian hybrid rice program has taken off again with full strength and strong support from the government. Bangladesh has strengthened its national capacity in breeding and seed multiplication and has already released two hybrid varieties for large-scale production. Sri Lanka has reshaped its hybrid rice program with additional Chinese assistance, but the current use of direct seeding in irrigated rice is an obstacle for adoption of this technology. However, the large-scale adoption of hybrid rice has been hampered by many constraints, notably the very high seed cost; low F 1 seed yield; expensive gibberellic acid (GA 3 ); less grain quality; inconsistent yield performance; a weak seed industry; the lack of effective linkage of research, F 1 seed multiplication and production, and technology transfer; and insufficient support from governments. The delay in finding appropriate solutions to these problems would lead to losing continued government support for hybrid rice activities in any country. International and national efforts are strongly needed to sustain a hybrid rice program at the initial stage. This paper thus focuses on the major issues that may seriously affect the process and efforts to introduce and disseminate hybrid rice. Main constraints to the wide adoption of hybrid rice Many valuable experiences in the development and technology transfer of hybrid rice were reported during several international and regional meetings and workshops. A review of these experiences would shed light on the future of the hybrid rice program in several interested countries. Especially, lessons learned from many years of work in a country would enhance the hybrid rice programs in other countries. The 3rd International Symposium on Hybrid Rice was jointly organized by the Indian Council on Agricultural Research (ICAR), IRRI, and FAO in Hyderabad, India, from 14 to 16 November In this Symposium, it was reported that many constraints hampered the wide spread of hybrid rice technology in some countries. Among others were a lack of proper facilities for producing promising parental lines and F 1 seeds (Paroda 1998), limited yield heterosis in commercial rice hybrids and low and inconsistent seed yields (Virmani 1998), the high cost of hybrid seed and 220 Dat Van Tran

206 less grain quality in India (Siddiq and Ahmed 1998), and the inadequate local F 1 seed supply in Vietnam (Nguyen et al 1998). The FAO-sponsored Regional Workshop on Progress in the Development and Use of Hybrid Rice Outside China was held in Hanoi, Vietnam, from 28 to 30 May The report of this workshop revealed several limitations to national hybrid rice research and development programs, above all the governments lower support and commitment regarding the need and importance of the programs and potential of hybrid rice technology to increase rice productivity and production (Tran 1997, Ahmed 1997) and the lack of effective linkage and coordination among research, F 1 seed production, and extension (Tran 1997). The number of commercially usable cytoplasmic male sterile (CMS) lines with stability for complete sterility, adaptability to the target environment, a good outcrossing rate, good general combining ability, and easy restoration is limited (Virmani 1997). Serious concerns were raised about three techniques genetic purification of parental lines and F 1 seed production, heading synchronization of parental lines, and expensive GA 3 for promoting panicle exsertion (Ahmed 1997, Yin 1997, Tran 1997). The IRRI-sponsored Workshop on Policy Support for Rapid Adoption of Hybrid Rice in Large-Scale Production in Asia was jointly organized by the Ministry of Agriculture and Rural Development and FAO in Hanoi, Vietnam, from 22 to 23 May 2001, with the aim to sensitize senior officers for their further commitment to national hybrid rice programs. Several high-level officers such as the minister of agriculture of Bangladesh, vice-minister of agriculture of Vietnam, assistant secretary of agriculture of the Philippines, agriculture commissioner of India, and directors-general and directors of Indonesia and Sri Lanka attended this workshop. Major concerns were raised on the holistic approach in the implementation of hybrid rice programs and the high costs of hybrid seeds, which farmers cannot afford. H.E. Matia Chowdhury, minister of agriculture of Bangladesh, said, The government of Bangladesh is convinced that hybrid rice is a viable and proven technology and is a new frontier for increasing rice production. The acceleration of hybrid rice research and development is one of its priorities. Major causes of concern are the supply of required F 1 seed production and reducing its cost, increasing grain quality, and developing pest resistance and tolerance for other abiotic stresses. The Inaugural Meeting of the ADB/IRRI Project on Sustaining Food Security in Asia Through the Development of Hybrid Rice Technology took place at IRRI in the Philippines from 18 to 20 March This meeting reviewed the activities and accomplishments of the first phase of the project on the Development and Use of Hybrid Rice Technology in Asia and found that the major problems related more to research than to development, especially unstable CMS lines, low F 1 seed yield, genetic impurity, and inadequate grain quality. Over the past decade, considerable progress was made in both research and dissemination of hybrid rice technology. In research, many hybrid combinations and CMS lines were released, but the stability of a few CMS lines was frequently questioned, yields were not quite consistent, and seed set was not high enough compared with line Bo A. For development, around 480,000 ha of hybrid rice were grown in Hybrid rice: how to go forward? 221

207 Vietnam, with a high proportion of imported F 1 seeds; 200,000 ha in India, with a slow annual growth rate; and more than 100,000 ha to be planted in the Philippines in However, F 1 seed yields were still low or less than 1.5 t ha 1, the heterotic advantage was often variable, and the optimal yield performance of hybrids was still questionable. The major development constraints to the wide adoption of hybrid rice are summarized below: A. High seed cost (5 6 times) 1. Not many available stable CMS lines. 2. Not many CMS lines with high out-pollination like Bo A lines outside China. 3. Expensive GA 3 for facilitating complete panicle exsertion. B. Complicated technology 4. Difficulty of maintenance of genetic purity of parental lines and F 1 seeds. 5. Unavailability of two-line hybrids, especially outside China. 6. Lack of proper facilities and/or organization for parental line multiplication and F 1 seed production of promising/released hybrids. 7. Heading synchronization of parental lines in different areas. 8. Grain quality requirement in a particular region. 9. Varietal adaptability in various agroecological systems in tropical conditions. C. Weak seed industry 10. Lack of a rice seed industry in many developing countries. 11. Not many private seed companies involved in F 1 seed production without incentives. 12. Limited exchange of genetic materials among national agricultural research and extension systems (NARES). D. Policy and coordination 13. Inadequate and not well-defined policies for supporting national hybrid rice programs. 14. Ineffective coordination and linkage of hybrid rice research, seed production, and extension at national and local level. 15. Inadequate trained manpower in heterosis breeding techniques, especially F 1 seed multiplication and production at all levels. Obviously, these constraints have slowed the hybrid rice programs in several countries and the existent hybrid technologies have not yet performed well at the field level to keep up with the public interest. In view of this, some national programs would stagnate in the near future if appropriate solutions to these problems do not arrive in time. It is therefore believed that a significant improvement in F 1 seed yield, maintaining consistent hybrid yield, acceptable quality, the availability of simplified 222 Dat Van Tran

208 Table 1. Hybrid rice seed yield and cost changes in China, Year Yield F 1 seed cost (t ha 1 ) (US$ kg 1 ) Source: Mao et al (1998). hybrid technology such as two-line hybrids, and the optimal performance of hybrid rice yield would stimulate governments interest and commitment and expedite the wider adoption of this technology in large-scale production. F 1 seed yield The cost of F 1 seeds, which is 5 to 6 times higher than that of conventional seeds, will discourage farmers from the new technology if governments do not provide adequate support to their hybrid rice programs. This phenomenon is mainly caused by the low F 1 seed yield and intensive crop management. In practice, hybrid seed production has reached about t ha 1 or less in tropical conditions, while China has achieved yield of 2 3 t ha 1. Could climatic conditions or technological development play a decisive role in determining the final yield of hybrid seed production in a particular area? It was observed that in Vietnam the Bo-type CMS line always has a high seed setting rate because of its spikelets possessing a long exposure of exserted stigma in tropical regions. However, its medium grain size is undesirable for grain quality of hybrid combinations. High expectations for the development of such a Bo-type CMS line with acceptable grain quality and suitable for tropical and humid conditions have been long voiced by many hybrid rice specialists. Chinese rice specialists have spent 20 years improving skills in F 1 seed production, resulting in an increase of F 1 seed yield from 0.3 to 2.25 t ha 1. This enables them to cut down the cost of seed production from US$5.96 kg 1 to $0.79 (Table 1) (Mao et al 1998). At present, other countries can obtain only 1 to 1.5 t ha 1 or less and seed costs vary from $1.50 to $2.50 kg 1. Seed costs would be reduced by half if the technical gap between China and other countries were significantly narrowed and current seed yield were doubled (Tran 2002). Research for a substitute for GA 3 becomes urgent to reduce the cost of F 1 seed production, which is expensive, particularly in countries that have no facilities and technology to produce this product locally. Nevertheless, GA 3 promotes panicle exsertion, contributing to high seed set. Boric acid (H 3 BO 3 ) is a potential candidate for replacing GA 3 in hybrid seed production. The use of boric acid after the first spray of GA 3 gave an average seed set of 29.5%, as this chemical induced increased stigma Hybrid rice: how to go forward? 223

209 life of IR28025A (Nguyen 2002). Other chemicals should be widely screened, tested, and disseminated to the interested countries. Yield heterosis of hybrid rice The yield heterosis of hybrid combinations is still limited and not often consistent in various planting seasons in a particular location. Their yield advantage also tends to decrease with time if no appropriate or timely action is taken to maintain the genetic purity of parental lines and F 1 seeds. It seems that this problem is still persistent and has often raised serious concerns for many rice scientists and extension workers, as this could cause a possible collapse of a national hybrid rice program. In fact, most concern for future hybrid rice programs is the long-term maintenance of the genetic purity of F 1 seed and parental lines: A, B, and R lines. Several reports have recently indicated that the purity of these lines is deteriorating in a few countries, thus affecting the heterotic characteristics and yield advantage of rice hybrids. According to Mao (1988) and Yin (1997), each percentage of genetic impurity in F 1 seed could reduce paddy yield by approximately 100 kg ha 1. Genetic impurity of parental lines of hybrid rice was reported in India (Ikehashi 2000), Indonesia (Krishnaiah 2001, Viraktamath 2001), and the Philippines (Pan 2000). Aggressive training programs and dissemination of information on techniques of purification and maintenance of parental lines should be urgently conducted by international agricultural research centers, NARES, and international agencies to ensure good heterosis and avoid the latent negative trend in the application of hybrid rice technology (Tran 2002). Furthermore, the development of CMS lines with high ability to maintain genetic purity is recommended. Grain quality Grain quality is a major factor limiting the large-scale adoption of hybrid rice. Frequent complaints about low grain quality of hybrid rice have been raised, although this aspect has been improved in China, India, and Vietnam. Greater efforts are still needed to overcome this problem of hybrid rice under local conditions. As economic conditions improve in Asia, consumers require diverse and better grain quality in different regions even within a country, especially in India. In a survey made in Andhra Pradesh, India, in 2001, the main weaknesses of hybrid rice were (1) its quality was not suitable for local consumers, hence there was no domestic demand for it, (2) its price was lower than that of inbred varieties, and (3) its low milling quality led to more broken grains (Rao and Desai 2001). In a survey of farmers who grew rice hybrids during the crop year in five major rice-growing states of India (Andhra Pradesh, Karnataka, Tamil Nadu, Orissa, and West Bengal), about 80 85% of the surveyed farmers reported that the grain quality of hybrid rice was inferior to that of the commercial inbred rice varieties. Another 66% of surveyed respondents thought that hybrid rice had an unpleasant smell after cooking. Consequently, the price of hybrid rice was 11% lower than the 224 Dat Van Tran

210 price of inbred varieties (Janaiah and Hossain 2002). The lower price of hybrid rice will seriously affect the yield advantage of hybrids. The potential tool: two-line hybrids Direct seeding has become very popular in many countries, especially where labor is in short supply and expensive and herbicides are less costly. This planting method is well deployed where rice cultivation is fully mechanized. Direct seeding requires a high seed rate varying from 80 to 200 kg ha 1, while transplanting needs only from 20 to 40 kg ha 1. In hybrid rice production, the seed rate is less than 30 kg ha 1 because of the high cost. For this reason, the direct-seeding method has impeded the wider adoption of hybrid rice production in Latin America, Europe, and a few Asian countries such as Sri Lanka, the southern part of Vietnam, etc. Some attempts to reduce the seed rate to kg ha 1 and introduce the Chinese seedling throwing method have been made in hybrid rice cultivation, but not yet successfully. Developers, extension workers, and farmers have high expectations of promising two-line hybrids, which have been developed and tested since the 1980s by using temperature- and photoperiod-sensitive genic male sterile lines (TGMS and PGMS). Research on two-line hybrids has advanced considerably in China, at IRRI, and in other NARES. This technology would further boost the adoption of hybrid rice as it has a yield advantage of 5% to 10% over commercially cultivated three-line hybrid varieties. In the two-line system, there is no need for maintainer lines. The field area ratio among CMS line multiplication, hybrid seed production, and commercial production of two-line hybrids is therefore much higher (1:100:15,000) visà-vis that of three-line hybrids (1:50:6,000). Consequently, this technology would lower the cost of F 1 seed production (Yuan 1996). However, more time is required to perfect two-line hybrid rice technology before introducing it to farmers outside China because of the T/PGMS genetic instability. Rice specialists, developers, and others concerned have high expectations of the two-line hybrids, which may result in low-cost F 1 seed production and facilitate the dissemination of hybrid rice in large-scale production. But when will this technology be available for dissemination? Optimal performance of hybrid rice: RICM and Ricecheck Rice integrated crop management Farmers usually take more care of hybrid rice cultivation to optimize yield and offset high seed costs. Under this condition, we wonder whether hybrid rice farmers always use best-practice management. In addition, the yield gap between the research station and farmers fields still exists in hybrid rice production (Fig. 1). The yield gap is probably less for progressive farmers than for ordinary farmers across agroecosystems, mostly because of gaps in farmers knowledge, management, and extension delivery services. The yield potential of hybrid rice is around t ha 1 in tropical condi- Hybrid rice: how to go forward? 225

211 For scientists to conceive and breed potential varieties Yield gap 0 Yield gap I Nontransferable technology Environmental differences Yield gap II Biological Variety Weeds Pests Problem soils Water Soil fertility Socioeconomic Costs Credit Tradition Knowledge Input Institutions Theoretical potential Experiment station yield Potential farm yield Actual farm yield Fig. 1. The concept of yield gap (based on Chaudhary 2000). tions while that of high-yielding varieties is t ha 1. The actual average yield of hybrid rice varies from 6 to 9 t ha 1. Hence, a sizable yield gap still exists. Hybrid rice is very sensitive to crop management, including planting season, land preparation, crop establishment, fertilizer application, pest management, water and weed control, and harvest. Out of these, fertilizer management is the most important factor for determining the final yield of hybrid rice, as it affects other factors such as crop establishment and pest and weed occurrence. Ordinary farmers usually do not handle the application of fertilizers well, especially nitrogen, in terms of methodology, rate, and timing. The rice plant needs nitrogen timely and in adequate amounts during tillering, panicle initiation, and the heading period. Farmers carry out several cultural operations when growing a rice crop. All these activities, both separately and collectively, affect all phases of crop development and yield components that ultimately determine yield. Management determines the degree of the effects and the interaction of these activities within an agroecological system. Integrated crop management systems are based on the understanding that 226 Dat Van Tran

212 limitations in production are closely linked (Clampett et al 2002). For example, stronger seedlings from high-quality seeds will not benefit yield if the crop is inadequately fertilized. Similarly, the crop cannot respond to improved fertility if weeds compete. In addition, to obtain high fertilizer efficiency, enough water is obligatorily required in time. The Ricecheck model The Ricecheck Model used in the Australian rice industry has introduced a framework for using integrated crop management and the evaluation of management results as a means to improve productivity and environmental outcomes. In this model, farmers are actively involved using discussion groups in a collaborative learning environment to improve management and yield (Clampett et al 2002). As a result, rice yield in Australia increased from 6 to 8.5 t ha 1 in the past 15 years and Australia was among the highest-yielding countries in the world. Briefly, Ricecheck is a system (1) to formulate appropriate recommendations for best-practice management with output targets, (2) to disseminate them through farmer discussion groups, and (3) to monitor and evaluate crop management areas (based on targets) for improvement of the following crop. Thus, the Ricecheck concept is similar to the farmers field school approach, but it requires more commitment from farmers and attention to input and output targets and monitoring of crop growth from land preparation to harvest for improvement of the following rice crop. For example, to achieve 8 t ha 1 requires, among others, a plant density of 250 tillers m 2, which, in turn, requires adequate good seeds and fertilizer and good management of pests, weeds, and water (Table 2). The conditions of these management areas should be recorded and discussed in farmer discussion groups. The Australian key checks (recommendations) for yield cover seven integrated crop management areas, which include one or more key checks (objective targets) as well as minor targets. These include The rice field irrigation layout: The field layout in regard to contour bank (bund) height and other specifications, such as contour interval, is described and measurements provided. Sowing time: Specific dates for an ideal sowing window are provided for each variety. A measure of the output of sowing time is panicle initiation date. Plant establishment: Uniform plant stand as plant number m 2 is the desired output. The techniques used for land preparation and sowing and seeding rates are inputs, which affect the result. Crop protection: Control of weeds and pests is carried out to avoid economic loss, along with management to avoid pesticide residues in drainage waters. Nutrition: Preflood nitrogen fertilizer is used to achieve growth index targets at panicle initiation and assess additional fertilizer needs. Soil testing is the basis of phosphorus inputs. Hybrid rice: how to go forward? 227

213 Table 2. Examples of relationships among input practices, growth, and management outputs and yield, grain quality, and environmental outcomes. Inputs Outputs Outcome (practices) (results) (final effects) Cultivation, vegetation Plants m 2 Yield control, leveling, seed rates, 100% uniform plant coverage Grain quality sowing techniques, water depths, time of sowing, pest control Cultivation; nitrogen rates, PI nitrogen uptake (kg N ha 1 ) Yield timing, and application; water Grain quality management; pest and weed control; time of sowing Field irrigation layout, banks (bunds), Specific water depths at Yield channels, water supply rates and growth stage depth control, drainage Cultivation, sowing time, Grain moisture at harvest Grain quality establishment, plant stand and uniformity, preharvest, draining, nitrogen supply, harvest time Field irrigation layout, cultivation, Pesticide residue levels in field Environmental effect time of sowing, establishment, drainage water field drainage, water management, pesticide use and timing Source: Clampett et al (2002). Water management: Specific depths for each growth stage are used, particularly to assist establishment and weed control and protect the crop against low temperature-induced sterility at early pollen microspore. A need exists to avoid permeable soil types. Harvest management: Grain moisture limits are used to maximize whole grain after milling and quality assurance practices are followed to ensure clean grain. The key checks are revised each year by farmers, researchers, and extension workers. These checks normally interact with each other. A change in management factors can affect the performance of one or more other factors (Fig. 2). How to establish the RICM under the Ricecheck concept Six major steps to establishing comprehensive rice integrated crop management (RICM) to close the existing yield gap under the framework of Ricecheck are as follows: 1. Identify key management areas: Study and prioritize all factors affecting the current rice yield gap and production in the selected or target location. 228 Dat Van Tran

214 Yield (t ha 1 ) Number of key checks achieved Fig. 2. Relationship between yield and the achievement of key checks of 2,100 commercial Amaroo rice crops over seven seasons, (Clampett et al 2002). 2. Quantify best management practices of progressive farmers: Survey and analyze the rice production technology and practices of farmers to identify differences in each key management area. 3. Review available technology and knowledge: Review current knowledge based on research results, practices, and experiences of researchers, extension workers, and farmers. 4. Develop interim best management practices: Based on steps 1, 2, and 3, collate, in conjunction with researchers, extension workers, and farmers, an appropriate RICM technological package (within the capabilities and resources of farmers) using the concept of RICM and inputs and outputs based on objective criteria to achieve outcomes. 5. Evaluate the best management practices: Test, demonstrate, and monitor the suitable package of RICM with farmer discussion groups and train extension workers and farmers. 6. Develop and expand a comprehensive RICM program at a district, regional, and national level, with the participation of all stakeholders under the framework of Ricechecks. Conclusions Over the past decade, considerable progress was made in several national hybrid rice programs, especially in breeding research, F 1 seed production, and technology transfer. However, some countries such as India and Vietnam, which had advanced pro- Hybrid rice: how to go forward? 229

215 grams in earlier years, appear to move forward slowly because of various limitations, above all technological constraints. However, hybrid rice programs will keep on moving forward with bright prospects if appropriate solutions can be found quickly for the following problems: 1. Reducing the cost of F 1 seed by half or more (twice that of normal seeds) by doubling current F 1 seed yield (2 2.5 t ha 1 ), with several CMS lines having high out-pollination and improved seed production systems (synchronization, GA 3 ). 2. Having many more stable CMS lines and maintaining the genetic purity of parental lines and F 1 seeds. 3. Expediting the availability of two-line hybrid technology with the aim of facilitating the use of hybrid rice in direct-seeding areas as well as reducing seed costs. 4. Improving grain quality to meet local demands. 5. Closing the yield gap to optimize the yield performance of rice hybrids and cut down on the cost of production. 6. Strengthening policy support and national capacity in hybrid breeding and F 1 seed production and creating effective linkages of research, seed production, and extension. References Ahmed MI Development and use of hybrid rice technology: lessons learned from the Indian experience. In: Proceedings of the Workshop on Progress in the Development and Use of Hybrid Rice Outside China, Hanoi, Vietnam, May p Chaudhary RC Strategies for bridging the yield gap in rice: a regional perspective for Asia. Int. Rice Comm. Newsl. (FAO, Rome) 49: Clampett W, Nguyen VN, Tran DV Paper prepared for the 20th Session of the International Rice Commission, Bangkok, Thailand, July. FAO (Food and Agriculture Organization of the United Nations) FAOSTAT Rome (Italy): FAO. Ikehashi H Consultancy Mission Report on Project UNDP/FAO/IND/98/140 Development and Large-Scale Adoption of Hybrid Rice Technology. FAO Mission, 28 August-16 September 2000, FAO, Rome, Italy. 19 p. Janaiah A, Hossain M Hybrid rice technology for India s food security: needs, current status and policy options. Econ. Polit. Weekly 36(3). (Forthcoming.) Krishnaiah K Mission report on strengthening the development and use of hybrid rice in Indonesia (TCP/INS/8921). Mission undertaken from 24 February to 10 March Rome (Italy): FAO. 40 p. Mao CX Hybrid rice seed production in China. In: Seed health. Los Baños (Philippines): IRRI. p Mao CX, Virmani SS, Kumar I Technological innovations to lower the costs of hybrid rice seed production. In: Virmani SS, Siddiq EA, Muralidharan K, editors. Advances in hybrid rice technology. Manila (Philippines): International Rice Research Institute. p Dat Van Tran

216 Nguyen TH, Nguyen NKK, Bui BB, Nguyen TT, Tran DQ, Nguyen QB Hybrid rice research and development in Vietnam. In: Virmani SS, Siddiq EA, Muralidharan K, editors. Advances in hybrid rice technology. Proceedings of the 3rd International Symposium on Hybrid Rice, November, Hyderabad, India. Manila (Philippines): International Rice Research Institute. p Nguyen TH Recent progress in hybrid rice research in Vietnam. In: Proceedings of the Workshop on Policy Support for Rapid Adoption of Hybrid Rice on Large-Scale Production in Asia, Hanoi, Vietnam, May Rome (Italy): FAO. (In press.) Pan XG Mission report on hybrid seed production of technical cooperation project. Mission undertaken from 15 Sept. to 28 Oct. 2000, FAO, Rome, Italy. 27 p. Paroda RS Hybrid rice technology in India: problems and prospects. In: Virmani SS, Siddiq EA, Muralidharan K, editors. Advances in hybrid rice technology. Proceedings of the 3rd International Symposium on Hybrid Rice, November, Hyderabad, India. Manila (Philippines): International Rice Research Institute. p 5-9. Rao PP, Desai GR Studies on promotion and adoption of hybrid rice in Andhra Pradesh, India. Report, FAO, Rome, Italy. Siddiq EA, Ahmed MI Ushering in an era of hybrid rice in India. In: Virmani SS, Siddiq EA, Muralidharan K, editors. Advances in hybrid rice technology. Proceedings of the 3rd International Symposium on Hybrid Rice, November, Hyderabad, India. Manila (Philippines): International Rice Research Institute. p Tran DV FAO global hybrid rice development programme. In: Proceedings of the Workshop on Progress in the Development and Use of Hybrid Rice Outside China, Hanoi, Vietnam, May p Tran DV Hybrid rice for food security: recent progress and issues in the large-scale production. In: Proceedings of the Workshop on Policy Support for Rapid Adoption of Hybrid Rice on Large-Scale Production in Asia, Hanoi, Vietnam, May Rome (Italy): FAO. (In press.) Viraktamath BC Mission report on strengthening the development and use of hybrid rice in Indonesia. Mission undertaken from 24 Feb. to 24 April 2001, FAO, Rome, Italy. 42 p. Virmani SS Opportunities and challenges in breeding CMS, maintainer and restorer lines for hybrid rice breeding in the tropics. In: Proceedings of the Workshop on Progress in the Development and Use of Hybrid Rice Outside China, Hanoi, Vietnam, May MARD, Hanoi, Vietnam. p Virmani SS Hybrid rice research and development in the tropics. In: Virmani SS, Siddiq EA, Muralidharan K, editors. Advances in hybrid rice technology. Proceedings of the 3rd International Symposium on Hybrid Rice, November, Hyderabad, India. Manila (Philippines): International Rice Research Institute. p Yin HQ Strategies and procedures used for nucleus, breeders and foundation seed production. In: Proceedings of the Workshop on Progress in the Development and Use of Hybrid Rice Outside China, Hanoi, Vietnam, May MARD, Hanoi, Vietnam. p Yuan LP Hybrid rice in China. In: Hybrid rice technology. Hyderabad (India): Directorate of Rice Research. p Yuan LP Hybrid rice development and use: innovative approaches and challenges. IRC Newsl. (FAO, Rome) 47:7-14. Hybrid rice: how to go forward? 231

217 Notes Author s address: Executive secretary, International Rice Commission, FAO, Rome, Italy. Citation: Virmani SS, Mao CX, Hardy B, editors Hybrid rice for food security, poverty alleviation, and environmental protection. Proceedings of the 4th International Symposium on Hybrid Rice, Hanoi, Vietnam, May Los Baños (Philippines): International Rice Research Institute. 407 p. 232 Dat Van Tran

218 Country reports

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220 Hybrid rice research and development in Bangladesh A.W. Julfiquar, M. Jamil Hasan, A.K. Azad, M. Anwar Hossain, and S.S. Virmani Hybrid rice technology has been recognized as a viable technology for meeting the increased demand for rice in Bangladesh. Research and development (R&D) of hybrid rice technology began in 1993 with the introduction of rice hybrids and parental materials from IRRI. The technology was initially constrained by (1) a lack of conviction by researchers, policymakers, and administrators about the economic viability of the technology, (2) a lack of trained human resources to develop and use the technology locally, (3) a lack of knowledge about its seed production technology, and (4) the poor performance of introduced hybrids over conventionally bred released varieties. Over the past few years, some of these constraints have been overcome and the government is convinced about the potential of this technology and is committed to giving priority to accelerating hybrid rice R&D. Several IRRI-developed hybrids were tested in preliminary to advanced yield trials during the last four years at different locations in Bangladesh. The objective was to test the yield potential of the IRRI-developed hybrids under ideal boro conditions and then select the best hybrids for Bangladesh. Two hybrids, IR69690H and IR68877H, were nominated in the National Hybrid Rice Trial in the boro season ( ) coordinated by the Seed Certification Agency and the Department of Agriculture Extension. After extensive evaluation, the National Seed Board recommended IR69690H as BRRI hybrid dhan-1, primarily in the Jessore and Barisal region, with a view to subsequent cultivation in farmers fields in other regions of the country. Seed production is an important component of the development and use of hybrid rice technology. Results of the past two years show the possibility of raising the yield of F 1 hybrid seed on average to 2.0 t ha 1. The Bangladesh Rice Research Institute (BRRI) maintains close collaboration with IRRI and receives technical support, including seed materials and training, to establish an effective hybrid rice program in Bangladesh. Nucleus and breeders seeds are being produced under the supervision of the plant breeder at BRRI and those are being supplied to public and private seed-producing agencies for commercial seed production. BRRI is sharing its parental materials of promising hybrids with NGOs to multiply seed of parental lines and produce F 1 hybrid seeds. 235

221 This paper reports on the progress of hybrid rice R&D in Bangladesh, including recent accomplishments in technology generation and seed production. The economy of Bangladesh is largely based on agriculture and is therefore predominantly agrarian. Situated in the delta of the Bay of Bengal, the country is agroclimatically well endowed for crop production. With a land area of 147,570 km 2, the country supports about 132 million people, with the population growing at 1.5% per annum. About 84% of the total population lives in rural areas and is directly or indirectly engaged in a wide range of agricultural activities. Agricultural development is therefore essential to Bangladesh, not only to reduce the prevailing poverty situation but also to raise the overall living standard of its population. In the last few years, Bangladesh has achieved dramatic economic growth in the agricultural sector and rice has played the most significant role in this process. Favored by many scientific, social, and political factors, rice production has been growing at a very impressive rate. As a result, Bangladesh has moved from chronic hunger to self-sufficiency in food. The use of high-yielding varieties developed at the Bangladesh Rice Research Institute (BRRI) and at other national rice research institutions and new agricultural policies, new agricultural extension policies, the timely supply of inputs, and a good harvest price for the product have been instrumental in stimulating our farmers to make use of their internal potential and resources to increase their rice production. Although the growth rate of rice is 2.2%, which is higher than the estimated population growth rate of 1.8% in 1990 (Hossain 1998), the population is projected to increase by another 50% in the first quarter of the 21st century from 120 million in 1995 to million by 2025 (Karim 1998). The increased demand for rice will have to be met with less land, less water, less labor, and less pesticide. Shifting the yield frontier in rice is also an important research goal for rice scientists around the world. One of the best options available to plant breeders is hybrid rice. Hybrid rice became highly successful in China and more than 50% of the rice area is reported to be under hybrid rice cultivation. Some other countries, such as India and Vietnam, are also developing and popularizing hybrid rice technology. Hybrid rice in China gave a yield advantage of t ha 1 (20 30%) over conventionally bred modern varieties (Yuan et al 1998, Yuan 1994). Currently, China covers about 50% of its total rice area (31 million ha) with hybrid rice varieties, which produce 60% of the total rice production. Hybrid rice helped China to produce 20 million t of additional paddy every year, which contributes to the country s food security. The technology has also allowed the country to save more than 2 million ha of agricultural land for other uses. Recently, India (Siddiq et al 1998) and Vietnam (Hoan et al 1998) have also reported that hybrid rice offers an economically viable option to increase rice yield beyond the level of high-yielding semidwarf inbred rice varieties. 236 Julfiquar et al

222 Hybrid rice technology could offer considerable opportunity for increasing rice productivity in Bangladesh, where the labor-land ratio is high, labor costs are reasonably low, land is becoming scarce, and population density is increasing at an alarming rate. Therefore, to go beyond the present yield ceiling of semidwarf modern varieties, hybrid rice seems to be an attractive alternative. It is expected to have positive sociopolitical implications for the food front under Bangladesh conditions. Research and development of hybrid rice technology began in 1993 with the introduction of rice hybrids and parental materials from IRRI. The technology initially was constrained by (1) a lack of conviction by researchers, policymakers, and administrators about the economic viability of the technology, (2) a lack of trained human resources to develop and use the technology locally, (3) a lack of knowledge about its seed production technology, and (4) the poor performance of introduced hybrids vis-à-vis that of conventionally bred released varieties. Over the past few years, these constraints have been overcome and the government is convinced about the potential of this technology and is committed to giving priority to accelerating hybrid rice research and development. The government released its first national hybrid, named BRRI hybrid dhan-1, for commercial cultivation by farmers in This paper reports on he status of hybrid rice research and development in Bangladesh, including recent accomplishments in technology development and seed production. The development of hybrid rice technology in Bangladesh Research and development of hybrid rice technology began in 1993 at BRRI in collaboration with the International Rice Research Institute (IRRI). But this was mostly academic in nature rather than goal-oriented. From 1996 onward, hybrid rice research gained momentum with the formation of a working group and technical support from IRRI. Initial work involved testing F 1 hybrids and evaluating CMS and restorer lines introduced from IRRI. The hybrids were early maturing but failed to outyield the local high-yielding varieties. Some CMS and maintainer lines tested showed sterility and other desirable traits. The IRRI-introduced CMS lines IR58025A, IR62829A, IR64608A, and IR68886A and an Indian CMS line (PMS10A) showed % pollen sterility and more than 95% spikelet sterility, with an outcrossing rate of 22 41% during the boro season (Table 1), during which 20 new CMS lines from IRRI were evaluated. Four CMS lines IR67684A, IR68281A, IR68275A, and IR66707A were found to be stable for both pollen and spikelet sterility and adapted under Bangladesh conditions. The outcrossing rate ranged from 22% to 43%. Some new CMS lines from IRRI were also evaluated recently. Some of them were also suitable and adapted under Bangladesh conditions. But only a few CMS lines were usable in terms of stability in sterility, a good outcrossing rate, and an adequate level of disease and insect resistance. The maintainers showed 80 90% spikelet fertility and many restorers were also identified. Some Chinese CMS lines, such as V20A and Zhen Shan 97A, were Hybrid rice research and development in Bangladesh 237

223 Table 1. Features of CMS lines found adaptable in Bangladesh. Season and line Plant Days to Pollen PER a SER OCR height 50% sterility (cm) flowering (%) Boro IR58025A IR62829A IR67684A IR68887A IR68890A IR68891A IR68881A PMS10A Boro IR69627A IR68902A IR68886A IR68897A IR68888A a PER = panicle exsertion rate, SER = stigma exsertion rate, OCR = outcrossing rate. evaluated along with their maintainers for adaptability and performance, but these were not adapted under Bangladesh conditions and were highly susceptible to diseases and insects. Since the introduced CMS lines from China were unusable under our conditions, the wild abortive (WA) cytosterility system from both IRRI-developed CMS lines and China was used to develop locally adaptable CMS lines. Several selected local varieties/lines were initially identified as maintainers and were backcrossed to their respective CMS sources, but their sterility varied in different seasons. Many high-yielding locally developed elite lines have been identified as good restorers. These are being purified and multiplied for use in producing experimental hybrids. The important ones are IR R, IR R, IR R, IR46R, IR R, IR R, IR R, IR10198R, BR R, BR R, BR R, and BR R. Several IRRI-developed hybrids were tested in the International Rice Hybrid Observational Nursery (IRHON), Preliminary Yield Trials (PYT), Coordinated International Hybrid Rice Trials (CIHRT), and multilocation yield trials during the past four years at different locations in Bangladesh. The objective was to test the yield potential of IRRI-developed hybrids under ideal boro conditions of Bangladesh and subsequently select the best hybrids for Bangladesh. In the CIHRT, hybrids IR68877H and IR69690H had a yield advantage of 0.68 to 1.87 and 0.30 to 1.25 t ha 1, respectively, over the check variety of the same duration. 238 Julfiquar et al

224 Yield (t ha 1 ) IR68877H BRRIdhan IR69690H BRRIdhan Gazipur Comilla Habiganj Rajshahi Locations Fig.1. Yield of hybrids IR68877H and IR69690H and their checks in on-station trials. Five hybrids outyielded the check varieties, with a significant yield difference, and were chosen for on-station verification in larger plots during the boro season. Hybrids along with early (BRRIdhan 28) and late (BRRIdhan 29) inbred check varieties were evaluated at Gazipur, Comilla, Habiganj, and Rajshahi during the boro season. Hybrid IR69690H outyielded BRRIdhan 29 by 1.66 and 0.88 t ha 1 at Gazipur and Comilla, respectively, and by more than 0.5 t ha 1 at Habiganj and Rajshahi (Fig. 1). Other hybrids failed to show a significant yield increase over the check varieties of the same duration. Release of a hybrid Two hybrids, IR69690H and IR68877H, were nominated in the National Hybrid Rice Yield Trial (NHRYT) in the boro season coordinated by the Seed Certification Agency (SCA) and carried out by the Department of Agriculture Extension (DAE) and research stations. Under this trial, six on-station and six on-farm trials were conducted. The objective for the National Seed Board (NSB) was to recommend the most promising entry for commercial cultivation by farmers. These two hybrids showed about a 1 t ha 1 yield advantage over the check variety of the same growth duration in both on-farm and on-station trials at some locations (Fig. 2). But the results were not consistent at all the locations. The technical committee of the NSB recommended conducting a pilot production program for those two proposed hybrids with a view to recommending them for commercial cultivation in the region where they consistently had a higher yield. Hybrid rice research and development in Bangladesh 239

225 Yield (t ha 1 ) 9 8 On-station On-farm IR68877H BRRIdhan28 IR69690H BRRIdhan25 IR68877H BRRIdhan20 IR69690H BRRIdhan20 IR69690H BRRIdhan29 IR68877H BRRIdhan20 IR69690H BRRIdhan29 IR68877H BRRIdhan20 IR69690H BRRIdhan29 IR68877H BRRIdhan20 IR69690H BRRIdhan29 IR68877H BRRIdhan29 Dhaka Mymensingh Jessore Comilla Rangpur Rajshahi Experimental site and variety Fig. 2. Yield of proposed hybrids IR68877H and IR69690H in on-station and on-farm trials conducted by the Seed Certification Agency. A pilot production program was launched in the boro season, using more than 20 plots for each hybrid around the country. In the pilot production program, IR69690H had an average yield of 8.4 t ha 1 and yield ranged from 7.25 to 9.45 t ha 1 in four regions (Table 2). Although IR68877H had good yield in some regions, it showed shattering and sterility in most regions. The NSB recommended IR69690H as BRRI hybrid dhan-1 primarily for the Jessore and Barisal regions, with a view to subsequent cultivation in farmers fields in other regions of the country. Meanwhile, the grain quality characteristics of these hybrids were evaluated and they were comparable with those of released inbred check varieties (Table 3). A comprehensive study on cultural management, fertilizer management, and pest and disease reaction of the promising hybrids and parental lines was and is being conducted at BRRI headquarters and its regional stations. 240 Julfiquar et al

226 Table 2. Region-wise results of pilot testing of hybrids IR68877H and IR69690H. Region Days to maturity Spikelet sterility (%) Yield (t ha 1 ) IR69690H IR68877H IR69690H IR68877H IR69690H IR68877H Jessore Barisal Comilla Rajshahi Range Mean Table 3. Grain characteristics of proposed hybrids IR68877H and IR69690H. Designation Milling Length- Size and Protein Amylose 1,000- recovery width shape a (%) (%) grain (%) ratio weight (g) IR68877H LS IR69690H LS BRRIdhan LS BRRIdhan LS a LS = long slender. Seed production Seed production is an important component of the development and use of hybrid rice technology. The effects of several seed production components on outcrossing and seed yield of CMS lines and F 1 seed were studied, including row ratio, flag-leaf clipping, gibberellic acid application at the heading stage, and supplementary pollination. The preliminary study revealed that a row ratio of 6:2 and 8:2 (female to male) was the optimum for obtaining good seed yields for CMS multiplication and F 1 seed production under Bangladesh conditions (Table 4). The highest outcrossing rate appeared in treatments that included flag-leaf clipping along with supplementary pollination + the application of gibberellic acid. Either flag-leaf clipping along with supplementary pollination or gibberellic acid application gave similar yields but there was a significant yield advantage over the control for applying all three or any of the above methods. However, the results need to be verified in larger plots and over several seasons. Results of the past two years showed the possibility of raising the yield of F 1 hybrid seed. Seed yield varied with hybrid and season and with the optimum seed production package. The seed yield of both A lines (Fig. 3) and F 1 hybrids was generally higher during the boro season (Fig. 4) than during the T. aman season (Fig. 5). Hybrid rice research and development in Bangladesh 241

227 Table 4. Seed yield of CMS multiplication (A/B) and F 1 seed production in different row ratios. A/B or F 1 hybrid Row ratio Yield OCR a Remarks (kg ha 1 ) IR58025A/B 2:6 1,124 a 32.8 Recommended 2:8 1,034 a : b 23.9 IR62829A/B 2:6 1,226 a 35.2 Recommended 2:8 1,097 b : c 25.7 IR67616H 2:6 438 b 23.5 Recommended 2:8 591 a : b 19.0 IR69690H 2:6 488 b 22.0 Recommended 2:8 671 a : b 17.5 a OCR = outcrossing rate. Yield (kg ha 1 ) 2,650 IR58025A/B boro season 2,250 IR58025A/B T. aman season 2,510 1,850 1,450 1,050 1,760 2, Years Fig. 3. CMS multiplication during boro and T. aman seasons. The government s initiative The government has adopted a comprehensive master plan on hybrid rice, indicating the target area, season, and mode of evaluation of the developed hybrids and also the seed production mechanism of the released hybrid. A national network for hybrid rice has been established in three tiers. A working group on hybrid rice has been formed, including a plant breeder, seed production specialist, plant pathologist, agronomist, entomologist, and applied research specialist. This program also includes collaborative research with other public research institutions, NGOs, and private agencies engaged in seed production in the country. The irrigated ecosystem (boro sea- 242 Julfiquar et al

228 Yield (kg ha 1 ) 2,500 2,000 1,500 1,000 IR69690H IR68877H 1,080 2,300 2,280 1, Boro Boro Boro Boro Season Fig. 4. F 1 seed production during the boro season. Yield (kg ha 1 ) IR69690H IR68877H Year Fig. 5. F 1 seed production during the T. aman season. son) has been identified as the target season and a time-bound goal-oriented work plan has been made. Research institutions and universities such as BRRI, BINA, BAU, and BSMRAU are joining together to develop a coordinated national research program on hybrid rice. Nucleus and breeders seeds are being produced under the supervision of the plant breeder at BRRI and these are being supplied to public and private seed-producing agencies for foundation seed production. Technical assistance and development of human resources BRRI maintains close collaboration with IRRI and receives technical support including seed materials and training to establish an effective hybrid rice program in Bangladesh. IRRI also provides consultancy services to develop effective hybrid rice research and development in the country. An FAO-funded technical cooperation project on hybrid rice was launched during through which two consultants from China were sent to assess the research facilities and seed production capacity of the country. At present, DFID, through the PETRRA subproject on hybrid rice, provides human resources and financial support, infrastructure development, and the operational costs of hybrid rice R&D in the country. Moreover, Bangladesh was in- Hybrid rice research and development in Bangladesh 243

229 cluded in the IRRI-ADB Hybrid Rice Network of the project Sustaining Food Security in Asia Through the Development of Hybrid Rice Technology, from which a moderate budget has been allocated to accelerate the transfer of technology in the country. As a part of the development of human resources in hybrid rice within the country, regular training programs are conducted from time to time. These training programs include scientists, managers, and other officers of research institutions and seed-producing agencies from public, private, and NGO organizations. Constraints and challenges In spite of the progress made so far, there are many constraints to overcome in the adoption of hybrid rice technology in Bangladesh. Among the policy issues, human resources deployed in hybrid rice are still inadequate. A national research team with a critical mass to undertake breeding, seed production, and agronomic research is urgently needed. Adequate research facilities should be developed and the funds required should be allocated. Institutional linkages among the research institutions and seed production agencies should be strengthened. The seed production infrastructure needs to be strengthened in the public and private sector and linked with hybrid rice breeding institutions so that responsibilities for seed production can be shared. Inherent hybrid vigor suggests a bright future for hybrid rice in Bangladesh. The foremost constraint to overcome by researchers is the identification of heterotic hybrid combinations that are adapted to Bangladesh conditions and that can consistently yield at least 1 t ha 1 more than conventionally bred high-yielding varieties. A cheaper seed production package should be developed to make the hybrids economically viable for commercial cultivation. The successful adoption of hybrid rice in Bangladesh will ultimately depend on the facilities and resources deployed for this technology. References Hossain M Recent developments in the Asian rice economy: implications for research prioritization. Paper presented at a workshop on prioritization of rice research in Asia, held at the International Rice Research Institute, Los Baños, Laguna, Philippines, April Hoan NT, Kinh NN, Bong BB, Tram NT, Qui TD, Bo NV Hybrid rice research and development in Vietnam. In: Virmani SS, Siddiq EA, Muralidharan K, editors. Advances in hybrid rice technology. Proceedings of the 3rd International Symposium on Hybrid Rice, November 1996, Hyderabad, India. Manila (Philippines): International Rice Research Institute. p Karim Z Overview of the problem and prospects of Bangladesh agriculture. Paper presented at the Symposium on Vision of Agricultural Research and Development, held at the Bangladesh Agricultural Research Council, 6 April Julfiquar et al

230 Siddiq EA, Ilyas Ahmed M, Viraktamath BC, Rangaswami M, Vijay Kumar R, Vidyachandra B, Zaman FU, Chatterjee SD Hybrid rice technology in India: current status and future outlook. In: Virmani SS, Siddiq EA, Muralidharan K, editors. Advances in hybrid rice technology. Proceedings of the 3rd International Symposium on Hybrid Rice, November 1996, Hyderabad, India. Manila (Philippines): International Rice Research Institute. p Virmani SS Prospects of hybrid rice in the tropics and subtropics. In: Virmani SS, editor. Hybrid rice technology: new developments and future prospects. Manila (Philippines): International Rice Research Institute. p Yuan LP Increasing yield potential of rice by exploitation of heterosis. In: Virmani SS, editor. Hybrid rice technology: new developments and future prospects. Manila (Philippines): International Rice Research Institute. p 1-6. Yuan LP Hybrid rice breeding in China. In: Virmani SS, Siddiq EA, Muralidharan K, editors. Advances in hybrid rice technology. Proceedings of the 3rd International Symposium on Hybrid Rice, November 1996, Hyderabad, India. Manila (Philippines): International Rice Research Institute. p Notes Authors addresses: A.W. Julfiquar, chief scientific officer and leader, Hybrid Rice Project, BRRI, Gazipur; M. Jamil Hasan and A.K. Azad, researchers, PETRRA subproject on hybrid rice, BRRI, Gazipur; M. Anwar Hossain, principal scientific officer and head, BRRI Regional Station, Barisal, Bangladesh. Citation: Virmani SS, Mao CX, Hardy B, editors Hybrid rice for food security, poverty alleviation, and environmental protection. Proceedings of the 4th International Symposium on Hybrid Rice, Hanoi, Vietnam, May Los Baños (Philippines): International Rice Research Institute. 407 p. Hybrid rice research and development in Bangladesh 245

231 Hybrid rice achievements and development in China Ma Guohui and Yuan Longping With the hybrid rice research begun by Professor Yuan Longping in 1964, only in the past 12 years did China become the first country to commercialize hybrid rice in the world. Now, the annual area under hybrid rice is about 15.5 million ha, 50% of the total rice area, and the production of hybrid rice occupies nearly 60% of the total rice production in China. Hybrid rice has a yield advantage of more than 20% over conventional rice. The technology of hybrid rice seed production was optimized in 1976, resulting in national average yield rapidly increasing from kg ha 1 in 1975 to 2.25 t ha 1 in Yield on a large scale can be t ha 1 and the area ratio of A line multiplication to F 1 hybrid seed production to F 1 hybrid commercial production is about 1:50:6,000, which greatly lowers the cost of hybrid seeds. China has made great achievements in research on two-line hybrid rice. Many viable PTGMS lines developed that possess the characteristics of low critical sterility-inducing temperature (CSIT) and safe hybrid seed production have been technically identified and approved. Up to now, more than 20 twoline hybrids have been released to farmers. The area of two-line hybrid rice increased to 2.67 million ha in 2001, about 17.2% of the total hybrid rice area and showing a yield increase of 5 10% compared with current three-line hybrid rice as well as better grain quality and good pest resistance. Some constraints exist in three-line hybrid rice, such as yield and area stagnation, the lack of japonica hybrid rice with strong heterosis, and the need for grain quality as well as sources of male sterility-inducing cytoplasm to develop better CMS lines. One of our breeding strategies is to use heterosis in hybrid rice breeding by means of systems such as the three-line, two-line, and one-line methodology and to improve heterosis by means of intervarietal, intersubspecific, and distant hybrids. 247

232 In the past decade, China became self-sufficient in basic food for the first time in modern history. This was a tremendous accomplishment when one considers that China, with almost 1.3 billion people, has 22% of the world s population but contains only 7% of its arable land. Hybrid rice is one of the greatest innovations and miracles in the world, which contributed much to Chinese food security in the past 20 years. Hybrid rice research was begun by Professor Yuan Longping in 1964 and a series of breakthrough achievements have been made in China. For the past 30 years, hybrid rice has undergone the stages of preliminary research, demonstrations, popularization, and extensive development. China became the first country to commercialize hybrid rice. In recent years, the yearly area under hybrid rice has been about 15.5 million ha, which accounts for 50% of the total rice area, and hybrid rice production occupies nearly 60% of China s total rice production. The yearly rice production added by hybrid rice can feed another 60 million people. From 1976 to 2001, the accumulated planting area of hybrid rice was 270 million ha and the added paddy from hybrid rice was 380 million tons. In addition, about 1,000 enterprises work with hybrid rice in China, which yearly provide 100,000 direct employment opportunities and 1 million indirect employment opportunities. Hybrid rice played a very important role in Chinese food security as well as improving rice farmers living conditions in the past and we are sure that China can feed its almost 1.3 billion people in the future. The introduction of three-line hybrid rice development Completion of the three-line hybrid rice system within 10 years In 1964, Yuan Longping first put forward the idea of using the heterosis in rice and began research on hybrid rice in China. In November 1970, a pollen abortive wild rice plant (called wild abortive, WA, thereafter) was discovered among the plants of common wild rice in southern China. This discovery resulted in the subsequent breakthrough in hybrid rice development. In 1972, the first group of cytoplasmic male sterile (CMS) lines such as Erjiunan 1A, Zhenshan 97A, and V20A was developed by using WA as the donor of male sterile genes. In 1973, the first group of restorer lines such as Taiyin 1, IR24, and IR661 was screened out through the direct testcrossing method. The first rice hybrid developed by Professor Yuan, Nanyou 2, was released in 1974 and showed very strong heterosis, with more than a 20% yield advantage over the best conventional rice varieties of that time. Rapid optimization of hybrid rice seed production for commercialization When the first rice hybrid was released, the technology of hybrid rice seed production was the key problem for commercialization because the yield of hybrid rice seed production was very low, and the average yield in China was only kg ha 1 in Fortunately, many agronomists shifted their focus to that problem and the tech- 248 Ma Guohui and Yuan Longping

233 Yield (t ha 1 ) China Hunan Province Year Fig. 1. Yearly variation in the yield of hybrid rice seed production in China and Hunan Province. nology package for high-yielding seed production was developed within a few years, resulting in the yield of hybrid rice seed production increasing greatly and rapidly (Fig. 1). In 1985, the national average yield increased to 1.66 t ha 1, then reached 2.25 t ha 1 in In Hunan Province, yield on a large scale increased to t ha 1 in the 1990s from 324 kg ha 1 in 1976 and the highest recorded yield of 7.4 t ha 1 was made on a small plot (0.113 ha) at Zixing City, Hunan Province, in With the yield increase in multiplication and seed production, the area ratio of A line multiplication to F 1 hybrid seed production to F 1 hybrid commercial production has changed from 1:30:1,000 in the 1970s to 1:50:6,000 in recent years, which greatly lowers the cost of hybrid seeds. Grain yield variation and differences between hybrid and conventional rice in China When hybrid rice was first popularized, its national average grain yield increased to 4.2 t ha 1 in 1976, which was 0.73 t ha 1 higher than the yield of conventional rice. The yield of hybrid rice in 1988 reached 6.6 t ha 1 in China (Fig. 2), 2.06 t ha 1 higher than the yield of conventional rice. For the national average yield in commercial production, hybrid rice has a yield advantage of more than 20% over conventional rice. Hybrid rice achievements and development in China 249

234 Yield (t ha 1 ) 7 China Hunan Province Year Fig. 2. Grain yield differences between hybrid and conventional rice in China. Total hybrid rice area variation in China With the technology of hybrid rice seed production basically developed in 1975, hybrid rice was rapidly and widely extended in China and its planted area rose steeply to 138,700 ha in 1976 from 373 ha in 1975 and 7 ha in 1974 (Fig. 3). After that, the area under hybrid rice continued to increase and its per unit area grain yield became higher and higher. In 1991, hybrid rice area reached 17.6 million ha, accounting for 54% of the total rice area. In recent years, the annual area under hybrid rice has been about 15.5 million ha, which accounts for 50% of the total rice area, and hybrid rice production occupies nearly 60% of the total rice production in China. From 1976 to 1999 (Fig. 3), the accumulated planted area of hybrid rice was 270 million ha. Up to now, hybrid rice has covered indica and japonica types with different maturity, which made hybrid rice production possible throughout China from Hainan in the south to Liaoning in the north and from Shanghai in the east to Yunnan in the west. Hybrid rice shows not only high yielding ability but also wide adaptability. Reasons for the success of hybrid rice in China First of all, the Chinese government paid much attention to developing hybrid rice and the heads of the government at all levels were very much committed to and 250 Ma Guohui and Yuan Longping

235 Area (million ha) Year Fig. 3. Hybrid rice area in China supportive of hybrid rice research and development, such as special budget and policy support provided when needed. Second, a very efficient organization and coordination had been established in the 1970s-1980s, such as the National Hybrid Rice Advisory Committee (NHRAC) and the hybrid rice leading group or office at different levels. NHRAC coordinated the relations between technology development and dissemination activities. At the same time, frequent conferences on hybrid rice were held to discuss and solve the serious problems encountered in the initial stages. Third, targeting the real technical problems, financially supported by the government and organized by the networks, and with applied and strategic studies, extension activities were carried out in each 5-year period. Training, yield trials, onfarm demonstrations, study tours, and propaganda for hybrid rice were extensively organized in the initial stage in the entire country. The government put large efforts into producing a large amount of hybrid rice seeds at Hainan Island in the off-season to widely demonstrate hybrid rice in the whole country. Hybrid rice technology was immediately incorporated into a textbook for agricultural education in the early 1970s. Last, the policy dealing with hybrid rice development played a very important role also, such as the subsidy in the initial time, the national standard of hybrid rice seed quality, and the hybrid seed law implemented. Hybrid rice achievements and development in China 251

236 Two-line hybrid rice development and progress The strategic breeding idea of using heterosis The first natural male sterile rice plant was discovered by Chinese scientist Shi Mingsun in Hubei in 1973 and he developed the rice male sterile line Nongken 58S with a dual purpose in The dual-purpose line is also called a photoperiod- and thermosensitive genic male sterile (PTGMS) line because its fertility expression is regulated by daylength and temperature. The discovery of the dual-purpose line or PTGMS line indicated a new way to use heterosis in hybrid rice with the two-line system. With reports on the discovery of the PTGMS gene and the discovery of a wide compatibility (WC) gene by Japanese scientists (Ikehashi et al) in 1984, Professor Yuan put forward the strategic breeding idea of using heterosis in hybrid rice breeding. The approach was to use heterosis by means of the systems from the three-line, two-line, and one-line methodology and from intervarieties to improve heterosis by means of intervarietal, intersubspecific, and distant hybrids. PGMS and TGMS development After the discovery of Nongken 58S, nationwide research was carried out on the mechanism of PTGMS and its application. Soon afterward, a group of japonica and indica PTGMS lines was transferred from the original Nongken 58S. Furthermore, some other germplasm materials with fertility alteration such as Annong S-1, 5460S, and Hengnong S-1 were found. Unfortunately, Nongken 58S, selected from a japonica variety, possessed the characteristics of both daylength and high-temperature sensitivity so that most of the PTGMS lines could not be used for commercial two-line hybrid rice development. At that moment, as a chief scientist and national coordinator, Professor Yuan focused on the most serious problems in research on two-line hybrid rice. He proposed a new and very efficient concept to develop TGMS lines with the characteristic of low critical sterility-inducing temperature (CSIT) for safe hybrid seed production and then he designed the process to produce the key foundation seeds of PGMS lines. Now, Chinese scientists have developed more than ten viable PTGMS lines in rice that possess the characteristics of low CSIT and safe hybrid seed production and that have been technically identified and approved. Progress in two-line hybrid rice With the two-line hybrid rice combination Peiliangyou Teqing (Pei ai 64S/Teqing) developed and released for commercial production in 1994 and the corresponding complete set of application techniques, including the procedure of foundation seed production and multiplication of TGMS lines with low CSIT, and high-yielding techniques for hybrid seed production matured, the success of two-line hybrid rice research was declared in 1995 and two-line hybrid rice began to be widely extended in commercial production in China. Up to now, more than 20 two-line hybrids have been developed and released to farmers fields. They show high yielding ability, bet- 252 Ma Guohui and Yuan Longping

237 Year , Area (000 ha) Fig. 4. Area of two-line hybrid rice in China. ter grain quality, and good pest resistance. Since the declaration of success for twoline hybrid rice research in 1995, the area planted to two-line hybrid rice has been increasing year after year (Fig. 4). For instance, the annual area of two-line hybrid rice was 73,000 ha in China in 1995, while in 2001 it increased to 2.67 million ha, about 17.2% of the total hybrid rice area. In general, two-line hybrid rice shows a yield increase of 5 10% compared with the current three-line hybrid rice. Furthermore, the two-line method shows promise for developing elite hybrids with both high yield and early maturity for early rice cropping and for developing hybrids with stronger heterosis for japonica rice, which would very likely overcome the bottleneck of stagnant yield and area in hybrid rice. In addition, Xiangliangyou 68, one of the examples, is an early two-line hybrid rice combination with high yield, fine grain quality, and early maturity, which was released for commercial production in It shows promising prospects for overcoming the great difficulty existing for a long time in developing early hybrid rice combined with high yield, good grain quality, and early maturity in China. Constraints and challenges The following constraints and challenges exist: The yield of three-line hybrids has been stagnant for years Since the yield of hybrid rice reached 6.6 t ha 1 in 1986, it has remained at the same level for many years (Fig. 1). It seems that it is very difficult to further increase the yield of hybrid rice if no new genetic resources are used and no new methods are exploited in hybrid rice breeding. Hybrid rice achievements and development in China 253

238 Strategies Area has been at a standstill for years In 1991, hybrid rice area reached its peak at 17.6 million ha, but after that the area decreased and has stayed at about 15.5 million ha. The main reasons are considered to be a decrease in the area of double cropping of early hybrid rice and japonica hybrid rice. Another reason is that the cropping system has been regulated by both the local government and farmers themselves to improve their farming income in the past 10 years such as cash crops or fish pools instead of growing rice. Lack of japonica hybrid rice with strong heterosis The planting area of japonica hybrid rice has been limited to around 100,000 ha, accounting for only 1 2% of total japonica rice in China for many years because of its relatively poor heterosis (about 10% over conventional japonica rice), the sterility of its CMS lines is not stable enough to produce high purity of F 1 seeds, and it has poorer grain quality. Grain quality of hybrid rice needs improvement With the increasing living standards of rice consumers in China, it is necessary to improve the grain quality of rice. In comparison with conventional rice, hybrid rice shows poorer grain quality in head rice recovery and chalkiness. How to develop rice hybrids with both high yield and good grain quality is still a challenge for breeders. The sources of male sterility-inducing cytoplasm for developing better CMS lines are poor Currently, more than 85% of the CMS lines used in commercial production belong to WA types. This dominant cytoplasm in the existing threeline hybrid rice could lead to incidence of a destructive pest. On the basis of studies of Chinese scientists, the following breeding strategy for developing hybrid rice has been put forward by Professor Yuan. First, in terms of breeding methodology, there could be three approaches: (1) the three-line method or CMS system, (2) the two-line method or PGMS/TGMS system, and (3) the one-line method or apomixis system. Second, from the viewpoint of increasing heterosis, its exploitation in rice could also be divided into three levels: intervarietal hybrids, intersubspecific hybrids, and interspecific or intergeneric hybrids (distant hybrids). Making three-line hybrid rice continuously play an important role The existing rice hybrids predominantly used in commercial production belong to the category of intervarietal hybrids by the three-line method. It has been proven that the three-line method is an effective way to develop hybrid combinations and it will continue to play an important role in hybrid rice development. 254 Ma Guohui and Yuan Longping

239 Developing intersubspecific hybrid rice or super hybrid rice Research findings have proven that intersubspecific hybrids have a much stronger heterosis than intrasubspecific hybrids because of the greater genetic distance between their parents. They generally have taller plants, larger panicles, and a stronger root system. Theoretically, the yield potential of intersubspecific hybrid rice is more than 30% higher than that of intervarietal hybrid rice. Therefore, the use of heterosis in intersubspecific hybrid rice is a promising way to further increase rice yield. However, there are some barriers, such as plants that have a low seed-setting rate, are too tall, mature too late, have poorly filled grains, and have poor grain quality commonly found in typical indica/japonica intersubspecific hybrids. For the first three problems, effective ways have been found to solve them: the use of wide compatibility (WC) genes, the transfer of the allelic dwarf gene, and the careful choice of crossing parental lines with suitable maturity. For the last two problems, the idea to emphasize developing indica/javanica hybrids in indica rice-growing regions and japonica/ javanica hybrids in japonica rice-growing areas rather than developing indica/japonica hybrids has been proposed. Since research on two-line intersubspecific hybrid rice was included in the key national program 863 Project in 1987, through more than ten years of research, great progress has been achieved in two-line intersubspecific hybrid rice. Some elite WC lines and some promising intersubspecific hybrid rice combinations have been developed and these hyrids are now under various trials and experimental production. On the basis of the great progress achieved in two-line intersubspecific hybrid rice breeding, Professor Yuan proposed the Chinese super hybrid rice breeding program in The ultimate target is to develop hybrid rice with a daily grain yield of 100 kg ha 1, that is, 12 t ha 1 for hybrids with a medium maturity of 120 days. He proposed a new model of plant type for super hybrid rice and the technical approaches to achieve the target. The most important in super hybrid rice breeding is to combine the ideal plant type with the use of heterosis of intersubspecific hybrids. Since the program began, great progress has been made. Up to now, promising hybrids with super-high yield potential such as Pei ai 64S/E32 and Liangyou Peijiu (Pei ai 64S/9311) have been developed. In 2000, only in Hunan Province, there were eighteen 6.7-ha locations and four 66.7-ha locations under Liangyou Peijiu, where the average yield was higher than 10.5 t ha 1. Another combination, Pei ai 64S/E32, had two 6.7-ha locations with average yield above 9.75 and 10.5 t ha 1 grown as late-crop rice and one 66.7-ha location with yield up to t ha 1 grown as a main crop plus ratoon crop in Hunan in A record yield of t ha 1 for single-crop rice was achieved under a small plot (487 m 2 ) at Yongsheng, Yunnan Province, in According to the results from the large-scale production demonstrations, super hybrid rice shows a large yield-increasing capacity, generally 2.25 t ha 1 higher than that of the existing three-line hybrid rice. Super hybrid rice is expected to play a very important role in making a leap ahead in increasing rice yield in the 21st century. Hybrid rice achievements and development in China 255

240 The use of favorable genes In 1995, in a cooperative research program with Cornell University, based on molecular analysis and field experiments, we identified two favorable quantitative trait loci (QTL) genes (yld1 and yld2) from the wild rice (Oryza rufipogon L.). Each of the QTL genes contributed to a yield advantage of 18% over the high-yielding hybrid V64 (one of the most elite hybrids in China, with a yield potential of 75 kg ha 1 day 1 ). By means of molecular marker-facilitated backcrossing and selection, creating near-isogenic lines carrying these two QTL genes is under way. Through large-scale genome sequencing, the genome working draft for indica and japonica has been obtained. Research on genome function has been conducted in cooperation by CNHRRD and the Huda Gene Research Institute since Developing one-line hybrid rice The utmost target in hybrid rice is to develop a one-line system of hybrid rice, which is to breed a true-breeding heterotic F 1 hybrid, that is, the nonsegregating F 1 hybrid, so that it is not necessary to conduct hybrid seed production year after year, which means fixing heterosis. At present, it seems that the use of apomixis to develop a true-breeding F 1 hybrid may be a more promising method. The research program on apomixis began in the late 1980s in China, but it is still in a tentative stage. The development of apomictic rice may require biotechnology as well as traditional breeding. Notes Authors address: China National Hybrid Rice Research and Development Center. Citation: Virmani SS, Mao CX, Hardy B, editors Hybrid rice for food security, poverty alleviation, and environmental protection. Proceedings of the 4th International Symposium on Hybrid Rice, Hanoi, Vietnam, May Los Baños (Philippines): International Rice Research Institute. 407 p. 256 Ma Guohui and Yuan Longping

241 Hybrid rice research and development in Egypt A.O. Bastawisi, H.F. El-Mowafi, M.I. Abo Yousef, A.E. Draz, I.R. Aidy, M.A. Maximos, and A.T. Badawi Egypt reached its ceiling in rice productivity in Although the national average yield in 2001 was 9.3 t ha 1, research trials showed an increase in the productivity of hybrids ranging from 15% to 20% in normal soils and from 20% to 30% in saline soils over inbreds. Five Egyptian hybrid combinations were recognized under multilocation yield trials; some of these are highly adapted to normal soils, some to saline soils, and others to both. The two-line system (PGMS and TGMS) is in the testcrossing stage. Nucleus seed for initial seed production and purification in three hybrid combinations was started. In the training of human resources, 32 researchers were trained on the scientific basis and applied technology for hybrid rice breeding and seed production. By the end of 2002, extension and seed production specialists, seed growers, and farmers will have been trained. The economic evaluation and advantage of hybrids vis-à-vis inbreds are discussed. Rice in Egypt is grown in the North Nile Delta, with an area ranging from 0.5 to 0.6 million ha annually under a controlled irrigation system. In 2001, the national average yield was 9.3 t ha 1, with a technology gap of 20% between attainable and actual national yield. Since 1987, Egypt has reached its productivity ceiling under research experiments by introducing high-yielding semidwarf rice varieties. To surpass this productivity ceiling, hybrid vigor was recognized as one realizable technological option. Our research trials on yield showed an increase in productivity of hybrids ranging from 15% to 20% in normal soils and from 20% to 30% in saline soils vis-àvis that of inbreds. A preliminary study showed that increasing the yield average by 20% would bring about extra net income of 50% to Egyptian rice growers. This increase in average yield would achieve food security and alleviate poverty. At the same time, since a hybrid variety is a product of two varieties, it is expected that the degree of resistance to and tolerance of biotic and abiotic stresses will be more than 257

242 that of the inbred variety, which allows less consumption of chemicals, which would lead to environmental protection. Hybrid rice research started in Egypt in 1982 using Chinese CMS lines and Egyptian japonica cultivars as male parents, which had poor restoring ability. The evaluation of exotic hybrids started in 1986 and these were inferior to the local inbreds mainly because of poor adaptation and lateness (Maximos and Aidy 1994). The systematic hybrid rice research program began in 1995 (Bastawisi 1998). The ATUT Project on Enhancement of Hybrid Rice Research and Development in Egypt (R 016), jointly sponsored by the Government of Egypt, the International Rice Research Institute (IRRI), and the USDA-ARS-University of California, added strength to the national initiative, resulting in the development of valuable breeding material in the following three years, ending with the recognition of good hybrid combinations. It was at this juncture that the program gained further strength through the FAO-sponsored project on Training in Hybrid Technology Through Technical Cooperation Between Developing Countries (TCP/EGY/8923) launched in 2000 to train human resources and formulate a medium-term program for the sustainable development and use of hybrid rice in Egypt. This paper summarizes the current status of research and development for hybrid rice technology in Egypt. Evaluation of experimental hybrids Hybrid combinations are evaluated under four kinds of experiments: advanced yield trials (AHRT), preliminary yield trials (PHRT), observational yield trials (OHRT), and combining ability experiments (CA). The best hybrid combinations in each experiment are promoted to the next yield trial in the following season. Under advanced yield trials in 2001, five hybrid combinations and the local inbred check variety Giza 178 were grown in a randomized complete block design (RCBD) in four replications at four locations in two environments: (1) normal soil at three locations Sakha, Gemmiza, and Zarzoura; (2) saline soil at one location El- Sirw. These four sites included four rice-growing governorates across the North Nile Delta. Yield, days to heading, height, and standard heterosis of these promising hybrids are presented in Table 1 for normal and saline environments. As we can observe, some hybrids are well adapted for normal soil, some are adapted for saline soil, and some are adapted for both. Two or three of these hybrid combinations are being promoted for demonstration fields in the 2002 season after passing grain quality and panel taste tests. Under the other yield trials, 16 hybrids (Table 2) were identified as being the most promising. After passing the evaluation for disease and pest resistance and grain quality, they will be promoted to the AHRT in the coming season. 258 Bastawisi et al

243 Table 1. Promising hybrids identified in advanced yield trials, Rank Hybrid/check Days Plant Yield Yield Standard to height (t ha 1 ) versus heterosis a heading (cm) local inbred (%) (t ha 1 ) Under normal conditions at three locations (Sakha, Gemmiza, and Zarzoura) 1 SK 2046 H ** 2 SK 2034 H ** 3 SK 2058 H ** 4 SK 2035 H * 5 SK 2029 H * Check Giza Under saline conditions (El-Sirw) 1 SK2035 H ** 2 SK 2029 H * 3 SK 2058 H SK 2046 H SK 2034 H Check Giza a Standard heterosis = [Hybrid local inbred check (Giza 178)] 100/local inbred check. *, ** = significant at 5% and 1% probability levels, respectively. Table 2. Hybrid combinations having an impressive crop stand in various trials during Designation SK 2003 H SK 2005 H SK 2010 H SK 2025 H SK 2029 H SK 2033 H SK 2034 H SK 2037 H SK 2046 H SK 2047 H SK 2051 H SK 2053 H SK 2058 H SK 2063 H SK 2069 H SK 2072 H Parentage G 46A/Giza 178R G 46A/Giza 181R G 46A/Giza 182R IR58025A/Giza 178R IR68888A/Giza 178R IR68902A/Giza 178R IR69625A/Giza 178R IR58025A/Giza 181R IR69625A/Giza 181R IR70368A/Giza 181R IR68885A/Giza 182R IR68888A/Giza 182R IR69625A/Giza 182R G 46A/IR25571R IR68888A/GZ 5121R IR68899A/GZ 5121R Country report for Egypt 259

244 Table 3. List of stable CMS lines being used for testcrossing and hybrid seed production. CMS line Cytosterility Grain Amylose Outcrossing source b type c content (%) (%) IR58025 A a WA L IR62829 A WA L IR75602 A Gambiaca M IR73796 A WA S IR68884 A WA M IR68885 A IR62829A Mutant L IR68886 A WA L IR68888 A* WA L IR68897 A WA L IR68899 A WA L IR68902 A WA L IR69625 A* WA M IR70368 A* WA M IR70369 A WA ML IR72079 A WA L IR75600 A Gambiaca M IR75601 A Gambiaca L D 297 A Dissi L CRMS 21 A Kalinga L G 46 A* Gambiaca MB Large Stigma A Kalinga LB a Highly adapted under Egypt environment. b WA = wild abortive. c L = long, M = medium, S = short, ML = medium long, MB = medium bold. Identification and development of parental lines All lines were testcrossed and screened from 1996 to Of these, 425 (28%) were found to be complete maintainers and 530 (35%) complete restorers. Year-wise from 1996 to 2001, the maintainers were, respectively, 15, 93, 78, 29, 119, and 91, whereas the restorers were 12, 28, 49, 127, 193, and 121. The availability of a high percentage of restorer lines is highly encouraging. The conversion of potential maintainers possessing desirable floral traits into new CMS lines was in progress. Out of 12 BC 5 containing 142 lines in the 2001 season, 11 crosses containing 120 lines were selected for the BC 6 in the 2002 season for the purpose of transferring cytoplasmic male sterility to our local Egyptian maintainers such as Giza 177, Sakha 101, GZ , GZ , and GZ Eleven CMS lines introduced from IRRI and elsewhere (Table 3) are now being used in testcrossing and are being multiplied for hybrid seed production. The maintenance, purification, and multiplication of such a large number of lines with limited resources are a difficult task. Therefore, based on grain shape, amylose content, agronomic characteristics, combining ability, floral traits for outcrossing, per- 260 Bastawisi et al

245 formance of their hybrids, and stability, four CMS lines IR69625A, IR68888A, IR58025A, and G 46 A have been selected for large-scale multiplication, purification, and use. Preference would be given to locally developed CMS lines in the background of popular varieties Sakha 101 (BC-5), Giza 177 (BC-5), GZ , GZ , and GZ (BC-5). Of these, Giza 177 A and Sakha 101 A are at the final stages of backcrossing. The other CMS lines having stable characteristics could be maintained in a CMS maintenance nursery using handcrossed seeds for eventual use. These 61 additional CMS lines from various sources are also being maintained. These need to be screened to select desirable lines. Some of the promising CMS lines were identified for the hybrid rice breeding program. The CMS lines identified are listed in Table 3. Two-line hybrid breeding Both the TGMS and PGMS systems of two-line hybrid breeding hold great promise in Egypt. Environmental conditions are highly favorable for both systems. However, this would require the development of much basic information before it could be translated into a commercial reality, such as determining the critical sterility/fertility temperature/daylength, determining the correct period for hybrid seed production and EGMS multiplication using meteorological data, and conducting experiments at prospective locations and converting desirable lines into usable TGMS/PGMS lines, preferably with the wide compatibility gene. The delta area having a low mean temperature is suitable for maintenance and seed multiplication of male sterile lines, whereas the new valley area having high temperature is suitable for hybrid seed production. In the delta area, where from 20 May to 20 July the daylength exceeds h and from 10 June to 30 June it is more than 14 hours, this period can be used for hybrid seed production. Similarly, from 23 August onward, the daylength is less than 13 h and the area can be used for the multiplication of PGMS lines. Some TGMS lines introduced from IRRI as well as some segregating populations with the Egyptian background, such as Giza 178, GZ , Giza 177, and Sakha 101 crossed with Norin PL 12 and TGMS line IR are now being screened simultaneously in both environments to identify appropriate lines. These will be studied for their critical sterility-fertility points. Of the 273 F 5 lines received from IRRI under the Egypt-IRRI shuttle breeding program, only in 13 lines have sterile plants been recovered. These will be further studied for their sterilityfertility transformation and other details using glasshouse and phytotron facilities. From among the 92 PGMS lines being screened at Sakha, 21 anther-derived lines have been found to be uniform and completely sterile and they are being studied for their fertility transformation. If they are suitable, the selected lines will be multiplied and used for testcrossing in the 2002 season. Country report for Egypt 261

246 Seed production of experimental hybrids and parental lines Eleven CMS lines and their maintainers were grown in isolated plots surrounded by maize and sunflower plots with 10 A:3 B, 8 A:2 B, and 6 A:2 B row ratios. Grain yield ranged from 1.5 to 3.8 t ha 1 and outcrossing % ranged from 15.6% to 35.0%. Twenty-two hybrid combinations using five restorers Giza 178R, Giza 181R, Giza 182R, GZ 5121R, and IR25571R and 10 CMS lines were grown in isolated plots with row ratios of 10 A:3 R, 8 A:2 R, and 6 A:2 R. The range of outcrossing and yield varied greatly because of the synchronization problem in some cross combinations. The ratio of outcrossing ranged from 10.5% to 50.0%, whereas yield ranged from 1.0 to 3.8 t ha 1. Initially, seed yields obtained for CMS multiplication and hybrid seed were less than t ha 1 but now routinely average 2.0 to 3.8 t ha 1. A maximum seed yield of 4 t ha 1 has been recorded in a seed production plot of IR69625A/B and IR69625A/GZ 5121R. The best hybrid combinations will be selected for preliminary and multilocation yield trials in the 2002 season. Seed production of commercial hybrids Three CMS lines, IR58025A, IR69625A, and IR70368A, along with their maintainers were purified and increased, along with three promising hybrid combinations IR58025A/Giza 178R, IR69625A/Giza 182R, and IR70368A/Giza 181R. Techniques for hybrid rice seed production were used such as adjusting flowering, roguing, GA 3 application, flag-leaf clipping, and supplementary pollination. The seed parent yielded 2.2 to 2.3 t ha 1 and had seed set of 34% to 42%, whereas the F 1 hybrid seeds yielded 2.4 to 3.6 t ha 1 and had seed sets of 31% to 50%. Training of human resources In 2001, 32 researchers and hybrid rice technicians were trained for 1 week on hybrid rice breeding and seed production under the supervision of Technical Cooperation among Developing Countries of FAO consultants (a breeder and a seed production specialist). By the next crop season (2002), we expect that some hybrids will be available for on-farm evaluation, wherein the role of extension and seed production specialists, seed growers, and farmers is important. Two courses will be included: hybrid rice seed production and hybrid rice cultivation. Economic evaluation Under the Egyptian experimental evaluation for inbred and hybrid rice, agricultural inputs such as fertilizer, herbicide, insecticide, and manual and mechanical power are the same. The only difference is the cost of seeds. The seed rates for rice inbreds are from 120 to 144 kg ha 1, whereas the expected seed rate under Egyptian condi- 262 Bastawisi et al

247 tions is 50 kg ha 1 for hybrids. If we remember that the cost of 1 kg of hybrid rice seed is five times that of inbreds, the seed cost of hybrids will be double that of inbreds. This difference can be covered by the 15 20% extra yield of hybrids. However, since demonstration and on-farm yield trials will start in the 2002 season, one agricultural economist will be assigned during the summer to study the economics of hybrid rice cultivation versus the cultivation of inbreds. At the same time, the economist will study the economics of hybrid rice seed production for seed companies and seed growers. References Bastawisi AO, Aidy IR, El-Mowafy HF, Maximos MA Research and development for hybrid rice technology in Egypt. In: Advances in hybrid rice technology. Proceedings of the 3rd International Symposium, Nov. 1996, Hyderabad, India. Manila (Philippines): International Rice Research Institute. p Maximos MA, Aidy IR Hybrid rice research in Egypt. In: Virmani SS, editor. Hybrid rice technology: new developments and future prospects. Manila (Philippines): International Rice Research Institute. p Notes Authors address: Agricultural Research Center, Rice Research and Training Center (RRTC), Sakha, Kafr El-Sheikh, Egypt. Citation: Virmani SS, Mao CX, Hardy B, editors Hybrid rice for food security, poverty alleviation, and environmental protection. Proceedings of the 4th International Symposium on Hybrid Rice, Hanoi, Vietnam, May Los Baños (Philippines): International Rice Research Institute. 407 p. Country report for Egypt 263

248 Hybrid rice development and use in India B. Mishra, B.C. Viraktamath, M. Ilyas Ahmed, M.S. Ramesha, and C.H.M. Vijayakumar Recognizing the potential of hybrid rice to enhance production and productivity, India launched a national program in 1989 for the development and large-scale adoption of hybrid rice to sustain self-sufficiency in rice production. As a result of intensive efforts to develop and evaluate hybrids during the last decade, 16 hybrids have been released for commercial cultivation. A systematic multilocational evaluation of released hybrids during three seasons indicated that six were high-yielding and widely adaptable. These, in the order of their performance, are KRH-2, PHB-71, Sahyadri, PA6201, NSD-2, and DRRH-1. A significant achievement has been the development of the first super-fine-grain aromatic hybrid, Pusa RH-10. This hybrid has a 40% yield advantage over the highest-yielding basmati check variety, Pusa Basmati- 1, with comparable grain and cooking quality characteristics. Many promising hybrids with enhanced magnitude of heterosis and better grain quality are in the pipeline. Farmer adoption of hybrid rice has been much slower than expected because of several constraints. India is now estimated to have around 200,000 hectares under hybrid rice annually. With the added emphasis on parental line improvement through recombination breeding and population improvement approaches by developing and using intra- and intersubspecific germplasm, more than 3,000 genetically diverse promising derivatives have been developed and are being used to develop experimental hybrids. In recent years, many promising hybrids identified are based on indigenously developed CMS lines such as PMS 10A, PMS 12A, Pusa 6A, CRMS 31A, DRR 2A, and APMS 6A. New TGMS lines developed through recombination breeding are at the final stage of evaluation (F 6 generation). Initial evaluation of intersubspecific hybrids involving indica/tropical japonica derivatives indicated enhanced heterosis of 5 10% over indica/indica hybrids. Refinement of the seed production package has resulted in average seed yields improved from 1.5 to 2.0 t ha 1 by experienced seed growers in large-scale seed production. Effective public- and private-sector partnership in seed production has been a key to the successful adoption of this technology. Of the estimated 3,000 t of hybrid seed produced in the country annually, more than 90% is produced by the private seed sector. 265

249 Efforts to transfer the technology already developed, through extensive compact block front-line demonstrations and training programs, has created awareness and motivated farmers to begin adopting hybrid rice in target states. Policymakers and senior research managers have been sensitized and the government has planned to popularize hybrids on a priority basis during the tenth five-year plan period ( ). Improving grain and cooking quality characteristics of hybrids, incorporating resistance to some major pests and diseases, enhancing the magnitude of heterosis, and further increasing average seed yields to 2.5 t ha 1 on a large scale to reduce seed costs are the research priorities during the next five years. Policy interventions by the government for increased support, aggressive popularization of hybrids, and assured procurement of hybrid rice at a minimum support price are needed. If these problems can be solved, hybrid rice could be cultivated on 3 4 million hectares in India during the next decade to partially sustain food security. The Hybrid Rice Program in India was launched in During the past decade, 16 hybrids have been released. Multilocational evaluation of released hybrids over three seasons indicated the hybrids KRH-2, PHB-71, Sahyadri, PA 6201, NSD-2, and DRRH-1 to be high yielding and widely adaptable. A significant achievement has been the release of the first super-fine-grain aromatic hybrid, Pusa RH-10. Area under hybrid rice is now estimated to be around 200,000 hectares annually. Specific breeding efforts for the development of parental lines with desirable characteristics have resulted in 500 genetically diverse derived lines. The frequency of restorers and maintainers in derived lines is considerably high. New diversified CMS lines have been developed in the cytoplasmic background of Oryza nivara and O. rufipogon. To enhance the magnitude of heterosis, research has begun on two-line breeding. It is estimated that annually 4,000 tons of hybrid seed are produced in the country, 95% of it by the private seed sector. Average seed yields are 1.5 to 2.0 t ha 1. To popularize hybrid rice, many compact block front-line demonstrations and training programs are being conducted. Improvement of grain and cooking quality characteristics, enhancement of the magnitude of heterosis, incorporation of resistance to major pests and diseases, and further increasing average seed yields to 2.5 t ha 1 are some of the research priorities for the next five years. During the past 50 years, rice area in India has increased from 30 million to 45 million hectares. The annual production of milled rice has gone up from 22 million to 88 million tons, while productivity has increased from 0.7 to 1.9 t ha 1. Despite this considerable progress, to sustain self-sufficiency in rice at the present rate of population growth, production needs to increase by 1.5 million tons of milled rice every year. 266 Mishra et al

250 The development and adoption of hybrid rice are one of the feasible approaches to achieving the targeted increase in production. If suitable hybrids are developed and popularized among farmers, at least 10 million ha out of 20 million ha of irrigated area in the country form a potential area for hybrid rice cultivation. Isolated, scattered, and academic research efforts on hybrid rice, initiated during the 1970s, were consolidated into a goal-oriented, time-bound mission-mode project by the Indian Council of Agricultural Research (ICAR) in Technical backstopping by the International Rice Research Institute (IRRI) and the Food and Agriculture Organization of the United Nations (FAO) and financial support from the United Nations Development Programme (UNDP) and Mahyco Research Foundation (MRF) considerably strengthened the project. Now, the hybrid rice program in India operates as a national network, comprising 12 research network centers across the country, and is being coordinated by the Directorate of Rice Research (DRR) in Hyderabad. Besides the 12 research centers, the network also involves public- and private-sector seed agencies, nongovernment organizations (NGOs), and departments of agriculture of the target states. Effective collaboration exists with international institutes/organizations. Progress made during earlier years has been described by Siddiq et al (1998). This report briefly covers the significant progress made during Hybrids released Five hybrids PHB-71, 6201, HRI-120, Pusa RH-10, and KRH-2 were released by the Central Variety Release Committee and six hybrids DRRH-1, KRH-2, Sahyadri, CORH-2, PSD-1, and NSD-2 were released by the respective State Variety Release Committees during the period. Table 1 gives the salient features of these hybrids. Pusa RH-10 was the first fine-grain, aromatic, basmati-like hybrid released in the country during This hybrid has a 40% yield advantage over the check Pusa Basmati-1, with comparable grain and cooking quality. Large-scale seed production of this hybrid has begun. This hybrid may occupy a sizable area in the traditional basmati zone of northwestern India. Among these hybrids, the promising ones such as KRH-2, PHB-71, Sahyadri, PA 6201, NSD-2, and DRRH-1 are being cultivated in target states. Based on the hybrid seed produced and sold, it is estimated that 200,000 hectares are planted annually to hybrid rice in the country. The adoption of hybrid rice has been much slower than expected because of several constraints, which are discussed elsewhere in this paper. Multilocational evaluation of released hybrids To make a comparative evaluation of hybrids released by the Central and State Variety Release Committees and to get information about their adaptability in different states, a multilocational evaluation of 11 released hybrids was made. These hybrids Hybrid rice development and use in India 267

251 Table 1. Hybrids released in India. Name of hybrid Year of Released Cross Duration Yield in OFT a (t ha 1 ) Yield Released for release by (d) advantage Hybrid Check over check (%) 268 Mishra et al DRRH DRR, Hyderabad R58025A/ Telangana and Rayalaseema IR40750 (Tellahamsa) regions of Andhra Pradesh KRH-2 b 1996 UAS, Bangalore IR58025A/ Irrigated regions of south, KMR-3 (Jaya) west, and eastern zones Pant Sankar Dhan GBPUAT, IR58025A/UPRI Western area and plains of Uttar Pantnagar (Pant Dhan-4) Pradesh and Uttaranchal CORH TNAU, Coimbatore IR58025A/ Irrigated regions of Tamil Nadu C20R (ADT 39) ADTRH TNAU, Aduthurai IR58025A/IR Irrigated regions of Tamil Nadu (ASD-18) Sahyadri 1998 KKV, Karjat IR58025A/ Konkan region of Maharashtra BR (Jaya) Narendra Sankar Dhan NDUAT, Faizabad IR58025A/ Eastern parts of Uttar Pradesh NDR3026 (Sarjoo-52) PHB 71 b 1997 POC, Hyderabad Haryana, Uttar Pradesh, Tamil (PR 106) Nadu PA 6201 b 2000 HRI, Hyderabad Eastern and some parts (Jaya) of southern India HRI-120 b 2001 HRI, Hyderabad Telangana region of Andhra (Jaya) Pradesh, Karnataka, Konkan region of Maharashtra, plains of Uttaranchal, eastern Uttar Pradesh, Orissa, and Tripura Pusa RH-10 b 2001 IARI, New Delhi Pusa 6A/PRR Haryana, Delhi, and Uttaranchal (Pusa Bas.1) a OFT = on-farm trials. b Hybrids released by CVRC.

252 Table 2. Mean grain yield (kg ha 1 ) of released hybrids in multilocation trials. Hybrid/ Wet season Dry season Wet season Mean check (125 locations) (64 locations) a (15 locations) (46 locations) KRH-2 5,538 (1) 6,146 (2) 5,285 (1) 5,518 (1) PHB-71 5,287 (2) 6,101 (3) 5,179 (2) 5,345 (2) PA ,221 (3) 5,980 4,920 (4) 5,201 (4) NSD-2 5,211 (4) 6,007 (5) 4,851 5,174 (5) Sahyadri 5,175 (5) 6,329 (1) 5,115 (3) 5,291 (3) DRRH-1 4,895 5,971 4,880 (5) 5,019 CORH-2 4,884 6,072 (4) 4,717 4,965 ADTHR-1 4,559 5,122 4,380 4,561 CNRH-3 4,256 5,168 4,158 4,329 PSD-1 4,546 5,851 4,401 4,649 APHR-2 4,875 5,377 4,747 4,888 Highest 4,465 5,219 4,620 4,613 national check (Jaya) (Sasyasree) (Jaya) Highest local 4,770 5,654 5,632 5,193 check a Numbers in parentheses indicate ranking. were evaluated during three seasons at 125 locations. Table 2 gives the results obtained. Based on overall performance, hybrid KRH-2 ranked first in the wet season of 1999 and In the dry season of 2000, it ranked second. On an overall basis, it ranked first. The hybrid PHB-71 ranked second in two wet seasons, whereas during the dry season it ranked third. On an overall basis, it ranked second. The hybrid Sahyadri ranked first in the dry season and third and fifth in the wet season of 1999 and wet season of 2000, respectively. On an overall basis, it ranked third. Evaluation of hybrids Evaluation of experimental hybrids at the network centers as well as at the research farms of the private seed sector and NGOs is the major activity of the coordinated evaluation in the Hybrid Rice Network. After promising hybrids are identified in Observational Yield Trials (OYT) by the respective centers, they are nominated for national evaluation in the Initial Hybrid Rice Trials (IHRT). Based on duration, three IHRTs are constituted: IHRT-Early, IHRT-Mid-early, and IHRT-Medium. In the IHRT, those hybrids that record a mean yield advantage of 15% and above over the yield of the highest check are promoted to the Advanced Varietal Trial-1 (AVT-1). Hybrids recording a yield advantage of 10% and above in the AVT-1 are promoted to the AVT-2. In AVTs, the promising hybrids are evaluated with the best inbred elite lines Hybrid rice development and use in India 269

253 Table 3. Hybrids evaluated during Year IHRTs a AVTs E ME M Total E ME M Total Total a IHRTs = International Hybrid Rice Trials, AVTs = advanced yield trials. E = early, ME = mid-early, M = medium. of the country, which is a unique system of combined evaluation of the best breeding material coming from hybrid and conventional breeding systems. At the AVT stage, agronomic trials as well as screening of entries for major pests and diseases are done. Those entries found promising for all aspects in the AVT-2 are identified for release at the time of the Annual Rice Research Workshop by a special committee constituted for the purpose. The proposals of identified hybrids are placed before the Central Variety Release Committee for deliberation and final approval. The number of experimental hybrids evaluated in IHRTs and AVTs during is given in Table 3. In total, 574 hybrids were evaluated during the six-year period. From 1990 onward, 1,188 hybrids were evaluated in 61 trials (38 in the wet season and 23 in the dry season). About half of these hybrids (47%) were developed and nominated by the national agricultural research and extension system (NARES), whereas one-fourth (24.5%) of these hybrids were nominated by the private seed sector and the rest (28.5%) were developed and nominated by the International Rice Research Institute (IRRI), Philippines. The percentage contribution of hybrids from IRRI was more in the initial years and has been declining over the years, whereas the reverse has been the trend with the hybrids nominated by the private seed sector. Promising hybrids in the pipeline are PAC and MPH-5401 in the medium-maturity group and EXPH-208, XR-593, and PR-122 in the mid-early group. Grain quality The large-scale adoption of hybrid rice depends on the profitability of hybrid rice cultivation, which in turn depends on the price offered by traders for the produce. The price offered is determined by consumer acceptability, which is primarily influenced by grain, cooking, and eating quality characteristics. Hence, quality considerations are of paramount importance for the popularization and large-scale adoption of hybrid rice. 270 Mishra et al

254 Table 4. Important grain quality characteristics of hybrids released by public sector (A) and those marketed by private-sector (B) agencies. Hybrid Grain quality characteristics a Milling (%) HRR (%) Grain type AC Alkali value Water uptake (mm) A APHR MS , APHR LS , KRH LS , CNRH LB , DRRH LS , KRH LB , PSD LS , CORH MS , ADTRH LS , Sahyadri LB , NSD MS , B PHB LS , PA LS , MPH LB , MPH LB , PAC LS , PRH LB , VRH LS , EXPH LS , Check varieties Rasi SB , Jaya SB , Vikas LS , S. Mahsuri 69.0 MS , Swarnadhan 60.6 SB , a HRR = head rice recovery, AC = amylose content, PSD = Pant Sankar Dhan, NSD = Narendra Sankar Dhan. MS = medium slender, LS = long slender, LB = long bold, SB = short bold. Consumer preferences are region-specific and differ greatly from place to place. Hybrids released in India were critically analyzed for grain and cooking quality (Table 4). It has been observed that, compared with the current popular inbred varieties, hybrids in general have lower head rice recovery. Most of the released hybrids have long slender grains and these get broken more frequently during the milling process than medium-slender-grain popular inbred varieties. Upon cooking, there is a mild aroma in all the hybrids based on the CMS line IR58025A and they are slightly sticky. Stickiness is not preferred in many parts of India. Hybrid rice development and use in India 271

255 Table 5. Grain yield of selected hybrids at different levels of N and K (wet season 2000). Treatments Mean grain yield (t ha 1 ) KRH-2 PHB-71 CORH-2 Mean T1 control T2 N90 + P60 + K T3 N90 + P60 + K T4 N90 + P60 + K T5 N120 + P60 + K T6 N120 + P60 + K T7 N120 + P60 + K T8 N150 + P60 + K T9 N150 + P60 + K T10 N150 + P60 + K Mean Hybrids are on a par with many of the cultivated varieties such as Jaya, IR64, IR36, and others, but cannot be compared for quality characters with premium-quality rice varieties such as basmati types, Mahsuri type (BPT 5204), etc. Hence, currently released hybrids are being specifically recommended for the areas where premium-quality rice is not grown. Simultaneously, efforts are being made to develop hybrids to cater to specific quality preferences of various regions. Nutrient management Appropriate nutrient management is essential to exploit the full potential of hybrids. Region-specific packages were developed for the cultivation of hybrids. The packages developed earlier are now being refined further. A trial was carried out to determine the interactions of varying levels of nitrogen, phosphorus, and potash on hybrid yield. In the first trial, phosphorus was kept constant (60 kg ha 1 ) while nitrogen was applied at 90, 120, and 150 kg ha 1 and potassium at 0, 40, and 80 kg ha 1. Three hybrids, KRH-2, PHB-71, and CORH-2, were used in this study. There was a good response to increased application of nitrogen and potash and the highest yield was obtained at 150 kg N and 80 kg potash ha 1 (Table 5). In another study, potash was kept constant (50 kg ha 1 ) while nitrogen was applied at 90, 120, and 150 kg ha 1 and phosphorus at 40, 60, and 80 kg ha 1 to the four hybrids PA-6201, VRH-704, PHB-71, and DRRH-1. Results indicated that the maximum mean yield of the four hybrids was obtained at 120 kg N and 60 kg P ha 1. There was no response for P beyond 60 kg ha 1 (Table 6). Further studies are in progress to refine and develop region-specific hybrid rice cultivation packages for various target states. 272 Mishra et al

256 Table 6. Grain yield of selected hybrids at different levels of N and P (dry season 2000). Treatments PA 6201 VRH-704 PHB-71 DRRH-1 Mean T1 control T2 N90 + P40 + K T3 N90 + P60 + K T4 N90 + P80 + K T5 N120 + P40 + K T6 N120 + P60 + K T7 N120 + P80 + K T8 N150 + P40 + K T9 N150 + P60 + K T10 N150 + P80 + K Mean Table 7. Hybrids a resistant to different pests and diseases. Pests b BPH WBPH Gall midge Hybrid KRH-2*, PHB 71, PA 6201, MGR-1, IR58025A/IR29723, IR58025A/IR RI 158, MTUHR 2037, MGR 1, HRI-126, IR58025A/IR29723 MGR-1, IR58025A/IR54742, IR58025A/ IR34686 Diseases Blast RTV c Sheath rot False smut Sheath blight HRI 126 2RI 158, KRH-2, DRRH-1, PA 6201, HKRH- 1005, NDRH-2, IR58025A/IR29723, IR58025A/IR21567 HKRH-1005, HKRH-1008, MTUHR-2036, MTUHR RI 158, NDRH-2, IR58025A/IR21567, IR58025A/IR29723, IR58025A/IR34686 IR58025A/IR21567, IR58025A/IR34686 a Released hybrids are given in bold. b BPH = brown planthopper. c RTV = rice tungro virus. Screening for resistance to biotic stresses The incorporation of resistance to major biotic stresses of the region is necessary for the successful adoption of hybrids. Hence, promising hybrids are regularly screened for resistance to major pests and diseases. Table 7 lists the hybrids found to be resistant to some of the major pests and diseases. Hybrid rice development and use in India 273

257 Table 8. Desirable traits a of promising CMS lines. CMS line DFF HT PN PL PE TS GT PS OC (cm) (cm) (%) (%) (%) APMS-6A* MS CRMS-31A* LS CRMS-32A* LS DRR-2A* LS Pusa 5A* LS PMS-10A* LS PMS-11A* LS PMS-14A* LB IR 68886A* MS IR 68888A* LS IR 68897A* LS IR 69628A* MS IR 58025A LS IR 62829A MS a DFF = days to 50% flowering, HT = plant height, PN = panicle number, PL = panicle length, PE = panicle exsertion, TS = total no. of spikelets, GT = grain type, PS = pollen sterility, and OC = outcrossing; * = promising CMS lines, + = standard checks, MS = medium slender, LS = long slender. Developing and evaluating CMS lines Outside China, the availability of commercially usable CMS lines is a major constraint to the development and use of hybrid rice. Among the several CMS lines developed at IRRI, only two lines, IR58025A and IR62829A, are commercially usable in India and elsewhere in the tropics. Of these two CMS lines, IR58025A is widely used in the tropics because of its higher outcrossing potential and better combining ability. Hybrids based on IR58025A possess mild aroma and long slender grains that tend to break during the milling process, thereby giving low head rice recovery besides being sticky on cooking. Because of these factors, there is no ready acceptance for IR58025A by consumers in some parts of India. Hence, there is an urgent need to develop CMS lines to meet specific Indian requirements. Work on developing CMS lines is actively done at five centers: Hyderabad, Delhi, Kapurthala, Cuttack, and Maruteru. Presently, promising maintainers are at various stages of conversion in the backcross nursery at each of these centers. Some of the promising CMS lines developed by these centers, along with those recently introduced from IRRI, were evaluated in multilocational trials for stable sterility and other desirable floral characters (Table 8). Six each of indigenous and exotic CMS lines were found to be promising in India and are being used to develop experimental hybrids for evaluation. Data on outcrossing percentage given in Table 8 were obtained from smaller plots, without supplementary pollination. For outcrossing potential among indigenous CMS lines, CRMS 32A and CRMS 31A are promising, along with exotic lines IR68888 and 274 Mishra et al

258 Table 9. Donors selected for desirable traits for recombination breeding. Donor Restorers Higher restorability IR10198, IR24, KMR 4, 9302, BR Better grain quality IR40750, Swarna Resistance to major pests IRBB 21, Suraksha, and diseases Triguna, IR40750 Maintainers Higher outcrossing Better quality 9601 B PMS 2B Partial maintainers Better quality BPT 5204 Better plant type 9314 and its derivatives IR All the abovementioned CMS lines are free from mild aroma, which is a desirable character for the majority of consumers in India. In India, greater emphasis is now being given to developing CMS lines with higher outcrossing, medium slender grains with intermediate amylose content, and better combining ability. Some of the genotypes under conversion in the backcross nursery possess these desirable characteristics. Parental line improvement In hybrid rice breeding, elite lines and released varieties from varietal improvement programs are now being used as parental lines. Besides narrow genetic diversity, the frequency of restorers and maintainers among these lines is 10 15% only. The remaining genotypes (70 80%) cannot be directly used in developing hybrids. Within the identified restorers and maintainers, the frequency of good combiners possessing desirable traits, such as outcrossing ability, good grain quality, and resistance to biotic stresses, is still lower. Hence, a specific parental line improvement program was undertaken by recombination breeding and genetic male sterility-facilitated population improvement. Improving restorers and maintainers through recombination breeding Restorers/maintainers possessing one or other desirable traits and partial restorers/ maintainers possessing outstanding desirable qualities such as premium grain quality, higher outcrossing, and resistance to biotic stress were selected and crossed in single and three-way crosses. Table 9 lists some of the original donors used in recombination breeding. In addition, several derivatives from these donors were also used in the crossing program. Selections were made for desirable segregants in the F 2 to F 6 Hybrid rice development and use in India 275

259 Table 10. Restorer and maintainer line development, DRR, Hyderabad. Type of No. of No. of lines in cross crosses segregating generations F 3 F 4 F 5 F 6 Restorer development A (R 1 R 2 ) R PR R R R 1 (R 2 R 3 ) R 1 (R 2 PR) Maintainer development B B B 1 (B 2 B 3 ) Table 11. Status of use of newly developed parental lines (DRR, Hyderabad). Type of No. of lines No. of lines No. of lines in Frequency of cross testcrossed identified BC a /OYT b M/R (%) as R/M a R 1 (R 2 R 3 ) A R A (R 1 R 2 ) R PR R R B B a 16 a (4 in BC 3 ) 74 a Indicates no. of maintainers. b OYT = observational yield trial. (Table 10). About 1,000 stabilized lines developed through this program are being used to make testcrosses with the CMS lines. Table 11 gives the status of the use of some of the derived parental lines. The frequency of restorers among the derived lines is 38 60% vis-à-vis 15 20% among the available elite lines from the varietal improvement program. Similarly, among the derived maintainer lines, the frequency of maintainers was as high as 74%. Sixteen newly developed maintainer lines are in the backcross nursery. All these lines possess very high stigma exsertion (70 80%) with the preferred medium slender grain type. The derived restorers also possess medium slender grains, with very good plant type with long panicles and high pollen dispersal. From the derived restorer lines, 25 restorers are already in OYT seed production and 14 are being used to produce experimental hybrids for the IHRT. 276 Mishra et al

260 Table 12. Development of composite populations of restorers and maintainers, DRR, Hyderabad. Name of population a No. of Special attributes b new lines introduced Restorers DRCP-101 (M) 12 Good grain quality, better plant type, BPH and BLB resistance. DRCP-102 (M) 10 Good plant type, restoration ability, and good grain quality. DRCP-103 (ME) 14 Good plant type, restoration ability, and good grain quality. DRCP-140 (ME) 8 Better plant type, good restoration, disease and insect pest resistance. Maintainers DRCP-104 (ME) 8 Better plant type, good grain quality, high outcrossing ability, stable maintenance of male sterility, and BPH and BLB resistance. DRCP-105 (M) 10 Better plant type, good grain quality, high outcrossing ability, stable maintenance of male sterility, and BPH and BLB resistance. a Letters in parentheses indicate maturity group: ME = medium-early, M = medium. b BPH = brown planthopper, BLB = bacterial leaf blight. Developing parental lines through population improvement In addition to crossbreeding methods, a genetic male sterility-facilitated recurrent selection program was undertaken to develop new parental lines. The main aim of the program is to develop heterotic gene pools of restorers and maintainers by incorporating genetically diverse and broad-based genotypes possessing good combining ability for grain yield, better grain quality, and disease and insect pest resistance, and to extract a large number of genetically diverse parental lines from improved populations from time to time. The base material for the population improvement program consisted of two composite populations of restorers IR69701-CP. 138 (early) and IR69702-CP.139 (late) and two maintainer populations IR71590-CP-140 (early) and IR71591-CP-141 (late) developed by using genetically diverse component lines at IRRI (Philippines). The original composite populations received from IRRI were multiplied at DRR and distributed to network centers for their use in region-specific breeding programs as per the mandate of each center. Table 12 gives the details on composite populations of maintainers and restorers, the number of new lines introduced, and their desirable characteristics. These populations have reached the fourth to sixth generation of random mating. This is a very innovative approach introduced to hybrid rice breeding. This activity has resulted in the development of genetically diverse parental lines, which are a prerequisite for developing hybrids with enhanced magnitude of heterosis. The results have been particularly impressive at DRR, Hyderabad, and at ARS, Maruteru. Hybrid rice development and use in India 277

261 Some of the lines extracted from these populations are already being used to develop new hybrids. Diversifying CMS sources and developing new restorers About 95% of the commercial hybrids in China and elsewhere are based on a single source of cytoplasmic male sterility: the wild abortive (WA) system. Dependence on a single CMS source in the long run may result in genetic vulnerability of hybrids to sudden outbreaks of diseases and insect pests or in their restricted adaptability. Although no relationship between susceptibility to any major diseases or insects pests and WA cytoplasm has been established, the vulnerability of hybrids tailored to a single male sterility source cannot be ruled out. Therefore, diversification of CMS sources was taken up as a part of hybrid rice breeding. Among the several approaches to developing a cytoplasmic genic male sterility system, interspecific hybridization followed by substitution backcrossing is the most commonly employed method. Many Asian, American, and African wild species belonging to the AA genome offer potential sources for diversifying the cyto-sterility source. Nevertheless, very limited efforts have been made so far to identify diverse sources of cyto-sterility. Therefore, it is necessary not only to diversify the cytosterility sources but also to find restorers for the new sources of cyto-sterility developed. Identifying diversified cyto-sterility sources and developing new CMS lines At DRR, Hyderabad, 132 interspecific hybrids involving cultivated rice Oryza sativa and closely related wild species of the A genome O. rufipogon, O. nivara, O. barthii, O. glaberrima, and O. longistaminata were produced and evaluated. The embryo rescue technique was employed to save hybrids and time in obtaining the F 1 plants. The F 1 hybrids with a pollen fertility of less than 30% and spikelet fertility not exceeding 30% were backcrossed to recurrent male parents. The BC 1 F 2 progenies were evaluated for pollen and spikelet fertility on an individual plant basis and those with less than 10% pollen/spikelet fertility combining desirable agronomic traits were used in further backcrossing to original male or female parents. By comparing the fertility/sterility status of plants in the backcross generations, the presence of sterility-inducing cytoplasm was detected. All the identified sources of sterility-inducing cytoplasm were from Asian species and none of the African species studied was found to possess such cytoplasm. Combinations exhibiting cytoplasmic genic interactions for male sterility were chosen for developing new CMS lines by substitution backcrossing. In all, six male sterility sources involving cytoplasm of either O. rufipogon or O. nivara were identified. The six sources were characterized based on pollen stainability status and restoration/maintenance reaction with known restorers of the WA source. Based on the above two criteria, out of six sources identified, three were found to be entirely different from the WA system. Among these three, two are gametophytic types and one is a sporophytic type. Among the other three sources, one is similar to MS 577A, 278 Mishra et al

262 Table 13. Characteristics of newly identified alternate CMS sources. Line Source Type of sterility Restorers Maintainers RPMS 1 O. rufipogon Gametophytic Nil IR66, IR70, PMS 2B, V20 B RPMS 2 O. nivara Gametophytic Nil IR66, IR70 RPMS 3 O. nivara Stained pollen IRBB 7 PMS 2B, (MS 577A-like) IR62829B RPMS 4 O. nivara Sporophytic Nil IR66, PMS 2B RPMS 5 O. nivara Sporophytic IRBB7, IR66, IR70 PMS 2B, IR62829B RPMS 6 O. nivara Sporophytic IRBB7, IR66, IR70 PMS2B, IR62829B while the other two resemble the WA type with respect to restoration and maintenance of male sterility (Table 13). These new CMS sources in the nuclear background of different maintainers were further characterized for pollen and spikelet sterility traits over the years. These observations clearly indicate that the cyto-sterility sources from O. rufipogon (VN1) and O. nivara (DRW 21039) possess a very high frequency of round sterile pollen grains and a lone source, RPMS 4, from O. nivara (DRW 21018) possesses a high degree of typical abortive pollen grains. All the new CMS lines were found to exhibit complete male sterility (Hoan et al 1997). All these new CMS sources were thoroughly characterized for pattern of pollen abortion and restoration/maintenance reaction. Based on these two criteria, two sources were categorized as sporophytic and one source was grouped as a gametophytic type. Among the six new CMS lines, RPMS 1-1, 1-2, 1-3, and 1-4 possess a source of male sterility from O. rufipogon and RPMS 2 and RPMS 4 possess a CMS source from O. nivara. One of the major drawbacks of WA systems is poor panicle exsertion (75 80%). However, the new CMS sources possessed better panicle exsertion (>95%) (Table 14). Besides the advantage of cytoplasmic diversification in preventing genetic vulnerability of hybrids to insect pests and diseases, the new CMS lines possess good grain quality, very good panicle and stigma exsertion, and high outcrossing potential, which will go a long way in maximizing hybrid seed yield without the use of GA 3, a costly input in hybrid rice seed production (Ramesha et al 1999). To identify restorers for MS 577A and IR64A CMS sources, for which no restorers are available so far, more than 100 interspecific hybrids involving different accessions of O. rufipogon, O. nivara, and O. sativa. f. sp. spontanea were produced and evaluated for spikelet fertility at DRR (Hyderabad). Some of the segregants from a cross between Pushpa A/VN2 (O. rufipogon) and Pushpa A/RPW (O. sativa f. sp. spontanea) possessed high pollen and spikelet fertility, thereby indicating the presence of restorer genes for MS 577A cytoplasm. Surprisingly, none of the accessions of wild species tested possessed restorer genes for the IR64 (O. perennis) CMS source. Hybrid rice development and use in India 279

263 Table 14. Morphological traits a and other desirable features of new CMS lines (DRR, Hyderabad). Nuclear PE SE OC CMS line donor DFF (%) (%) (%) GT e RPMS 1-1 b IR MS RPMS 1-2 b V20B MB RPMS 1-3 b IR LB RPMS 1-4 b PMS 2B MS RPMS 2 c IR MS RPMS 4 d IR MS a DFF = days to 50% flowering, PE = panicle exsertion, SE = stigma exsertion, OC = outcrossing, GT = grain type. b O. rufipogon source. c O. nivara (sporophytic) source. d O. nivara (gametophytic) source. e MS = medium slender, LB = long bold. Introgressing restorer genes and other useful traits from wild species of rice More than 300 introgressed lines from the F 2 generation of Mangala A and Pushpa A with as many accessions of O. rufipogon and O. sativa f. sp. spontanea were tested and screened for better restoration ability, pollen shedding ability, and good agronomic traits. Some of the promising iso-cytoplasmic restorer lines possessing good restorer characters and with many other undesirable traits, such as grain shattering, poor plant type, and presence of awns, etc., were backcrossed for two generations with IR40750 and PMS 2B and selection was carried out in segregating generations. Some promising lines possessing a relatively high degree of restoration, better plant type, large panicle size, and other restorer traits were developed into new restorers for the MS 577A (O. perennis) CMS source (Table 15). The use of these restorers will go a long way in using some of the highly stable and popular CMS lines such as Mangala A, Pushpa A, Pragati A, and others to produce highly heterotic rice hybrids. The two new restorer gene sources that were identified in O. sativa f. sp. spontanea and O. rufipogon were transferred into the genetic background of many elite breeding lines. The iso-cytoplasmic restorer gene source from Pushpa A O. rufipogon (VN 2) was transferred to Jaya and a large number of fertile iso-cytoplasmic segregants possessing different plant type characters and a high level of spikelet fertility (80 91%) were selected and stable lines were developed by the pedigree method. Similarly, the iso-cytoplasmic restorers isolated from a cross of Mangala A O. sativa f. sp. spontanea (RPW 2001) were crossed with PMS 2B and 9302 and a large number of segregants were selected and advanced to further generations. Apart from this, the two restorer gene sources that were not allelic were combined by crossbreeding and many segregants from the cross were selected and advanced to further generations. More than 60 stable restorers developed by the three methods were extensively evaluated for spikelet and pollen fertility. Based on both criteria, restorers were classified into three categories:(1) high pollen and high spikelet fertility types, (2) low pollen and high spikelet fertility types, and (3) low pollen and low spikelet fertility types. Table 16 gives the details of the improved restorers developed. 280 Mishra et al

264 Table 15. New iso-cytoplasmic restorers for MS 577A (O. perennis) source. Restorer gene source Elite lines used No. of Days to Spikelet restorers maturity fertility developed range (%) O. rufipogon (VN2) MRR. 10/IR O. rufipogon (VN2) MRR. 22/IR O. sativa f. sp. spontanea (RPW 2001) PRR 10/PMS 2B O. sativa f. sp. PRR 12/PMS 2B spontanea (RPW 2001) Table 16. Improved iso-cytoplasmic restorers for MS 577A source. Restorer gene source Cross No. of lines Days to Spikelet combination developed maturity fertility (range) (%) O. rufipogon (VN 2 ) RI Jaya RI Jaya O. sativa subsp. DNR 3P spontanea Combination of two DNR 3P 1 RI sources Developing and evaluating thermosensitive genic male sterile (TGMS) lines To begin a two-line heterosis breeding program in India, introduced and indigenously identified TGMS lines were evaluated and characterized. To develop new TGMS lines with better plant type and adaptability, a specific breeding program started. A few two-line hybrids were developed and a preliminary evaluation was conducted. Six introduced and indigenously identified TGMS lines were evaluated under field conditions for sterility/fertility transformation by sequential sowings during the wet seasons of at Hyderabad, Coimbatore, and Pantnagar. Among the six lines, two, TGMS-1 and TGMS-5, did not show clear transformation from fertility to sterility and vice versa. The remaining four TGMS lines became fertile when sown in the second half of July to the end of August. When sown during the rest of the year, they remained sterile. One line, TGMS-6, was a very low critical sterility point (CSP) line, remaining sterile most of the period. It became fertile when the critical stage occurred during November/December only. The four TGMS lines identified and evaluated, though they showed clear transformation from fertility to sterility and vice versa, do not possess the desirable plant type. They are shy tillering types with smaller panicles and a lower number of spikelets. To transfer the TGMS gene(s) to an elite agronomic background, the line TGMS- 6, which was the most promising among the available ones, was crossed with 58 Hybrid rice development and use in India 281

265 Table 17. Performance of two-line hybrids. Hybrid Yield Yield (t ha 1 ) advantage (%) over check TGMS-2/IR TGMS-3/Krishna Hamsa DRRH-1 (check) 6.8 released varieties/elite breeding lines. Desirable genotypes were selected in segregating generations. The TGMS character has been effectively incorporated into an elite agronomic base and the material is in the F 5 generation. Preliminary evaluation of 15 two-line hybrids was done at Hyderabad. Two hybrids, TGMs-2/IR64 and TGMS-3/Krishna Hamsa, outyielded the hybrid check by 4 9% (Table 17). The rest of the 13 two-line hybrids yielded lower than DRRH- 1, the three-line hybrid check. With the presently available TGMS lines, which do not have the desirable plant type, it is difficult to get two-line hybrids that outyield significantly the released three-line hybrids. Hence, TGMS lines with better plant type, adaptability, higher outcrossing potential, and better combining ability need to be developed and used. Seed production Economic and efficient large-scale seed production is a prerequisite for popularizing hybrid rice in the country. India has a strong seed industry in both the public and private sector. Involvement of the seed sector in the hybrid rice network right from the beginning has paid rich dividends. Seed production personnel from the public and private sector, trained within and outside the country, are successfully shouldering the responsibility of large-scale seed production. The seed production package developed earlier has been further refined in recent years, particularly for synchronization parameters and economization of GA 3, and seed yields of t ha 1 are routinely obtained. The seed industry, particularly in the private sector, is confident of successfully producing any quantity of seed, provided demand is regular. Seed yields as high as 4.0 t ha 1 have been obtained in smaller plots ( ha) by some private seed companies. During 2002, an estimated 4,000 tons of hybrid seed were produced, 95% of it by the private sector. The public seed sector has to be motivated to become involved in large-scale seed production in a bigger way. Seed cost is now US$2.00 to $2.50 kg 1. This cost has to be brought down to around $1.50 kg 1 to make it affordable for resource-poor marginal and small rice farmers. Table 18 gives the names of the major private- and public-sector seed agencies engaged in large-scale hybrid rice seed production in India. 282 Mishra et al

266 Table 18. Agencies engaged in large-scale hybrid rice seed production in India. Private-sector seed agencies Public-sector seed agencies Hybrid Rice International Ltd., National Seed Corporation, New Delhi Hyderabad State Farms Corporation of India, New Delhi PHI Seed Ltd., Hyderabad Andhra Pradesh State Seed Development Mahyco Ltd., Mumbai Corporation, Hyderabad Parr Monsanto India Ltd., Karnataka State Seed Corporation, Bangalore Bangalore Syngenta Seeds Ltd., Pune Maharashtra State Seed Corporation, Akola Indo-America Hybrid Seed Ltd., West Bengal Seed Development Corporation, Bangalore Kolkatta Advanta India Ltd., Bangalore Uttar Pradesh Terai Seed Development Nath Seed Company Ltd., Aurangabad Corporation, Pantnagar J.K. Agrigenetics, Hyderabad Amareshwara Agri Tech Ltd., Hyderabad Seed purity of parental lines and hybrids was a cause of concern in initial years. However, the situation has improved over the years. All the research centers that have developed public hybrids are producing nucleus and breeders seed by following prescribed procedures and an adequate quantity of pure seed of parental lines is now available. To test the genetic purity of hybrid seed, the grow-out tests (GOT) generally employed are laborious and time-consuming. Recently, DNA marker technology for determining hybrid seed purity was developed at DRR (Hyderabad). This procedure involves the use of microsatellite markers. The entire procedure from DNA extraction to gel documentation takes 4 5 hours. DNA samples are collected from young seedlings. Thus, within 7 10 days of harvest, seed purity can be assessed vis-à-vis 3 4 months time required for GOT. A sample of 400 seeds representing 1 10 t of seed material costs around $100. Transfer of hybrid rice technology Having developed hybrids during the early 1990s and optimized the seed production and cultivation packages, the next logical step was to transfer effectively the technology developed to the rice farmers in the target states. Hence, this aspect received major emphasis during the second phase ( ) of the ICAR/UNDP project. Compact-block front-line demonstrations, training programs at various levels for different clienteles, interstate farmer exposure visits, the publication of extension literature in local languages, the preparation of videos for training purposes, etc., were the major activities for transferring hybrid rice technology to farmers. Compact-block front-line demonstrations To create awareness about the advantages of the adoption of hybrid rice among rice farmers and to impart knowledge and skills for cultivation and seed production of Hybrid rice development and use in India 283

267 Table 19. Performance of hybrids in front-line demonstrations ( ). State Hybrid No. of Mean yield (t ha 1 ) demonstrations Hybrid Advantage over check (%) Andhra Pradesh DRRH PHB Karnataka KRH-2 1, Maharashtra Sahyadri Orissa PA Tamil Nadu CORH Eastern Uttar Pradesh NSD West Bengal PHB Total 2,418 hybrid rice, extensive compact-block front-line demonstrations were conducted in the target states. Table 19 gives details on the number of demonstrations conducted, hybrids evaluated, and the yield advantage of hybrids over the check varieties over three years. In total, 2,418 front-line demonstrations were conducted during the period. The yield advantage of the hybrids over the check varieties ranged from 0.9 to 1.9 t ha 1. Farmers were convinced of the yield advantage of the hybrids. A slightly lower price was offered for the produce of hybrids compared with that of the check varieties in the southern region of the country. Field days were organized in which many farmers participated and interacted with extension workers. Conducting compactblock front-line demonstrations on hybrid rice cultivation and seed production proved to be a very effective strategy for popularizing hybrids in the country. Training program To transfer effectively the knowledge and to impart the skills necessary for hybrid rice cultivation and seed production, several training programs were organized at various centers for different clienteles such as farmers, farm women, seed growers, seed production personnel of public, private, and NGO sectors, and officials from departments of agriculture from the target states and state agricultural universities. Table 20 provides details of the training programs conducted and number of persons trained. In all, 150 training programs were conducted on hybrid rice seed production and 94 programs were conducted on hybrid rice cultivation and more than 9,000 participants were trained on various aspects of hybrid rice technology during the period. 284 Mishra et al

268 Table 20. Training programs conducted at different centers during Center Hybrid rice seed Hybrid rice Interstate production cultivation farmers visits No. of training No. of No. of training No. of No. of No. of programs participants programs participants visits participants Hyderabad Mandya Coimbatore Maruteru Pantnagar Chinsurah Karnal Faizabad KVK (Gadipally) Karjat Total 150 4, , Eight interstate farmers exposure visits were arranged in southern and western India. In total, 356 farmers participated in these tours, seeing the developments in hybrid rice cultivation and seed production in the states of Andhra Pradesh, Karnataka, Tamil Nadu, and Maharashtra. These exposure visits were extremely useful and educational for the farmers. Future outlook Rice hybrids were developed and released for the first time in India in The adoption of hybrid rice has been slower than expected during the past seven years because of the lack of ready acceptance by consumers on account of grain quality considerations in some parts of India, the marginal profitability in the cultivation of hybrid rice, the poor quality of hybrid seed and high seed cost, inconsistency in the performance of hybrids, the lack of resistance to some of the major pests and diseases, and the lack of awareness because of inadequate transfer of technology efforts. To overcome these constraints, hybrid rice research has been reoriented to Develop hybrids with acceptable grain quality to meet the specific requirements of different regions. Enhance the magnitude of heterosis to 20% and above by developing twoline and intersubspecific hybrids. Insulate hybrids against the major pests and diseases of the target region. Enhance seed yields beyond 2.0 t ha 1 to bring down the seed cost. In addition to the above, efforts on technology transfer have been intensified through the conduct of a large number of front-line demonstrations and training pro- Hybrid rice development and use in India 285

269 grams to create awareness about the benefits of hybrid rice among rice farmers and consumers across the country. Policy interventions by the government for increased support, aggressive efforts to popularize hybrids, and the assured procurement of hybrid rice produce at a minimum support price are needed at this juncture. It is envisaged that, if the teething problems encountered now are solved effectively, hybrid rice is likely to be cultivated in a sizable area of 2 3 million ha in India during the next decade to help sustain food security. References Hoan NT, Sarma NP, Siddiq EA Identification and characterization of new sources of cytoplasmic male sterility in rice. Plant Breed. 116: Ramesha MS, Viraktamath BC, Ilyas Ahmed M, Vijayakumar CHM New CMS sources with stable male sterility and better outcrossing trait in rice (O. sativa L.). Indian J. Genet. 59(4): Siddiq EA, Ilyas Ahmed M, Viraktamath BC, Rangaswamy M, Vijaykumar R, Vidyachandra B, Zaman FU, Chaterjee SD Hybrid rice technology in India: current status and future outlook. In: Virmani SS, Siddiq EA, Muralidharan K, editors. Advances in hybrid rice technology. Manila (Philippines): International Rice Research Institute. p Notes Authors address: Directorate of Rice Research, Hyderabad , India. Citation: Virmani SS, Mao CX, Hardy B, editors Hybrid rice for food security, poverty alleviation, and environmental protection. Proceedings of the 4th International Symposium on Hybrid Rice, Hanoi, Vietnam, May Los Baños (Philippines): International Rice Research Institute. 407 p. 286 Mishra et al

270 Hybrid rice research and development in Indonesia Suwarno, N.W. Nuswantoro, Y.P. Munarso, and M. Direja The plateauing trend of rice production and the success of the development and commercial use of hybrid rice technology outside China, including India, Vietnam, and the Philippines, encouraged the government of Indonesia to intensify research on and development of hybrid rice in Some hybrid rice varieties, including two public and five private ones, were released recently. The public hybrids, Rokan and Maro, yielded t ha 1 higher than the inbred variety IR64 in suitable conditions. The hybrids are susceptible to the major pests brown planthopper, bacterial leaf blight, and rice tungro virus, and may not always express heterosis across locations. Cultivation of the hybrids should be followed by application of integrated pest management, but cultivation is not recommended in the endemic area of the pests. The breeding program of developing better hybrids is conducted by public as well as private institutions and some promising hybrids have been developed. The development of CMS lines resistant to the major pests is also in progress. A program for disseminating hybrid rice technology has begun in 15 districts identified as having potential for hybrid rice cultivation. The total area of hybrid rice cultivation during this season is about 60 ha and will be increased gradually to at least 500,000 ha in the wet season of A seed system suitable for hybrid rice, including seed production, inspection, and certification, is being established. Rice cultivation is the major source of income of millions of farmers in Indonesia. As the staple food, rice is not only considered as an economic crop; it also has an important role in politics in the country. Increasing rice production to support food security has therefore been given a high priority in agricultural development in Indonesia. Rice yield with the available varieties and cultivation technology, however, has exhibited a plateauing trend during the past decade. Moreover, agricultural land for rice cultivation is limited to meet the challenge of producing 68.8 million tons of rice for the estimated population of 265 million by 2025 from the present 203 million people. 287

271 Hybrid rice technology, which exploits the heterotic effect, has been widely practiced in China to increase rice yield by 15 20% over that of the high-yielding inbred variety (Yuan 1994). Indonesia has about 5 million hectares of irrigated lowland cultivated to rice, which is the third largest in the world after China and India. Most of the irrigated land is distributed in the densely populated area offering high labor availability. Intensive rice cultivation is practiced and farmers in the area obtain about 5.1 t ha 1 average yield. This situation offers potential for implementing hybrid rice technology (Lin and Pingali 1994). Hybrid rice technology has also been commercially used outside China in India, Vietnam, the Philippines, and some other countries (Paroda 1998, Hoan et al 1998). The success of the development and use of hybrid rice technology to increase rice yield has encouraged the government of Indonesia to intensify research and development on hybrid rice. Institutions involved are the Research Institute for Rice of the Central Research Institute for Food Crops, Directorate of Seed Development, Directorate of Food Crop Production, and some state-owned and private seed companies. Collaboration with the International Rice Research Institute (IRRI) on hybrid rice research has also been intensified through the ADB-funded project on the Development and Use of Hybrid Rice in Asia since Moreover, a Technical Cooperation Project sponsored by FAO was completed on strengthening the development and use of hybrid rice in Indonesia. Some hybrid rice varieties developed by the public and private sector have been released recently. The release was announced by the President of Indonesia during the Rice Week held at Sukamandi in March The dissemination program for the technology, including the establishment of seed production and a quality management system and the development of a hybrid cultivation area, is being implemented. This paper reports on the progress of hybrid rice research and development. Developing hybrid rice varieties It was assumed that hybrid rice technology, which had been practiced widely in China, was suitable only for temperate regions, and that the superiority of hybrid rice became less in the regions closer to the equator. The fact that parental lines introduced from China were not adapted to Indonesian conditions and skepticism about the practical feasibility of hybrid seed production slowed down the progress of hybrid rice development in the country. Though the research on hybrid rice began in 1983, the activity focused only on evaluating introduced hybrids and parental lines from IRRI, China, and other countries. Encouraged by the success of hybrid rice development in India, Vietnam, and the Philippines, the breeding program for hybrid rice has been intensified since 1998 with the following strategy: For the short term, evaluation of introduced hybrids for their yielding ability and other desirable characteristics to identify the superior ones for release. 288 Suwarno et al

272 Table 1. Average yield of Maro and Rokan in multilocation yield trials. Location Rokan Maro IR64 (t ha 1 ) Yield a Standard Yield Standard (t ha 1 ) heterosis (t ha 1 ) heterosis Dry season 2000 Sukamandi Muara 7.40* * Kuningan 7.48* Cianjur 7.38* Purwokerto 7.46* * Subang Kulonprogo 6.12* Klaten Ngawi Kediri * Av Wet season Ciamis Lampung Timur Batang Sleman * Kudus 6.37* * Nganjuk 6.56* * Pasuruhan 6.98* * Av Total av a Values with * and underlined are significantly higher and lower than the corresponding check, respectively. For the medium term, the identification of restorer lines among the breeding materials developed in Indonesia to develop more adapted hybrids with the existing introduced CMS lines. For the long term, the development of CMS lines with the genetic background of locally developed and more adapted breeding lines for developing rice hybrids suitable for Indonesian conditions. Released hybrids Two introduced hybrids, IR58025A/IR53942 and IR58025A/BR827-35, were released officially, named Maro and Rokan, respectively. Five other hybrids, Intani 1, Intani 2, Miki 1, Miki 2, and Miki 3, were released by private seed companies. The hybrids Maro and Rokan produced t ha 1 more yield than the high-yielding inbred variety IR64 in suitable conditions. The hybrids could not always express yield higher than that of the inbred check variety IR64 across locations in the yield trials conducted during the dry season (DS) of 2000 and wet season (WS) of (Table 1). Rokan, with an average yield of 6.82 t ha 1, appeared as the top yielder in Hybrid rice research and development in Indonesia 289

273 Table 2. Number of experimental units where the hybrids yielded higher than, similar to, and lower than the check variety IR64. Variety Number of experimental units Average yield (t ha 1 ) Higher Similar Lower Total Higher Similar Lower Rokan IR Yield increase (%) Maro IR Yield increase (%) Table 3. Reaction of rice hybrids Maro and Rokan to the major pests compared with inbred variety IR64. Pest a Rokan b Maro IR64 BPH Biotype 2 S S R Biotype 3 S S S BLB Strain III S MR MR Strain IV S MR S Strain VIII S S S RTV S S S a BPH = brown planthopper, BLB = bacterial leaf blight, RTV = rice tungro virus. b R = resistant, MR = moderately resistant, S = susceptible. the trials conducted during the DS of In the same trials, Maro had an average yield of 6.22 t ha 1 and IR64, as the inbred check variety, yielded 6.11 t ha 1. During the WS of , Maro yielded 6.26 t ha 1 and appeared as the top yielder, whereas Rokan and IR64 yielded 5.86 and 5.38 t ha 1, respectively. Table 2 shows the number of experimental units in which the hybrids produced higher, similar, and lower yield than that of the check variety. Rokan and Maro produced significantly higher yield over IR64 in 8 and 7 units out of 17 or about 47% and 41%, respectively. The released hybrids are susceptible to the major pests and diseases in Indonesia, including brown planthopper (BPH), bacterial leaf blight (BLB), and rice tungro virus (RTV) (Table 3). Dissemination of the hybrids should therefore be conducted only in suitable conditions that are not an endemic area for the major pests. Promising and identified hybrids Replicated yield trials of promising hybrids conducted at Sukamandi showed that five hybrids (Maro, Rokan, IR58025A/C20, IR58025A/IR65515, and IR58025A/ B4070) showed heterosis of more than 10% (Table 4). IR58025A/C20, IR58025A/ IR65515, and IR58025A/B4070, which showed heterosis of 17.2%, 19.7%, and 290 Suwarno et al

274 Table 4. Performance of some hybrids in advanced yield trials conducted at Sukamandi, wet season Hybrid Plant No. Maturity Yield Heterosis height of tillers (d) (t ha 1 ) (%) Rokan Maro IR58025A/MTU IR58025A/C IR58025A/IR IR62829A/MTU IR58025A/S IR58025A/RCN-B IR58025A/RHS IR58025A/B IR58025A/B IR Table 5. Selected hybrids from preliminary yield trials conducted at Muara and Cianjur, wet season Hybrid Height Maturity Yield (t ha 1 ) Standard (cm) (d) heterosis Muara Cianjur Av (%) IR58025A/IR IR62829A/IR IR58025A/B IR58025A/B IR58025A/B IR58025A/B IR58025A/IR IR62829A/B IR68897A/B IR64 (check) %, respectively, showed higher heterosis than that of the newly released hybrids Maro and Rokan and will be further evaluated. Among 30 hybrids evaluated in preliminary yield trials at Muara and Cianjur, nine were selected for further evaluation (Table 5). The selected hybrids yielded at least 1 t ha 1 higher that IR64 at one location and showed resistance or moderate resistance to BLB. IR68897A/B9775 appeared as the top yielder, followed by IR58025A/B10393, and they had 35.7% and 26.5% heterosis, respectively. Some restorer lines producing high-yielding hybrids with IRRI-bred cytoplasmic male sterile (CMS) lines have been identified. In the observational trial, most of the hybrids derived from the selected restorers yielded beyond 7 t ha 1, whereas the check varieties IR64 and Widas yielded 6.20 and 6.45 t ha 1, respectively (Table 6). The hybrids showed lower spikelet fertility but more filled grains per panicle than Hybrid rice research and development in Indonesia 291

275 Table 6. Selected hybrids derived from IRRI-bred CMS lines and restorers from locally developed breeding lines, Bogor, wet season Hybrid Duration Plant Panicles Spikelet Filled Yield (d) height hill 1 fertility grains (t ha 1 ) (cm) (%) panicle 1 IR58025A/B IR58025A/B IR58025A/S IR58025A/S IR58025A/S IR58025A/B IR58025A/B IR58025A/B IR58025A/B IR Widas Table 7. Selected hybrids from testcross nursery for observation nursery, wet season Hybrid Maturity Seed Weight (g) Standard (d) set heterosis (%) Panicle Grains plant 1 (%) IR58025A/BP IR58025A/BP IR5025A/BP IR58025A/BP IR58025A/BP IR68897A/B IR68897A/B IR58025A/BP IR58025A/BP IR58025A/BP IR Memberamo the check varieties. In addition, since most of the selected restorers are resistant or moderately resistant to BPH and BLB and those resistances are controlled by dominant genes, the hybrids must have resistance to the pest and disease. Some hybrids showed outstanding performance and resistance to BLB at the testcross nursery (Table 7). The seed of the hybrids is being increased for observation trials and other evaluations that follow the breeding method for hybrid rice published by IRRI (Virmani et al 1997). Most of the restorers are resistant to BPH and BLB; therefore, it is expected that the hybrids will also be resistant to these major pests. 292 Suwarno et al

276 Table 8. Selected lines to be converted into CMS lines and their important characteristics. Line CMS source Characteristics a Backcross S5097 IR58025A Resistance to BLB, RTV, BPH BC 5 S3393 IR58025A Resistance to BLB, RTV, BPH BC 5 B10385 IR58025A New plant type BC 5 B7830 IR62829A Resistance to BLB, BPH, GLH BC 4 S3385 IR62829A Resistance to BPH, grain quality BC 4 BP302 IR58025A New plant type BC 3 BP143 IR68897A Resistance to BPH, BLB BC 3 B7809 IR66707A Resistance to BLB, Fe toxicity BC 3 BP303 IR66707A Resistance to BLB BC 3 B10384 IR58025A Resistance to BPH BC 2 B7830 IR68897A Resistance to BPH BC 2 BP68 IR58025A Resistance to BPH, BLB BC 2 B9701 IR68897A Resistance to BLB BC 2 B10177 IR58025A Resistance to BPH, BLB BC 2 a BLB = bacterial leaf blight, RTV = rice tungro virus, BPH = brown planthopper, GLH = green leafhopper. It was indicated that the frequency of restorers in the breeding material developed in breeding for inbred varieties was very low. The fact that, of more than 2,000 hybrids made for the testcrosses in the WS , only 10 hybrids were selected for observation trials and 43 for further evaluation, or a total of 53 hybrids were selected, indicated the low frequency of good restorers. Breeding for restorer lines through R R and A R crosses has begun and some F 3 generations have been developed. Developing CMS lines Most of the existing CMS lines are susceptible to BPH, BLB, and RTV. The development of CMS lines more suitable for Indonesian conditions should therefore consider resistance to major pests. Maintainer lines identified in testcross trials that have the desirable characteristics for CMS lines, such as dwarfness, exserted panicle, exserted stigma, resistance to major pests, and good grain quality, are selected to be converted into CMS lines. Backcrosses to develop CMS lines are in progress (Table 8). Developing rice varieties with the new plant type is the other approach for increasing yield potential. The breeding program is in progress and some promising breeding lines have been developed. It is expected that a 10 20% increase in yield potential will be obtained through the program. A further increase in yield potential could be obtained by exploiting heterotic effects through the development of hybrids using the new-plant-type lines. Some backcrosses to develop CMS lines with the new plant type have begun. The development of breeding material for CMS through B B crosses also began. Hybrid rice research and development in Indonesia 293

277 AARD-RIR Breeder seed Provincial seed farm State-owned company (PT. SHS, Pertani) Hybrid seed Parental line seed BPSB (Seed Inspection and Certification Agency) Farmer Seed grower Hybrid seed Fig. 1. System of seed production and quality control for publicly developed hybrid rice. AARD = Agency for Agricultural Research and Development, RIR = Research Institute for Rice. Dissemination of hybrid rice technology Rice is basically a self-pollinated crop, whereas hybrid seed should be produced through cross-pollination. As a result, the production of high-quality seed is one of the major constraints to disseminating hybrid rice technology. In addition, the technology is new in Indonesia. The seed system for conventional inbred varieties is well established in Indonesia (Fig. 1). About 60% of the total rice cultivation area uses certified seed, mostly in irrigated lowlands. Two state-owned seed companies, PT. Sang Hyang Seri and PT. Pertani, produce about 80% of the certified seed of rice marketed in Indonesia and the rest is produced by some public seed farms and some private seed growers. Seed certification is conducted by the agency of seed inspection and certification, which has an office in each district. The system could be used for hybrid rice seed; however, since seed production for hybrid rice is different from that for inbreds, some adjustment is needed. Figure 1 shows the proposed seed system for seed production and quality control for hybrid rice. This system is for public hybrids, whereas, for private hybrids, all of the system is controlled by the seed company as the owner of the hybrids. PT. Sang Hyang Seri began to produce the released public hybrids Rokan and Maro in a total area of 3 ha for the A line and 25 ha for the hybrids. PT. BISI is producing its released hybrids Intani 1 and Intani 2 in an area of at least 50 ha. No information is available for the seed production of the other hybrids released by PT. Kondo, Mitsui 1, 2, and Suwarno et al

278 Development of cultivation area The existing released hybrids are susceptible to BPH, BLB, and RTV, the major rice pests in Indonesia. The hybrids could not express heterosis at some test locations perhaps because of the effect of genetic environment interactions. Therefore, the program for developing a cultivation area of hybrid rice began in 15 districts considered as the major rice-growing area, with the following criteria: (1) the area has fertile soil, (2) the area has no problem of irrigation water, (3) it is not an endemic area of the major pests, and (4) the farmers are responsive to new technology or are progressive farmers. Hybrid rice cultivation is being implemented in a total area of 60 ha or 4 ha in each identified district during this dry season. The area will be increased to a total of about 1,000 ha distributed in the suitable districts during the wet season of It is expected that the hybrid rice area will gradually increase to at least 500,000 ha in the WS. Hybrid seed is provided freely to farmers for the 2002 DS and WS cultivation. Pesticide will also be provided and distributed to farmers freely when it is needed. For the following season, hybrid seed will be provided as a revolving fund or credit to be paid after harvesting. The extension officer in charge will guide farmers on hybrid rice cultivation supervised by a trained technician who will stay at each site. References Hoan NT, Kinh NN, Bong BB, Tram NT, Qui TD, Bo NV Hybrid rice research and development in Vietnam. In: Virmani SS, Siddiq EA, Muralidharan K, editors. Advances in hybrid rice technology. Proceedings of the 3rd International Symposium on Hybrid Rice, Nov. 1996, Hyderabad, India. Manila (Philippines): International Rice Research Institute. p Lin JY, Pingali PL Economic assessment of potential for hybrid rice in tropical Asia. In: Virmani SS, editor. Hybrid rice technology: new developments and future prospects. Selected papers from the International Rice Research Conference. Manila (Philippines): International Rice Research Institute. p Paroda RS Hybrid rice technology in India. In: Virmani SS, Siddiq EA, Muralidharan K, editors. Advances in hybrid rice technology. Proceedings of the 3rd International Symposium on Hybrid Rice, Nov. 1996, Hyderabad, India. Manila (Philippines): International Rice Research Institute. p Virmani SS, Viraktamath BC, Casal CL, Toledo RS, Lopez MT, Manalo JO Hybrid rice breeding manual. Los Baños (Philippines): International Rice Research Institute. 151 p. Yuan LP Increasing yield potential in rice by exploitation of heterosis. In: Virmani SS, editor. Hybrid rice technology: new developments and future prospects. Selected papers from the International Rice Research Confernce. Manila (Philippines): International Rice Research Institute. p 1-6. Hybrid rice research and development in Indonesia 295

279 Notes Authors addresses: Suwarno, Y.P. Munarso, and M. Direja, Research Institute for Rice, Bogor; Suwarno, Directorate of Seed Development, Jakarta, Indonesia Citation: Virmani SS, Mao CX, Hardy B, editors Hybrid rice for food security, poverty alleviation, and environmental protection. Proceedings of the 4th International Symposium on Hybrid Rice, Hanoi, Vietnam, May Los Baños (Philippines): International Rice Research Institute. 407 p. 296 Suwarno et al

280 Hybrid rice research and development in the Democratic People s Republic of Korea Ri Tae Sik and Rim Yun Uk In recent years, the development of hybrid rice has focused on intersubspecific hybrids for high-yielding rice varieties. Yield per hectare in intersubspecific hybrids may increase by 30 50% over that of the best check variety because of its high heterosis. Many constraints and challenges exist, however, for the development of intersubspecific hybrids. The main problem in breeding a hybrid between two subspecies by the three-line method is to solve the problem of unstable sterility, F 1 semisterility, and delay of heading date and to improve hybrid seed production. Hybrid rice research and development began in the Democratic People s Republic of Korea in the mid-1970s. In the early stage, maintainer lines were tested and restorer lines suitable for CMS-boro II cytoplasm were selected with consecutive backcrossing to develop sterile lines. In 1989, the first japonica hybrid rice, Donghae 1, was developed and released in several areas. But this hybrid rice was not released in the early 1990s because of the higher yield of conventional (check) varieties. Because of the low heterosis of japonica hybrids, the breeding program for intersubspecific hybrids has been carried out since then. The major problems for the breeding of intersubspecific hybrids were the unstable sterility of boro-type (BT) cytoplasm, F 1 sterility, delay of heading date, and cold susceptibility. Most varieties or pure lines developed in Korea DPR are maintainers. The main factor affecting the sterility of cytoplasmic male sterile (CMS) lines is considered to be the nuclear background of maintainers. Only when each individual within a maintainer is tested and selected can the stable sterile line be developed. Some combinations of intersubspecific hybrids show delay of heading date. Such a phenomenon is mainly controlled by three multiple alleles, Lj, Li, and Ln, and the allele Ln shows dominance. Once japonica carrying Ln or Li and indica carrying Lj are developed, delay of heading date in intersubspecific hybrids can be overcome. 297

281 Because the maintainer and its family developed in Korea DPR are wide compatibility (WC) lines, their intersubspecific hybrids are fertile. Using the S-5i gene is more suitable at the Rice Research Institute (RRI). F 1 sterility varies with the relationship of parents and is controlled by a major gene. Japjong 1, 2, and 3, the intersubspecific hybrids developed in 1993, show yield potential of more than 10 t ha 1, but they have some defects. 319/96 and 322/96, intermediate combinations tested in recent years, have a yield potential of 10 t ha 1. Materials and methods Using the different japonica and indica varieties, including CMS lines, maintainers, and restorers developed at RRI, research work for developing a high-yielding hybrid was conducted. Each plant of the CMS line was isolated by bags when flowering occurred. Crossing work was conducted in the greenhouse and F 1 hybrid combinations were grown in the field. They were transplanted with a planting space of a single seedling per hill and 120 plants per 3.3 m 2. Nitrogen fertilizer was supplied at kg ha 1 and soil conditions were more fertile. Development of a stable sterile line by BT cytoplasm Effect of boro-type (BT) cytoplasmic instability on yield Many researchers reported that BT cytoplasm is generally more unstable than the wild abortive (WA) type under a different environment. According to the fluctuation of environmental conditions, some CMS plants have little fertility themselves. This phenomenon, however, does not seriously affect the yield of hybrid rice and is reversible. In fact, the problem was to discover which semifertile individuals were similar to apparent CMS lines. Off-type plants were increased by geometrical progression according to the foundation seed multiplication of CMS lines for several years (Table 1). As Table 1 shows, purity of the newly developed CMS foundation seed was 98.6% in the first year, but decreased to 47.6% after three years. Such a phenomenon directly affects the yield of hybrid rice and the results showed a negative effect (Table 2). Therefore, a yield advantage of hybrid rice cannot be demonstrated if the occurrence of off-types is not prevented. Factors affecting the stability of CMS lines CMS lines developed by using different original maintainers with BT cytoplasm showed different rates of off-types (Table 3). CMS lines developed by maintainers derived from japonica intervarietal progenies showed stable sterility, while those from the intersubspecific progeny are unstable. In general, japonica varieties act as maintainers on BT cytoplasm whereas indica varieties act as restorers. Because the restorer gene R 1 f 1 and many minor genes contained in indica rice act on BT cytoplasm, the subsequent generation shows a 298 Ri Tae Sik and Rim Yun Uk

282 Table 1. Rate of off-types according to CMS line multiplication. Year Off-type Purity of (%) CMS (%) Foundation seed Table 2. Change of hybrid yield according to rate of off-type plants. Off-type Replication yield (t ha 1 ) Mean Difference (%) (t ha 1 ) (%) I II III IV Table 3. Stability of CMS lines according to nuclear background of maintainers. Type Line Normal Off-type Stability pollen (%) (%) (%) Intervarietal progeny Intersubspecific progeny normal distribution. From this, we can consider that the instability of maintainers derived from intersubspecific progenies is due to the minor genes of indica rice (Table 4). CMS line A14 is a sterile one with stability. When A14 was crossed with a japonica maintainer, the stability of F 1 sterility was confirmed. The stability of the F 1, however, decreased in crossing with maintainers derived from intersubspecific progenies. To explain such a result, we investigated and analyzed the fertility of each plant within the unstable CMS line using individual isolation (Table 5). As shown in Hybrid rice research and development in the Democratic People s Republic of Korea 299

283 Table 4. Stability in different F 1 combinations (A/B). Combination a B line derived from Seed set Stability (%) (%) A14/B163 Intervarietal progeny A14/B162 Intervarietal progeny A14/B465 Intervarietal progeny A14/B504 Intervarietal progeny A14/B206 Intersubspecific progeny A14/B131 Intersubspecific progeny A14/B319 Intersubspecific progeny A14/B203 Intersubspecific progeny a A14 is a japonica CMS line. Table 5. Pollen fertility and self seed setting of individual plants in CMS lines. Plant Pollen Self seed Plant Pollen Self seed fertility setting (%) fertility setting (%) (%) (%) Table 5, half of the lines showed pollen fertility and self seed setting among 20 plants selected randomly within CMS line 237A. After ratooning these lines, they were crossed with other random individuals of maintainers and their F 1 self seed setting was investigated (Table 6). Among 20 combinations, only 9 showed complete sterility and 11 showed self seed setting, so the frequency of complete sterility was lower than in Table 5. For example, CMS plant numbers 5, 8, and 10 had complete sterility in Table 5, but they showed self seed setting in the F 1 crossed with another random individual maintainer. Thus, it must be true that instability is due to a nuclear maintainer background. For a more exact identification, we analyzed the self seed setting of F 1 s by crossing a stable CMS plant, A, with all of the B-line plants (Table 7). Although CMS line A was already a stable line, some combinations showing self seed setting were revealed according to the families of B lines. This shows that the instability of CMS lines is related to maintainers. Therefore, stable CMS lines can be developed if promising B lines with genetic purity are selected. 300 Ri Tae Sik and Rim Yun Uk

284 Table 6. Self seed setting in F 1 CMS plants crossed with other random B plants. Combination Self seed Combination Self seed setting (%) setting (%) 237-1A/237-1B A/237-11B A/237-2B A/237-12B A/237-3B A/237-13B A/237-4B A/237-14B A/237-5B A/237-15B A/237-6B A/237-16B A/237-7B A/237-17B A/237-8B A/237-18B A/237-9B A/237-19B A/237-10B A/237-20B 18.0 Table 7. F 1 self seed setting in crossing A with all the B- line plants. Combination Self seed Combination Self seed setting (%) setting (%) A/237-1B A/237-11B A/237-2B A/237-12B A/237-3B A/237-13B A/237-4B A/237-14B A/237-5B A/237-15B A/237-6B A/237-16B A/237-7B A/237-17B A/237-8B A/237-18B A/237-9B A/237-19B A/237-10B A/237-20B 13.9 The characteristics of stable CMS line 237A developed in such a way appear in Table 8. Growth delay Heading date in some combinations of intersubspecific hybrids was much later than that of the parents (Table 9). The heading of F 1 between japonica P-33 and japonica varieties, however, was late, whereas that between P-33 and indica varieties was normal (Table 10). This indicates that japonica P-33 has a heading gene derived from its parent, indica Nanjing 11. A similar phenomenon also appeared in some combinations using indica variety M23. Heading of the F 1 between M23 and indica varieties showed a delay, whereas the F 1 between M23 and japonica varieties was normal (Table 11). Hybrid rice research and development in the Democratic People s Republic of Korea 301

285 Table 8. Characteristics of CMS line 237A. Line Culm length (%) Panicle length (%) Panicles plant 1 (no.) Fertility (%) M a S CV M S CV M S CV (%) (%) (%) 237B A a M = mean, S = significant difference, CV = coefficient of variation. Table 9. Delay of heading date in intersubspecific F 1 combinations. Combination Type a Heading date No. of days delay compared with those P 1 P 2 F 1 of late parent P15/70 J/I 27 July 1 Aug 11 Sep 40 70/6 I/J 31 July 28 July 16 Sep 47 70/H37 I/J 31 July 16 July 10 Sep 40 R4/250 J/I 19 July 30 July 1 Sep 32 H37/250 J/I 17 July 30 July 9 Sep /H41 I/J 31 July 31 July 20 Sep 50 a J = japonica, I = indica. Table 10. Heading date in F 1 combinations that used japonica P-33. Combination Type a Heading date No. of days delay compared with those P 1 P 2 F 1 of late parent P33/70 J/I 28 July 31 July 28 July 3 P33/250 J/I 26 July 30 July 28 July 3 P15/p33 J/J 27 July 22 July 30 Aug 34 P33/H37 J/J 22 July 17 July 30 Aug 39 R-4/p33 J/J 19 July 24 July 30 Aug 37 P33/H47 J/J 22 July 30 July 4 Sep 36 a J = japonica, I = indica. Therefore, we can assume that indica M23 has a heading gene derived from its parent, japonica Suwen 232. Indica IR54, however, showed a normal heading date in the F 1 of all combinations, unlike the above varieties (Table 12). Putting all the data together, we suppose that the major gene delaying the heading date of intersubspecific F 1 exists among genes concerning heading. To test this supposition, the segregation ratio of the late and normal plants was investigated in the BC 1 F 1 and F 1 generations (Table 13). 302 Ri Tae Sik and Rim Yun Uk

286 Table 11. Heading date in F 1 combinations that used indica variety M23. Combination Type a Heading date No. of days delay compared with those Parent Parent F 1 of late parent P15/M23 J/I 27 July 2 Aug 1 Aug 1 P10/M23 J/I 30 July 2 Aug 8 Aug 6 H37/M23 J/I 18 July 2 Aug 29 July 5 P4/M23 J/I 8 Aug 2 Aug 7 Aug 5 97/M23 I/I 20 July 2 Aug 19 Sep 48 70/M23 I/I 31 July 2 Aug 10 Sep 39 a J = japonica, I = indica. Table 12. Heading date in F 1 combinations using IR54. Combination Type a Heading date No. of days delay compared with those Parent Parent F 1 of late parent R14/R54 J/I 19 July 9 Aug 31 July 9 B204/IR54 J/I 23 July 9 Aug 7 Aug 2 P15/IR54 J/I 27 July 9 Aug 7 Aug 2 H41/I/54 J/I 30 July 9 Aug 7 Aug 2 250/IR54 I/I 31 July 10 Aug 20 Aug 10 V20/IR54 I/I 20 July 10 Aug 15 Aug 5 70/IR54 I/I 31 July 10 Aug 15 Aug 5 a J = japonica, I = indica. Table 13. Segregation of heading date in B 1 F 1 and F 1. Generation No. of No. of plants X² P combinations Normal Delayed Total BC 1 F F , As shown in Table 13, late and normal plants in BC 1 F 1 and F 1 segregated at a 1:1 ratio. Therefore, we designated genes controlling heading as follows: Lj japonica gene, Li indica gene, Ln normal gene. This gene has three multiple alleles, Lj, Li, and Ln. Here, Ln has dominance. Thus, phenotypes of heading according to genotype are as follows: LjLj normal LiLi normal LnLn normal LjLn normal LiLn normal LiLj delayed Hybrid rice research and development in the Democratic People s Republic of Korea 303

287 ZO (indica) O. spontanea O-1 (japonica) 595 (japonica) Peidi (indica) 172 (japonica (F 3 ) Fig. 1. Breeding path of maintainer Table 14. Fertility in intersubspecific F 1 combinations. Cross type a Combination Spikelets Filled grains Seed panicle 1 panicle 1 set (%) (no.) (no.) I/J 123/P J/I P-9/ I/J 123/P J/I P-15/ J/I P-15/IR J/I P-9/IR I I IR J P J P a I = indica, J = japonica. From this, it is expected that japonica P-33 has Li, whereas indica M23 and IR54 have Lj and Ln, respectively. The delay in heading in intersubspecific F 1 hybrids may be overcome, once japonica carrying Ln or Li and indica carrying Lj are developed. F 1 sterility Development of wide compatibility (WC) lines Path for breeding. The breeding path of WC line with exserted stigmas appears in Figure 1. Test of wide compatibility. Generally, F 1 combinations between japonica and indica show semisterility. We selected parents of combinations showing F 1 sterility in intersubspecific hybrids for WC testing of maintainer (Table 14). In the F 1 between japonica 304 Ri Tae Sik and Rim Yun Uk

288 Table 15. Results of wide compatibility gene test on Combination Cross type Seed set (%) /123 S-5n/I /P-15 S-5n/J /123 S-5n/I /P-9 S-5n/J /123 S-5n/I /P-15 S-5n/J /123 S-5n/I /P-15 S-5n/J 96.0 N22/IR9711 S-5n/I 92.5 N22/P-9 S-5n/J 85.2 Dular/123 S-5n/I 81.3 Table 16. Range of seed set in intersubspecific F 1 hybrids. Combination Type a Seed set (%) /V20 J/I /6 I/J /H37 I/J 15.8 H37/250 J/I /IR20 J/I 16.9 P-15/90 J/I 72.0 M23/6-5-4 I/J 69.3 M23/P-15 I/J /M54 J/I 67.3 a J = japonica, I = indica. varieties (P-9 and P-15) and indica (123 and IR9711), seed set ranged from 43.4% to 67.9% irrespective of reciprocal crossing. Thus, P-9 and P-15 can be used as the japonica testers and 123 and IR9711 as indica ones. The results of the WC gene test on families appear in Table 15. F 1 seed set between testers and was above 80% irrespective of combination type. Therefore, undoubtedly has the WC gene. Also, it is thought that these lines had effects similar to those of WC gene lines known already. F 1 sterility depending on genetic distance between parents According to the WC gene theory, the seed setting rate of intersubspecific F 1 hybrids should be distributed in a range of 50%. In our experiment, however, seed was distributed in a wide range (Table 16). Considering the seed set of indica/japonica F 1 hybrids and the relationship of parents, it can be noted that seed set varies with genetic distance between parents (Table 17). Hybrid rice research and development in the Democratic People s Republic of Korea 305

289 Table 17. F 1 seed set according to relationship between parents. Line Origin Analyzed Average combinations seed set (%) 90 Intersubspecific IR8 Intersubspecific M54 Intersubspecific IR58 Intervarietal V20 Intervarietal IR20 Intervarietal Plants (no.) Fertility (%) Fig. 2. Distribution of seed set in F 1. Table 18. F 1 seed setting between intracombination lines and indica lines. Combination Seed set (%) Combination Seed set (%) /IR / /IR / /IR / /IR / /IR / / Hybrid sterility showed a normal distribution, varying with relationship between parents. Based on the WC gene theory, seed setting in indica/japonica results in semisterility and the ratio of semisterile to fertile plants is 1:1 in the F 1. Variation of seed set in the F 2 revealed a normal distribution in our experiment (Fig. 2). Such a phenomenon also occurred in the B 1 F 1. In the F 1, between intracombination lines of BC 1 F 1 and indica, fertility showed a different level (Table 18). The seed setting rate of the F 1 between intracombination lines and IR58 ranged from a maximum of 59.2% to a minimum of 30.0%. Similarly, the F 1 seed setting rate of combinations using indica 90 ranged from a maximum of 95.3% to a minimum of 306 Ri Tae Sik and Rim Yun Uk

290 73.1%. On the other hand, F 1 fertility between maintainers in the BC 1 F 1 and indica rice showed a strong difference according to the individual plants (Table 19). As shown in Table 19, the differences in F 1 fertility between the pure lines (4465 and 1644) and indica 90 were only 5 6%, but that between maintainers in BC 1 F 1 and indica was %. Therefore, we can recognize that F 1 fertility of indica/japonica crosses is controlled by minor genes in addition to a major gene. When the parents with a moderate relationship are used, not only can higher heterosis be expressed, but also the spectrum of compatibility is wider than with the use of indica varieties (Table 20). When using restorer 90, lines showing a normal fertility of the F 1 occurred more than with the use of another indica. Thus, it is more possible that the combinations showing normal fertility can be selected in large numbers among F 1 combinations with restorer 90. Use of S-5i allele According to the WC gene theory, lines derived form indica/japonica have the S-5i allele (Fig. 3). Japonica varieties derived from indica/japonica show a higher fertility in crossing with indica (Table 21). Thus, using these lines, we can develop intersubspecific hybrids. According to our experiment, intersubspecific F 1 combinations using japonica (S-5i) showed a higher performance (Table 22). Therefore, once the sterile line and restorer with S-5i are developed, the promising intersubspecific hybrid can be developed. Development of intersubsepecific hybrids The yield potential of hybrids Japjong 1, 2, and 3 appears in Table 23. Hybrids Japjong 1, 2, and 3 as intersubspecific combinations yielded at least 10 t ha 1. Standard heterosis over the check variety showed a range of 24 61%. As mentioned earlier, CMS lines 129A and 237A are WC lines and CMS line 203A is a japonica from intersubspecific hybrids having the S-5i allele. Restorer lines 90 and 78 and the newly developed indica and CMS lines 129A and 237A have exserted stigma rates of 42% and 51%, respectively. In the multiplication field, 129A showed an outcrossing rate of 40% and 237A had 45%, whereas 203A with nonexserted stigma had 18.5%. Seed production in the multiplication field of 230A, however, was 1,030 kg ha 1. Hybrid seed production per hectare of 129/90, 230/90, and 203/78 was 508, 856, and 403 kg, respectively. According to the year, yield stabilities of these hybrids changed and grain quality was poor. These hybrids also showed a lower filled grain rate and susceptibility for disease, so they were not released on-farm. Hybrid rice research and development in the Democratic People s Republic of Korea 307

291 Table 19. Seed set per plants of F 1 between maintainers in BC 1 F 1 and indica lines. Plant Range Mean Variance Standard combinations deviation /V /IR /IR / / / Table 20. Test of WC gene according to origin. Taxonomy Use of restorer 90 Use of another indica Normal Segregating Semi fertility fertility fertility Combinations (no.) Rate (%) Ri Tae Sik and Rim Yun Uk

292 Parents S-5j/S-5j x S-5j/S-5i F S-5j/S-5i (semisterility) 1 F 2 S-5j/S-5i (fertility) S-5j/S-5i (fertility) F 3 S-5j/S-5i (semisterility) S-5j/S-5i (fertility) S-5j/S-5i (fertility) Fig. 3. Segregation of seed set in progeny of indica/japonica. Table 21. Seed set of F 1 combinations using japonica lines (S-5i) derived from intersubspecific hybrids. Combination Type Seed set (%) 10/53 Japonica/indica /49 Japonica/japonica /53 Japonica/indica /49 Japonica/japonica /53 Japonica/indica /49 Japonica/japonica /53 Japonica/indica /49 Japonica/japonica /53 Japonica/indica /53 Japonica/indica /53 Japonica/indica /53 Japonica/indica 87.1 Table 22. Performance of F 1 combinations using japonica (S-5i). Hybrid Hybrid advantage over check a t ha 1 % 241/ / / / / a The check is a japonica, P-33. Hybrid rice research and development in the Democratic People s Republic of Korea 309

293 Table 23. Yield potential of hybrid rice. Combination Hybrid Heading Culm Panicles Spikelets 1,000- Seed Yield Difference date length plant 1 panicle 1 grain set (t ha 1 ) (t ha 1 ) (%) (cm) (no.) (no.) weight (%) (g) P-33 Check 30 Jul /90 Japjong-1 12 Aug /78 Japjong-2 6 Aug /90 Japjong-3 17 Aug Ri Tae Sik and Rim Yun Uk

294 Table 24. Yield potential of hybrids 319/96 and 322/96. Hybrid Heading Culm Panicles Spikelets 1,000-grain Seed Yield date length (cm) per 3.3 m 2 panicle 1 weight (g) set (%) (kg ha 1 ) (no.) (no.) 319/96 15 Aug , /96 18 Aug ,473 Development of intermediate hybrids To overcome defects (instability of yield, poor grain quality) appearing in the development of intersubspecific hybrids, we developed an intermediate hybrid recently. CMS lines 319A and 322A from intersubspecific progenies were developed by a maintainer carrying the S-5i allele. Restorer line 96 as an intermediate has the S-5i allele (Table 24). These hybrids have a yield potential of around 10 t ha 1. Their hybrid seed production showed a range of 700 1,000 kg ha 1 and outcrossing rate of CMS lines was about 25%. Conclusions Completion of the hybrid system is important for increasing rice production in Korea DPR. In research over the long term for hybrid development, we solved some problems arising in the breeding of intersubspecific hybrids. The genetic and breeding research work was conducted widely and some combinations with a high yield potential were selected. In recent years, research to combine heterosis and ideotype has been conducted on the basis of achievements obtained earlier. For research work in this field, ideotype is an important criterion for setting up the breeding method. Notes Authors address: Ri Tae Sik is the director and Rim Yun Uk is the chief of hybrid rice, Rice Research Institute, Academy of Agricultural Sciences, Democratic People s Republic of Korea. Citation: Virmani SS, Mao CX, Hardy B, editors Hybrid rice for food security, poverty alleviation, and environmental protection. Proceedings of the 4th International Symposium on Hybrid Rice, Hanoi, Vietnam, May Los Baños (Philippines): International Rice Research Institute. 407 p. Hybrid rice research and development in the Democratic People s Republic of Korea 311

295 Hybrid rice research and current status in Korea S.J. Yang, Y.C. Song, and H.P. Moon Hybrid rice research in Korea started in the early 1970s, but the national hybrid breeding program started in the early 1980s. The Korea-IRRI collaborative research project has played a significant role in the progress of the hybrid rice program. Hybrid breeding was active in 1985, when some wild abortive (WA)-type cytoplasmic male sterile (CMS) systems were introduced. Since then, tongil-type (derived from indica/japonica) cultivars Samgangbyeo, Taebagbyeo, Cheongcheongbyeo, and Milyang 23 and some breeding lines have been identified as maintainers. Most tongil-type cultivars showed good fertility restoration for WA-type lines. Several experimental hybrids yielded 4 34% more than elite cultivars until However, Korean consumers do not prefer their high chalkiness and high-amylose grain, so the tongil-type hybrid rice breeding program was reduced. Nevertheless, WA-type lines, maintainers, and restorers are being continuously developed. Japonica CMS lines were developed by transferring the boro-type CMS source into elite Korean cultivars and breeding lines in 1994 and backcrossing is being used to develop japonica restorer lines with some success. Forty CMS lines have been developed during the past six years by transferring WA types and COA types into leading Korean cultivars and breeding lines. Twenty lines are tongiltype, derived from indica/japonica hybridization, and 17 lines have the japonica genetic background. Commercial rice hybrids are not yet available to farmers because poor grain quality and labor-intensive seed production technology are major constraints. We hope to have 10 t ha 1 of milled rice with the use of hybrid rice in the 21st century; therefore, hybrid rice research in Korea restarted in the early 2000s. Korea has one of the highest rice yields per unit area in the world. The estimated average grain yield for Korea in 2001 of japonica rice was almost 6.5 t ha 1 and that of super-yielding rice was almost 9 t ha 1. Rice yields have increased by developing super-yielding varieties, but with the grain quality of current japonica rice. Exploiting heterosis is seen as a means of further increasing yields. The country has to maintain its current level of growth in rice production. Increasing productivity per unit 313

296 Table 1. New cytoplasmic genic male sterile lines developed in Suweon and Milyang, CMS line Ecotype a Origin of CMS line Cheonmabyeo A J Toyonishiki A Sobaegbyeo A J V 20 A Sobaegbyeo A J Toyonishiki A Hwaseongbyeo A J Reimei A Cheolweon 34 A J Reimei A Samnambyeo A J Reimei A Yeongdeogbyeo A J Reimei A IR58025 A I IR62829 A I IR64608 A I IR68886 A I IR68897 A I IR69628 A I Samgangbyeo A I/J IR58026 A Hangangchalbyeo A I/J IR58025 A Suweon311 A I IR58025 A Suweon311 A I IR58026 A Pusao A I HepcheongjoA I a I = indica, J = japonica. area is becoming more important. The use of high-yielding varieties and improved crop management practices is being vigorously pursued to achieve this objective. One of the approaches is the development of hybrids. The objective of the research was to explore the prospects and problems of using hybrid rice in Korea. The research included studies on grain quality, heterosis, male sterility and fertility restoration systems, hybrid seed production techniques, and the environmental genic male sterility (EGMS) system. The successful development of hybrid rice in Korea depends largely on the availability of good cytoplasmic male sterile and restorer lines with sustainability. This paper reports the current status of and problems encountered in hybrid rice research in Korea. New cytoplasmic male sterile lines Nineteen new CMS lines were bred possessing wild abortive (WA) and BT cytoplasm (Table 1). The WA-CMS of V 20A, Zhen Shan 97A, and V 5-20A was transferred into some selected tongil-type maintainers by backcrossing. Some japonica CMS lines were also developed by transferring the CMS system of V 20A, ReimeiA, and COA-ms into some elite japonica cultivars. While developing male sterile lines of Hwaseongbyeo AKeumobyeo *3 2 and IR68886 Adaesanbyeo *3, sterility was found to be stable (Table 2). 314 Yang et al

297 Table 2. New cytoplasmic male sterile lines developed in Suweon and Milyang, Combination Ecotype a Generation Source of cytoplasm b Hwaseongbyeo A/Keumobyeo 2 J BC 3 BT Hwaseongbyeo A/Ilpumbyeo J BC 3 BT Milyang 96 A/YR14750Acp 30 J BC 6 BT Gwanakbyeo A/YR14750Acp 30 J BC 6 BT Hyeobcheongjo A/Taebackbyeo I/J BC 3 WA Joseongtongil A/IR I BC 5 WA V 20 A/Yongmunbyeo I/J BC 3 WA IR62829 A/Hangangchalbyeo I/J BC 4 WA IR68886 A/Daesanbyeo I/J BC 4 WA IR68886 A/milyang 161 I/J BC 2 WA IR68888 A/Milyang 23 I/J BC 4 WA IR69628 A/Andabyeo I/J BC 4 WA IR69629 A/Iksan 449 I/J BC 2 WA a I = indica, J = japonica. b WA = wild abortive, BT = boro type. Table 3. Restorer lines identified in Suweon and Milyang, Cultivar Ecotype a Tester CMS lines Cheongcheongbyeo (Milyang 46) I/J Tongil-type A Nampungbyeo (Suweon 294) I/J Taebaegbyeo (Suweon 287) I/J Suweon 318 I/J Jangseongbyeo (Iri 362) I/J Yongmoonbyeo (Suweon 332) I/J Yongjubyeo (Suweon 325) I/J 1470SF2 J Sobaegbyeo A, Cheolweon34 A 14714F2 J HB474-3 J HB J a I = indica, J = japonica. Developing restorer lines Restorer lines were selected from testcross nurseries based on the fertile reaction of the F 1 plants. Breeding lines and cultivars developed locally were crossed with Z 97A, V 20A, and/or Cheongcheongbyeo, Nampungbyeo, Taebaebyeo, Suweon 318, Jangseongbyeo, Yongmoonbyeo, and Yongjubyeo, which were identified as strong restorers (Table 3). In developing japonica restorer lines of Sangju 1R *8 /Sangju 1, Gihobyeo R *8 / Gihobyeo, and Koshi R *7 /Koshi, spikelet fertility, measured at 87%, 80%, and 85%, respectively, was recovered successfully (Table 4). Hybrid rice research and current status in Korea 315

298 Table 4. Developing restorer lines of japonica male sterile lines in Milyang, Combination BC generation Spikelet fertility (%) BT-C/AR-3//Paldal BC 9 85 BT-C/AR-3//Hwajinbyeo BC 9 90 BT-C/AR-3//Sangju 1 BC 8 87 BT-C/AR-3//Gihobyeo BC 8 80 BT-C/AR-3//Suseong BC 8 82 BT-C/AR-3//Kosl BC 7 85 BT-C/AR-3//Milseong BC 7 79 Table 5. Yield performance and standard heterosis (SH) of the best experimental hybrid rice evaluated in Suweon and Milyang, Hybrid Year Yield (t ha 1 ) SH a (%) Test location Mikao A/Dasanbyeo Milyang Hangangchalbyeo A/Nampungbyeo Suweon Hepcheongjo A/Yongjubyeo Suweon Samgangbyeo A/Cheongcheongbyeo Suweon Samgangbyeo A/Namcheonbyeo Milyang Z97 A/Taebackbyeo Milyang IR69628A/Taebackbyeo Milyang IR58025A/Cheongcheongbyeo Milyang IR68897A/Andabyeo Milyang IR68897A/Namcheonbyeo Milyang IR69628A/Cheongcheongbyeo Milyang PMS4 A/Andabyeo Milyang Mean a Yield index when compared with the best check variety at the tested location. Yield ability of hybrids In a yield trial, several experimental F 1 hybrids have significantly outyielded the best inbred check cultivars (Table 5). Grain yield was highest in the best experimental rice nurseries. Heterosis for grain yield over that of the best inbred check cultivars ranged from 4% to 32%, with an average of 14%. The most promising rice hybrid was IR58025A/Cheongcheongbyeo, which yielded 12.4 t ha 1. Suweon hybrid 1 and Suweon hybrid 2 were developed as the first hybrid rice lines at the NCES (National Crop Experiment Station). The hybrids showed field resistance to major diseases and insects (Table 6). Several F 1 hybrids were introduced from IRRI and were evaluated in Korea. Until 2000, all F 1 hybrids showed yield comparable with or inferior to that of highyielding commercial inbred varieties, when the best check was a high-yielding commercial variety, Namchenbyeo (Table 7). To test the combining ability and produc- 316 Yang et al

299 Table 6. Super high-yielding hybrid rice developed by using cytoplasmic genic male sterility. Designation Combination Growth Culm Grain yield (milled) Remarks b duration a length (d) (cm) Potential Index to (t ha 1 ) standard variety Suweon- SR16284A/ Both are Jabjong 1 Yongmoonbyeo multiresistant to BL, BB, Suweon- SR16283A/ SV, and BPH Jabjong 1 Taebaegbyeo a Growth duration indicates the days from seedling planting to harvest. b BL = blast, BB = bacterial blight, SV = stripe virus, BPH = brown planthopper. Table 7. Combining ability test and performance of introduced IRRI CMS lines in Milyang, Combination No. of Ripening Yield Standard spikelets hill 1 rate (%) (t ha 1 ) heterosis a (%) IR58025A/Cheongcheongbyeo IR58025A/Taebaegbyeo IR62829A/Cheongcheongbyeo IR62829A/Taebaegbyeo IR64608A/Cheongcheongbyeo IR64608A/Taebaegbyeo IR68886A/Taebaegbyeo IR68888A/Cheongcheonbyeo IR68888A/Taebaegbyeo IR68897A/Cheongcheogbyeo IR68897A/Taebaegbyeo IR69628A/Taebaegbyeo Namcheonbyeo (check) a Compared with that of Namcheonbyeo. tive capacity of hybrid rice, IR58028A/Cheongcheongbyeo and IR58028A/ Taebaegbyeo yielded 9.1 and 7.6 t ha 1, respectively. This is a 34% and 12% increase in yield over that of the standard variety Namcheonbyeo, which yields 6.8 t ha 1. IR64068A/Cheongcheongbyeo, IR68888A/Cheongcheongbyeo, and IR68897A/ Cheongcheongbyeo showed an increase in yield of about 21 28%. Negative standard heterosis ranging from 11% to 36% in the hybrids was mainly due to their poor adaptability to the Korean environment. Hybrid rice research and current status in Korea 317

300 Table 8. Hybrid seed production under different row ratios between CMS restorers and maintainers in Suweon, Hybrid Flag-leaf Row ratio between CMS restorers and maintainers Av clipping at 10% 1:1 2:1 3:1 4:1 6:2 8:2 heading SR16282A/ No Nampungbyeo Yes IR62829A/ No Nampungbyeo Yes Av No Yes Hybrid seed production Two new CMS lines developed in IRRI and Korea IR62829A and SR16282A were evaluated for seed production with flag-leaf clipping and different row ratios (Table 8). IR62829A had the highest seed production, with 2.2 t ha 1 with flag-leaf clipping and a row ratio of 1:1 CMS lines to maintainers. SR16282A produced seed yields of around 1.5 t ha 1. IR62829A flag-leaf clipping at 10% heading generally gave higher seed yields, regardless of the CMS line or row ratio, but SR16282A had a low seed yield. Major constraints The following factors were responsible for the slow development of hybrid rice technology in Korea: Lower research priority in the hybrid breeding program because of economic value Low rice quality (appearance and palatability) compared with Korean japonica and even tongil-type varieties Japonica restorer lines with partial fertility restoration Inadequate natural condition of TGMS response Limited yield heterosis (japonica: 5 10%) in commercial rice hybrids Slow breeding progress in commercially usable CMS and TGMS lines during the initial stages Inadequate human resources in terms of number and quality of personnel available for hybrid rice research and seed production Lack of collaboration among national programs involved in hybrid rice research and development 318 Yang et al

301 Future plans Future plans for hybrid rice in Korea include the following: Developing new CMS lines with diverse cytoplasmic and/or nuclear genetic backgrounds Attempting to transfer the CMS factor(s) and restoring-ability gene(s) and wide compatibility gene(s) to Korean tongil-type varieties Developing maintainers and restorers among elite lines wlth the new plant type and a tropical japonica background Producing CMS seed Producing hybrid seed Testing heterotic rice hybrids in national programs Evaluating IRRI-bred TGMS lines in NYAES fields to test the stability of their performance and transferring the TGMS gene(s) to local materials Conserving yield vigor in F 1 hybrids through the anther culture technique and improving production practices to maximize hybrid rice seed production Conclusions Hybrid rice in Korea yielded 10.7 t ha 1 on average in experiment plots, a yield advantage of 21% over inbred tongil-type cultivars. However, commercial hybrid rice has not yet been distributed in farmers fields because of low seed production and no preference by Korean farmers. Poor grain quality and labor-intensive seed production technology are the major constraints. We need to focus on overcoming these major constraints by activating hybrid rice research in Korea through RDA/IRRI collaborative research. Notes Authors addresses: S.J. Yang and Y.C. Song, National Yeongnam Agricultural Experiment Station, Rural Development Administration (RDA), Milyang ; H.P. Moon, National Crop Experiment Station, RDA, Suweon , Korea. Citation: Virmani SS, Mao CX, Hardy B, editors Hybrid rice for food security, poverty alleviation, and environmental protection. Proceedings of the 4th International Symposium on Hybrid Rice, Hanoi, Vietnam, May Los Baños (Philippines): International Rice Research Institute. 407 p. Hybrid rice research and current status in Korea 319

302 Hybrid rice breeding in Russia I.K. Gontcharova and S.V. Gontcharov Research on hybrid rice problems has been carried out for a long time in Russia, but it is mainly theoretical. Hybrid rice research faces several problems in Russia: (1) the lack of suitable restorers in japonica rice, which is mainly grown in Russian conditions; and (2) the low level of allogamy does not allow using hybrid seed for direct sowing; and (3) combining ability evaluations and screening for hybrid combinations with high productivity or heterosis. During the first period of research, we studied available CMS sources for possible use. Wild abortive (WA) cytoplasmic male sterility was transferred into our local lines. All tested elite lines appeared to be maintainers. So, several CMS lines were successfully developed and a restorer line breeding program started. Rf genes were transferred from IR54. Studying the genetics of fertility restoration showed that at least three different Rf genes are found in the japonica background: two genes described in the literature and the last one with regulation activity the dominant allele of this gene allows other Rf genes to act. We think that indica WA-CMS lines have this dominant allele too. The low level of cross-pollination in rice is a serious problem in Russia. Only direct sowing is used in commercial rice production. Therefore, we studied floral behavior and allogamy-connected traits. Studying the inheritance of allogamy-connected traits showed that the inheritance of floral and flag-leaf traits was determined by polygenes in many cases. Additive variance prevailed in a population for all traits except for two characteristics (stigma exsertion and stigma diameter). Gene interaction (complementary epistasis) was also noticed for all traits studied. For stigma diameter, dominance was higher; for all other traits, dominance was lower. As proved by ANOVA, the number of recessive genes in varieties is correlated with the character value for all traits except for flag-leaf length. There was overdominance in the locus of floral traits. Length and breadth of the flag leaf were partially dominant. We analyzed the correlation between all studied allogamy-connected traits and only stigma traits were closely correlated with each other. Therefore, when breeding lines with increased stigma characters, it is enough to measure only one parameter because of the high correlation between them. 321

303 Different patterns of flowering behavior during the day have a large effect on outcrossing because usually the peak of flowering of CMS lines and fertile lines does not match. We found three different patterns of flowering behavior among CMS lines and one of them was the best but the least frequent among the lines. During two years, we studied our germplasm collection for allogamyconnected traits to screen and develop lines with allogamy good enough for industrial hybrid seed production. Many samples selected as donors of allogamy traits also have good productivity traits and a suitable vegetative period. This allows us to recommend the use of the given samples for hybridization and in the search for hybrid combinations with high heterosis. Combining ability evaluation and screening of hybrid combinations for the highest productivity are obligatory in hybrid breeding. We studied 51 hybird combinations. Among these, 15 showed 15% heterosis for the following productivity traits: panicle length, spikelet number per panicle, panicle weight, plant height, and others. We can conclude that the development of hybrid rice for moderate climatic conditions (south of Russia) is quite possible, although this will require strong breeding efforts. Research on hybrid rice problems has been carried out for a long period in Russia, but it is mainly theoretical. The use of hybrid rice in Russia faces several problems: (1) a lack of suitable restorers in japonica rice, which is mainly grown in Russian conditions, (2) the low level of allogamy does not allow us to use hybrid seeds for direct sowing, and (3) the low level of heterosis in japonica rice. At the All-Russian Rice Research Institute, we conduct hybrid rice research in the following areas: (1) cytoplasmic male sterility (CMS) and the fertility restoration system, (2) the inheritance of allogamy-connected traits, (3) germplasm collections with the aim of developing lines with an allogamy level good enough for industrial hybrid seed production, and (4) combining ability evaluations to screen for hybrid combinations with high productivity or high heterosis. CMS and the fertility restoration system During the first period of research, we studied available CMS sources for possible use in our country. We studied two types of sterile cytoplasm: WA (wild abortive) and BT (boro). For BT CMS, we could only prove the results described earlier (Shinjyo 1969, 1984): one dominant gene with gametophytic action controls fertility restoration. The situation with WA-CMS is not so clear. Many researchers think that two independent dominant genes control fertility restoration (Virmani and Edwards 1983, Young 1983, Young and Virmani 1984, Li and Yuan 1986). According to this model, any one dominant allele of the Rf genes is enough to restore fertility (partially, at 322 Gontcharova and Gontcharov

304 Table 1. Proposed model of gene interaction in fertility restoration for WA-CMS in japonica rice (Krasnodar, Russia, 1991). a Genotype (only dominant Theoretical Experimental Spikelet alleles are shown) frequency frequency fertility (%) Rf 1 Rf 3 and Rf 2 Rf ( ) 0 10 for 74 plants (including all other fertile genotypes) Rf 2 Rf 3 Rf Rf 1 Rf 3 Rf Rf 1 Rf 2 Rf 3 and Rf 1 Rf 2 Rf 3 Rf a The X 2 test showed good correspondence between the theoretical and empirical results. least). However, we suppose that this is true for indica rice only. We transferred WA- CMS to japonica germplasm. After that, we started breeding to develop japonica Rf lines. All our elite cultivars appeared to be maintainers, so we crossed some of them with the indica restorer line IR54. After three backcrosses, we used self-pollination to eliminate hybrid sterility. Crossing the material obtained with our japonica WA-CMS lines resulted in a very limited share of plants with restored male fertility in the progeny. We then calculated a possible model of gene interaction. We obtained and analyzed 2,940 F 1 plants for pollen fertility. Only 11 plants had complete pollen fertility restoration, 21 plants were partially male fertile (30 60% viable pollen), 75 plants were partially sterile (less than 30% viable pollen), and all others were completely sterile. All plants were self-pollinated. We obtained seeds from 96 plants only (3.3% of all the population) because plants with less than 10% pollen fertility were sterile. For spikelet fertility, we divided all 96 plants into four classes and calculated a theoretical model with four Rf genes: Rf 1 and Rf 2 important for fertility restoration, Rf 3 a gene with regulation activity (the dominant allele allows Rf 1 and Rf 2 genes to act), and Rf 4 a gene modifier. We supposed that any gene from IR54 would have a frequency of 1/8 in the obtained population, so the combination of two independent genes would be 1/64 and the combination of three genes 1/512 (Table 1). This model was confirmed by F 2 analysis. The expected segregation ratio was 27:18:19 (fertile:partially fertile:sterile plants) or 27:9:9:19 (fertile:partially fertile:partially sterile:sterile plants) for fertile plant progenies. This segregation was confirmed experimentally. Partially fertile plant progenies segregated into two classes, 9 partially fertile:7 sterile plants, as was expected. All segregation rates proved the existence of the Rf 3 gene because of the high presence of sterile plants in all crosses. We suppose that this gene is present in all indica CMS lines but is absent in our CMS lines. Additionally, we saw that sterile pollen grains of indica CMS lines (V-20A and V-41A) were spherical and alive (though without starch) at the time of anthesis, but anthers of our WA CMS-lines had only empty deformed shells of pollen grains. Hybrid rice breeding in Russia 323

305 Therefore, we concluded that fertility restoration of japonica WA-CMS lines is controlled by two pairs of independent dominant genes and a dominant regulator gene (which is usually present in indica restorer lines and CMS lines). Gene-modifiers take part in fertility restoration also. Allogamy studies In 1999, we studied 1,200 samples from rice germplasm collections with the aim of developing lines with allogamy good enough for industrial hybrid seed production. We selected 17 samples from the local and world collection, with stigma length of more than 1.7 mm, and we used them to create new sterile lines. Seventeen samples with big anthers (more than 2.7 mm) were selected from the working collection and nine from the world collection; in the future, they will be used to create fertility restorer lines and sterility maintainers. In 2000, we studied 460 samples of a collection from the Institute of Plant Industry (St. Petersburg, Russia) and 600 samples of the world and our Institute collections for the following traits: plant height, stigma and anther length, heading, and number of spikelets per panicle. Nineteen lines with big anthers (more than 2.5 mm) and 28 lines with a big stigma (more than 2.0 mm) and 11 samples with both traits were selected among the samples of the collection from the Institute of Plant Industry. Some 59 lines that were donors of the trait big anthers, 60 lines that were donors of the trait big stigma, and 35 samples characterized by a good combination of both traits have been selected. In total, 90 samples from our Institute collection and 39 samples from the collection of the Institute of Plant Industry have been selected. Many samples selected in collections as donors of allogamy traits also have good productivity traits. The vegetative period of these lines is practically the same as that of the majority of locally bred varieties. This allows us to recommend using these samples for hybridization and in a search for hybrid combinations with high heterosis. Different patterns in the time of day of flowering for fertile and sterile plants have a large effect on outcrossing because most plants of fertile lines have a maximum of opened spikelets from 1230 to 1300 in our conditions, though Indian researchers reported a peak of opening from 0900 to 1130 on sunny days. They also reported a second flowering flush from 1730 to 1830, although we couldn t find a second flush of flowering in fertile samples. Most sterile plant spikelets opened after 1400 and 1530 and some sterile lines had a peak of flowering in our conditions at 1100 and finished flowering at Sterile lines begin to flower at 0900 and finish flowering at The peak of flowering of all sterile samples was at a different time. We found three different patterns of flowering in sterile lines. The most usual pattern is sterile plants having a maximum number of opened spikelets at 1430 and even at The second pattern is sterile plants having two peaks of opened spikelets: the first at 1300 and the second at The best pattern, a simultaneous peak of both fertile and sterile lines, was the least frequent among the 324 Gontcharova and Gontcharov

306 CMS lines. We suggest selecting sterile and fertile samples with simultaneous flowering times from the beginning of the breeding process. We find that, in undesirable environmental conditions (low temperature, cloudy sky), fertile samples show a pattern of flowering similar to that of the sterile analogs. We can use this characteristic to forecast the pattern of flowering when developing sterile lines and to determine plants that will have in the sterile cytoplasm a synchronous peak of flowering with the fertile lines. Studying the inheritance of allogamy-connected traits A low level of allogamy is a breeders problem in hybrid rice. Because of this, it was important to study the inheritance of allogamy-connected traits. For diallel crossing, we selected five varieties (Panosa, Victoria Tarsio, Pervocvet, Rodnik, and Spalchik) from the collection of our Institute with different values of outcrossing-connected traits. We studied the genetics of the following traits: anther length, stigma length, length and diameter of receptive stigma parts, stigma exsertion, and length and width of the flag leaf. The results obtained were analyzed by the Hayman method. Results showed that inheritance of floral and flag-leaf traits in many cases was controlled by polygenes, but in some crosses it resembled digenic inheritance, with a segregation ratio of 9:7. In floral traits (anther length, stigma length, length and diameter of receptive stigma part, stigma exsertion), inherited additive dispersion was predominant over nonadditive dispersion. In flag-leaf traits (length and width of flag leaf), inherited additive dispersion was predominant. Additive variance prevailed in a population for all traits, with the exception of two characteristics stigma exsertion and stigma diameter. Gene interaction (complementary epistasis) was also noticed for all traits studied. For stigma diameter, the higher expression of the trait was dominant; for all other traits, a lower expression was dominant. ANOVA showed that the number of recessive genes in varieties is correlated with the character value for all traits, with the exception of flag-leaf length. There was overdominance in the locus of floral traits. Length and width of the flag leaf were partially dominant in the varieties used in diallel crossing. Genetic analyses of F 1 and F 2 hybrids were carried out to confirm the results obtained in Hayman diallel crossing. Segregation in the F 2 population for all traits studied was in correspondence with theoretically expected models for additive gene interaction (cumulative polymery) and complementary epistasis. These results confirm conclusions made on the basis of the diallel cross analyses. Thus, additive and nonadditive effects were important in the inheritance of allogamy-affected traits (Table 2). Analyses of variance showed that the genotype of the pollinated line is more important for cross-pollination (18%) than the genotype of the fertile line (16%). The interaction effect is 22.7%. Efficiency of cross-pollination is mainly determined by the genotype of the parents (57.8%). Hybrid rice breeding in Russia 325

307 Table 2. Genetic system of allogamy-affected traits. Trait Gene interaction Interaction inside Direction of locus dominance Anther length Complementary epistasis Overdominance Less value Length of receptive Complementary epistasis Overdominance Less value parts of stigma Stigma exsertion Complementary epistasis Overdominance Less value Stigma length Complementary epistasis Overdominance Less value Stigma diameter Complementary epistasis Overdominance Higher value Flag-leaf length Complementary epistasis Partial dominance Less value Flag-leaf width Complementary epistasis Partial dominance Less value Angle between flag leaf Complementary epistasis Additive action Less value and stem Angle between first, Complementary epistasis Overdominance Less value second, and third leaves and stem Panicle exsertion Complementary epistasis Partial dominance Less value Table 3. Correlation coefficient between allogamy-connected traits. Traits Panicle Flag-leaf Flag-leaf Anther Stigma exsertion length breadth length length Angle between flag leaf and stem Panicle exsertion Flag-leaf length Flag-leaf width Anther length We analyzed the correlation between the next traits: anther length, stigma length, length and diameter of receptive stigma parts, stigma exsertion, plant height, panicle exsertion, flag-leaf length and width, and angle between the flag leaf and stem (Table 3). Only stigma trait expression was closely correlated. The correlation coefficient between stigma length and length of receptive stigma parts was 0.812, between stigma length and stigma diameter it was 0.794, and between length of receptive stigma parts and stigma diameter it was (Table 4). So, in developmental lines with increased stigma character expression, it is enough to measure only one parameter because of the high correlation between them. Heterosis studies Combining ability evaluation and screening of hybrid combinations for the highest productivity are obligatory in hybrid breeding. We studied 51 hybrid combinations. 326 Gontcharova and Gontcharov

308 Table 4. Correlation coefficient between allogamy-connected traits. Traits Anther Stigma Length Stigma Stigma length length of PPS a diameter exsertion Stigma length Length of PPS Stigma diameter Stigma exsertion Plant height a PPS = receptive parts of stigma. Table 5. Heterosis values for some important traits (Krasnodar, Russia). Hybrid combination Grains Spikelets Main Additional Grain Av panicle 1 panicle 1 panicle panicle yield (no.) (no.) weight (g) weight (g) plant 1 (g) VNIIR7718/VNIIR VNIIR7718/Fontan VNIIR7718/Narciss Narciss/Fontan Narciss/VNIIR Liman/Fontan VNIIR7718/Liman Among them, 15 showed 15% higher heterosis for the productivity traits panicle length, spikelet number per panicle, panicle weight, plant height, and others (Table 5). In general, we can conclude that developing hybrid rice for moderate climatic conditions (south of Russia) is quite possible, though it will require a lot of breeding efforts. References Li C, Yuan LP Genetic analysis of fertility restoration in male sterile lines of rice. In: Rice genetics. Los Baños (Philippines): International Rice Research Institute. Shinjyo Ñ Cytoplasmic genetic male sterility in cultivated rice Oryza sativa L. II. The inheritance of male sterility. Jpn. J. Genet. 44: Shinjyo C Cytoplasmic genetic male sterility and fertility restoration in rice having genome A. In: Biology of rice. Tokyo. p Virmani SS, Edwards IB Current status and future prospects for breeding hybrid rice and wheat. Adv. Agron. 36: Young J Cytoplasmic-genetic male sterility and fertility restoration in rice. M.S. thesis. Manila (Philippines): International Rice Research Institute. 156 p. Young J, Virmani SS Inheritance of fertility restoration in a rice cross. Rice Genet. Newsl. 1: Hybrid rice breeding in Russia 327

309 Notes Authors address: All-Russian Rice Research Institute, Krasnodar, Russia. Citation: Virmani SS, Mao CX, Hardy B, editors Hybrid rice for food security, poverty alleviation, and environmental protection. Proceedings of the 4th International Symposium on Hybrid Rice, Hanoi, Vietnam, May Los Baños (Philippines): International Rice Research Institute. 407 p. 328 Gontcharova and Gontcharov

310 Hybrid rice research and development in Myanmar Khin Than Nwe, Myint Yee, Hmwe Hmwe, Myint Aung, and Aye Aye Myint Rice production is the backbone of the national program on economic development as well as for attaining rice self-sufficiency in Myanmar. To increase rice production, hybrid rice technology is considered as a new approach. Under the Myanma Agriculture Service (MAS), a hybrid rice research and development program started at the Central Agricultural Research Institute (CARI) in 1991 with collaboration from the International Rice Research Institute (IRRI) and strengthened its activities with cooperation and collaboration from seed companies from China and Japan and technical support of the Food and Agriculture Organization (FAO). The hybrid rice breeding program focuses on developing parental lines better adapted to local environmental conditions. Two cytoplasmic male sterile (CMS) lines, IR58025A and IR62829A, were initially introduced from IRRI and IR58025A was found to be more adaptable to local conditions. Two more lines, IR68897A and IR68887A, were selected as good CMS lines from further evaluation. Some prospective maintainers and restorers were identified and some were used in the backcross nursery. Yield tests were carried out at CARI and different research stations to evaluate the yield and agronomic characters of experimental hybrids and introduced hybrids. Research to determine the optimum male:female row ratio, the gibberellic acid dosage test, and a study on the best time for the use of GA 3 for CMS lines are being done at CARI. Some hybrid combinations from the International Rice Hybrid Observational Nursery seem to be promising. Commercial F 1 seed production was handled by MAS and Chinese seed companies. At present, the area planted to hybrid rice has decreased because Chinese hybrids were observed to have some constraints: poor grain quality and susceptibility to disease. In Myanmar, hybrids must show about 20% yield heterosis over existing inbred cultivars with acceptable grain quality. Myanmar also became a member country of the IRRI-ADB hybrid rice project phase II. 329

311 Rice is the staple food for about 48 million people in Myanmar. In addition, rice production in Myanmar is the main pillar of the national program on economic development. About 6 million hectares are planted to rice, including summer rice. In the early 1940s, the country occupied first place among the rice-exporting countries of the world. A period of stagnation followed. The major rice ecosystems in the country are the rainfed lowland (67%), irrigated (20%), deepwater (6%), and upland (4%). Favorable rice lands are found in irrigated areas and greater yield potential can be expected there than in the other rice environments. National average yield is about 3 t ha 1. The current population is million and the population is growing at 2.02% per annum. Rice production in Myanmar has increased since the mid-1970s by the adoption of high-yielding varieties. Although rice production has increased, the country still needs to meet the government policy of food security through rice self-sufficiency and to export the surplus. Hybrid rice technology helps China to produce 20 million tons of additional paddy every year. The successful development and use of hybrid rice in Vietnam and India have occurred also. Hybrid rice technology is recognized as a new approach for increasing rice production. Status of the hybrid rice program The hybrid rice research program started in Myanmar in Before the national hybrid rice research and development program, rice hybrids from China were cultivated in the northern part of the country. Myanma Agriculture Service (MAS) strengthened the hybrid rice program through collaboration with the Food and Agriculture Organization (FAO) of the United Nations in A technical cooperation agreement among MAS and Marubeni, SIMAR Company, and Shoufang Company was implemented in The objectives were to evaluate the adaptation of their rice hybrids to identify the best hybrids from commercial seed production by establishing a joint venture company with MAS. Feasibility studies were carried out for some elite rice hybrids initially at the research institute and research stations and later in farmers fields. The best rice hybrids selected were 6201, R501, FU2, FU4, FU5, and FU6, which showed yield superiority over the best inbred cultivar Manawthukha by 13% to 17%. Commercial F 1 seed production of these hybrids was handled by MAS and seed companies. Rice hybrids from China were observed to have some constraints: poor grain quality and susceptibility to diseases. Rice hybrid 6201 developed from Hybrid Rice International Co. Ltd., India, was found to be promising for eating quality. Hybrid rice cultivation Large-scale hybrid rice cultivation began in 1998 with imported F 1 seed. Later, under the technical cooperation agreement on hybrid rice development between MAS and 330 Khin Than Nwe et al

312 Table 1. The total amount of F 1 seed produced and imported in Myanmar, Company Hybrid Seed Remark amount (kg) Simar R501 53,019 Produced R ,393 Imported Total 947,412 Shoufang FU2 8,100 Produced FU4 16,371 Produced FU5 55,044 Produced FU6 729 Produced Total 80,244 Marubeni Hybrid ,551 Imported Hybrid ,000 Imported Total 74,551 Table 2. Area planted to rice hybrids in Region Sown area (ha) Ayeyawady 13,328 Bago 2,169 Kachin 264 Kayah 30 Kayin 71 Sagaing 19,808 Shan (South) 138 Shan (North) 15 Shan (East) 100 Tanintharyi 215 Mandalay 8,000 Magwe 3,400 Mon 299 Yakhine 116 Yangon 2,350 Total 50,305 foreign private seed companies, F 1 seed production was undertaken on the government research and seed farms. Table 1 shows the total amount of F 1 seed production and imported seed from 1998 to Large-scale cultivation contributed about 0.084% of the total rice area with average yield of 6.4 t ha 1 (wet season, WS) and 6.5 t ha 1 (dry season, DS) and 5.0 t ha 1 (WS) and 6.4 t ha 1 (DS) of hybrids 6201 and R501, respectively. The various rice hybrids were grown throughout the country (Table 2). Most of the hybrid rice area is in three divisions of Ayeyawady, Sagaing, and Mandalay, which are the largest Hybrid rice research and development in Myanmar 331

313 Table 3. Area planted to hybrid rice in different years and seasons. Year and season a Sown area (ha) 1998, DS , WS , DS , WS 2, , DS 16, , WS 28, , DS , WS 2,150 Total 50,305 a DS = dry season, WS = wet season. rice-growing regions in the country. About half of the total irrigated rice area is in Mandalay and Sagaing divisions. The sown area of hybrid rice increased until 2000 and decreased starting in 2001 (Table 3). This is the reason that Chinese hybrids had some constraints to grain quality. Hybrid rice research Hybrid rice research began with collaboration from IRRI in 1991 at the Central Agricultural Research Institute (CARI) in Yezin. To strengthen the national hybrid rice research and development program, FAO assisted through a technical cooperation project on Training in Hybrid Rice Technology in Chinese hybrid rice scientists were employed to assist and train national scientists on hybrid rice breeding and F 1 seed production. Under the hybrid rice research and development program, the following activities have been carried out: evaluation of cytoplasmic male sterile (CMS) lines, development of parental lines (A, B, and R), production of experimental hybrids, yield evaluation of hybrids, multiplication of CMS lines, parental line purification and seed multiplication, demonstration trials of F 1, seed production technology research, and fertilizer management. CMS evaluation Two CMS lines, IR58025A and IR62829A, were initially introduced from IRRI and IR58025A was found to be more adapted to local conditions. IRRI continuously provided more CMS lines and these were evaluated at CARI (Table 4). Two more CMS lines, IR68897A and IR68887A, were selected as good CMS lines for further evaluation. Two Chinese CMS lines, Zhen Shan 97A and Bo A, were evaluated and found to have stable sterility, but their performance was poor. 332 Khin Than Nwe et al

314 Table 4. Characteristics of IRRI CMS lines at CARI, Yezin. CMS line Days to 50% Total spikelets Seed Panicle flowering per panicle set (%) exsertion (%) IR58025A IR62829A IR68887A IR68888A IR68897A IR68899A IR68902A IR69682A IR70369A IR70959A Table 5. Frequency of maintainers and restorers from the testcross nursery. Year Total Frequency (%) testcrosses Maintainers Restorers Identification of maintainers and restorers Restorers and maintainers were identified. The prospective restorer and maintainer frequencies are % and %, respectively (Table 5). Some promising restorers from the International Rice Hybrid Observation Nursery (IRHON) were selected for further testing. Most of the good restorers are IRRI materials. Prospective restorers were evaluated for combining ability and maintainers were used in a backcross nursery. Experimental F 1 seed production and CMS multiplication Promising CMS lines and restorers were selected to make experimental hybrids in an isolation-free plot. IRRI restorer lines and locally identified restorers will be used to produce experimental hybrids. One hybrid combination has been developed and will also be included in experimental hybrid seed production. CMS IR58025A seed multiplication was done at CARI and a seed yield of 0.5 t ha 1 was obtained. Further studies are needed to improve seed yield. Hybrid rice research and development in Myanmar 333

315 Table 6. Yield and agronomic traits of IR58025A in the row ratio test. Row ratio Tillers per hill Total spikelets Seed Yield (t ha 1 ) set (%) 2: : : : : F test (1%) CV % 7.15 LSD Table 7. The best hybrids from F 1 evaluation. Hybrid Yield (t ha 1 ) % over check Origin Year tested IR72834H IRRI 1997 IR70411H IRRI 1997 IR73877H IRRI 1998 Hybrid India 1997 Seed production technology and research Research on seed production technology such as the optimum row ratio of male and female, the effect of gibberellic acid dosage to maximize outcrossing, the identification of optimum sowing time for CMS and F 1 seed production, and nutrient management was carried out. The results indicated that the optimum male and female row ratio in CMS IR58025A was 1:8 (Table 6). F 1 evaluation Introduced rice hybrids from China and India have been evaluated at different locations since IRRI hybrids in the IRHON have been evaluated. Hybrids from IRRI and India seemed to be adapted to local conditions (Table 7). Training and workshops Eight hybrid rice in-country training courses have been conducted since 1997 at CARI, Yezin, with a total of 151 participants from research institutes, research farms, and the extension division. The first in-country training course on hybrid rice was conducted in collaboration with Chinese hybrid rice scientists in The national workshop on hybrid rice technology under the TCP/MYA/6612 was held at Myanma Agriculture Service, Yangon, in National researchers were sent to IRRI and China for training in hybrid rice breeding and seed production. 334 Khin Than Nwe et al

316 Future prospects Hybrid rice is an alternative way to increase rice production. Therefore, the Government of Myanmar has been exploring the prospects of hybrid rice technology to increase rice yield. Since hybrid rice technology was introduced to farmers, F 1 combinations with higher yield and good grain quality have been in great demand. Existing varieties still have a high yield potential and higher market price; therefore, hybrid rice with a 20 30% yield advantage over the best local cultivar could be successful in a hybrid rice program in Myanmar. Myanmar became a member country of the IRRI-ADB hybrid rice development project phase II. The hybrid rice research and development program will be strengthened. The program will continue to focus on developing suitable hybrids and economical seed production systems. We expect that hybrid rice can cover about 10% of the total irrigated area in the next five years. Myanmar will also continue to develop and deploy more trained people coupled with the allocation of more resources to hybrid rice research and development in the future. Notes Authors addresses: Khin Than Nwe, deputy general manager and head, Myint Yee and Hmwe Hmwe, deputy supervisors, Myint Aung, assistant manager, Rice Division, Central Agricultural Research Institute; Aye Aye Myint, farm manager, Kyauktada Research Farm, Maddaya Township, Myanmar. Citation: Virmani SS, Mao CX, Hardy B, editors Hybrid rice for food security, poverty alleviation, and environmental protection. Proceedings of the 4th International Symposium on Hybrid Rice, Hanoi, Vietnam, May Los Baños (Philippines): International Rice Research Institute. 407 p. Hybrid rice research and development in Myanmar 335

317 Hybrid rice research and development in Japan T. Takita Shinjo (1969) proposed the idea of hybrid rice first, and then the idea spread elsewhere. China then developed hybrid rice first in the world. Unfortunately, hybrid rice has not been popular in Japan because of two major problems: one is the high cost of seed production and the other is that high-yielding japonica hybrids with commercially acceptable good quality have never been developed. To improve hybrid rice seed production, Shinjo (1975) developed cytoplasmic male sterile (CMS) lines and restorer lines (BT) derived from Chinsurah Boro II. The male sterile (ms) line is probably better than the wild abortive (WA) ms line, which is used widely in indica hybrid rice, because the BT ms line does not have problems such as delayed heading time and bad panicle exsertion, which are shown in WA lines (Virmani and Sharma 1993). Various aspects were studied for seed production in Japan and thermosensitive genic male sterile (TGMS) lines were developed by mutation breeding (Maruyama 1989). Since the sterility of the TGMS lines is not stable in Japanese weather conditions, we need more studies on the use of the TGMS lines. There are three important points in the improvement of seed production: simultaneous heading time between ms lines and restorer lines, taller plant height in restorer lines, and stigma exsertion in ms lines (Kato and Namai 1987). In addition, Takita and Yamaguchi (2002) have found that cold-tolerant varieties can produce more seeds. Kariya et al (1986) showed that cold-tolerant varieties have larger anthers and more well-ripened pollens than susceptible varieties. In this sense, coldtolerant varieties seem to scatter more pollens. In addition, if an indica maintainer line is available, it is obviously suitable for seed production in indica-japonica hybrid seed production because the flowering time of indica is earlier than that of japonica. However, indica maintainer lines have never been developed because most indica varieties have restorer genes for the ms line derived from Chinsurah Boro II. Kobayashi and Fukuta (1991) reported a tight linkage between the indica plant type and the restorer genes. However, Takita (2000) developed a semidwarf indica maintainer line. 337

318 Table 1. Pyramiding effect of important traits in japonica-indica hybrids (Takita et al 2000) Type Tolerance Plant Sink Length Leaf Yielding of low type size of grain senescence ability temperature ripening Hybrid (J/I) tolerant long late stable Japonica tolerant long late Indica susceptible short early = Very good, = good, = moderate, = poor. THR 1 showed the highest yield of brown rice every year in tests over three years ( ). It yielded 7.0 t ha 1 of brown rice on average, 37% higher than that of japonica check variety Nipponbare, while the paternal indica variety Habataki showed 15% higher yield than the japonica variety. However, the indica variety yielded lower than the japonica variety in the cool-weather year. The other combinations also showed similar patterns. According to observations before and during heading time, the japonica-indica hybrids showed a good plant type, large panicles, and large sink size like the paternal indica variety. In addition, after heading, those hybrids showed lodging resistance and a long grain-ripening period showing later leaf and panicle senescence like the maternal japonica variety. We can conclude that the japonica-indica hybrid can be high yielding because of the pyramiding effect of the good characteristics of both japonica and indica varieties (Table 1). Current status of hybrid rice in Japan Rice consumption is still decreasing in Japan and the price is becoming cheaper. About 30% of the rice-planting area has been changed into other crops and the riceplanting area in 2001 was only 1,700,000 ha. In this sense, our interest must be on grain quality and production costs. Last year, Mitsui Chemical Company released two hybrid rice varieties that have very high yields and acceptable eating quality. Oka and Konno (2000) reported that one of the hybrids has very high yielding ability because it has a good plant type, large root system, and desirable panicle shape. It is said that there are too many expensive rice varieties like Koshihikari in Japan while markets need more cheaper rice varieties. Hence, the hybrids may be accepted in the cheaper rice market. In addition, some large companies such as Toyota and Japan Tabako are interested in hybrid rice or have started research on it. Although research on hybrid rice may be decreasing in universities and national institutes, private companies are becoming major participants in this research. 338 Takita

319 Prospects of hybrid rice in Japan We know that hybrid rice is the best variety except for its seed price. So, the most important work is to decrease the seed price. Takita and Yamaguchi (2002) recently proposed that we should use cold-tolerant varieties that have more fully ripened pollens. In this aspect, we have many genetic resources because we are developing cold-tolerant varieties that are important in northern Japan. In addition, China lately improved seed production techniques significantly. We are convinced that the seed production problem will be solved in the near future. To improve yields, we can go for indica-japonica hybrids, which have a great possibility. To improve eating quality, we have low-amylose genes, which are obviously effective in improving eating quality (Sato et al 2002). We conclude that hybrid rice has a large potential and we have many materials for improving it. It depends on our efforts whether hybrid rice will be used or not in Japan. References Kariya K, Satake T, Koike S Relationship between anther size and cold tolerance in rice. Jpn. J. Crop Sci. 55(Suppl.2): Kato H, Namai H Intervarietal variations of floral characteristics with special reference to F 1 seed production in japonica rice (Oryza sativa L.). Jpn. J. Breed. 37: Kobayashi A, Fukuta Y Relationship between restorer genes and plant type in japonicaindica hybrids obtained through anther culture. Hokuriku Sakumotsu Gakkaiho 26: Maruyama K Hybrid rice breeding. Jpn. J. Agric. Technol. 44: Oka M, Konno T Characteristics of hybrid rice MH2005 compared with the highyield rice varieties. Tohoku J. Crop Sci. Jpn. 43: Sato H, Suzuki Y, Sakai M, Imbe T Molecular characterization of Wx-mq, a novel mutant gene for low-amylose content in endosperm of rice (Oryza sativa L.). Breed. Sci. 52: Shinjo C Cytoplasmic-genetic male sterility in cultivated rice, Oryza sativa L.: the inheritance of male sterility. Jpn. J. Genet. 44: Shinjo C Genetic studies of cytoplasmic male sterility and fertility restoration in rice, Oryza sativa L. Sci. Bull. Coll. Agric. Univ. Ryukyus 22:1-57. Takita T Selection of indica maintenance line for indica-japonica hybrid rice. Tohoku J. Crop Sci. Jpn. 43: Takita T, Terashima K, Yokogami N, Kataoaka T Stable high yielding ability of japonicaindica hybrid rice. In: 4th International Rice Genetics Symposium. Abstract. p 176. Takita T, Yamaguchi M Varietal differences of paternal ability of japonica maintainer lines for hybrid rice seed production. Breed. Res. 4(Suppl.1):101. Virmani SS, Sharma HL Manual for hybrid rice seed production. IRRI. Hybrid rice research and development in Japan 339

320 Notes Author s address: National Institute of Crop Science, Kannondai, Tsukuba, Ibaraki, Japan. Citation: Virmani SS, Mao CX, Hardy B, editors Hybrid rice for food security, poverty alleviation, and environmental protection. Proceedings of the 4th International Symposium on Hybrid Rice, Hanoi, Vietnam, May Los Baños (Philippines): International Rice Research Institute. 407 p. 340 Takita

321 Hybrid rice research and development in Sri Lanka S.W. Abeysekera, S.N. Jayawardena, K.D.S. Kiriwaththuduwage, and D.S. de Z. Abeysiriwardena Hybrid rice (HR) technology is considered as the best option to overcome yield stagnation in rice in Sri Lanka. In Sri Lanka, the research and development (R&D) program on hybrid rice began in late Initially, this program was academic rather than goal-oriented. However, in 1996, the hybrid rice R&D program was revised to be goal-oriented in collaboration with the International Rice Research Institute (IRRI). The R&D program on hybrid rice has three main components: developing and evaluating F 1 hybrids, developing locally adapted management practices for seed production, and developing locally adapted management practices for HR cultivation. Of the 60 IRRI, Indian, and locally bred CMS lines evaluated, 48 were adaptable and stable under local conditions. Some well-adapted varieties were identified as maintainers or good restorers for the wild abortive cytosterility system. Some elite maintainers identified were BR168-2B-23R, BR R, IR R, IR R, Bg99-350, and H4. Two CMS lines, BgCMS 1A and BgCMS 2A, have been developed at the Rice Research and Development Institute (RRDI). These lines have small round (samba) grain type with 100% sterility and 100% panicle exsertion rate (PER%). BgCMS 1A and BgCMS 2A are producing 14 and 18 panicles per plant and 203 and 243 spikelets per panicle, respectively. Three hybrid rice varieties, BgHR1, BgHR6, and BgHR12, developed at RRDI had significantly higher yields than the standard inbred varieties Bg 357 and Bg 403. The standard heterosis of these hybrids ranged from 28.9% to 58.6%. Grain quality characteristics of these hybrids were superior to those of inbred checks. Similarly, out of the 21 experimental hybrids evaluated in yield trials during the 2001 minor season, eight lines outyielded the standard checks Bg 359 and Bg 403 by more than 1 t ha 1. Grain quality characteristics of these hybrids were also superior to those of the check varieties. Of the 11 IRRIbred hybrids tested during the 2001 minor season, two, IR69686H and IR75217H, outyielded the local and international check varieties of the same growth duration by more than 1 t ha 1. Grain quality characteristics of the tested hybrids were comparable with those of the recommended check varieties. 341

322 Comprehensive studies on cultural management, fertilizer management, and reaction to pests and diseases of the promising hybrids are being conducted at RRDI to develop an appropriate package of management practices for hybrid rice cultivation. Average seed production in large-scale CMS plots, in small-scale CMS plots, in large-scale F 1 plots, and in small-scale F 1 plots was t ha 1, t ha 1, t ha 1, and t ha 1, respectively. Rice is the staple food of Sri Lanka and rice production is the most important agricultural subsector. Rice occupies 28% of the arable land and provides 25% of the employment in the total labor force. Assuming an annual population growth of 1.2% and per capita rice consumption of 100 kg y 1, the projected annual rough rice consumption by 2005 and 2010 would be 3.23 and 3.46 million tons, respectively. To reach these targets, the national average yield of rough rice must increase from the present level of 3.7 t ha 1 to 4.5 t ha 1 by Furthermore, during the past two decades, the rice sector has faced unprecedented challenges: diminishing profitability because of the increasing cost of production, low and stagnating yields leading to low returns to investments, declining rice area because of abandoning or converting rice lands for other purposes, and declining labor for rice. Hybrid rice technology has been identified as one possible option for solving the problem of low rice yield in the country (Abeysiriwardena et al 2000). Hybrid rice research in Sri Lanka began in 1994 at the Rice Research and Development Institute (RRDI) of the Department of Agriculture Sri Lanka (DOASL). Initial work involved testing F 1 hybrids and evaluating cytoplasmic male sterile (CMS) lines and restorer lines from the International Rice Research Institute (IRRI). From 1996 onward, hybrid rice research gained momentum with collaboration from IRRI under the Asian Development Bank-funded project on Development and Use of Hybrid Rice in Asia. The Ministry of Agriculture, Sri Lanka, identified this as a priority area and established a national research and development (R&D) network on hybrid rice across the country involving public, private, and nongovernmental institutions (Abeysekera et al 2000). Improving parental lines to enhance hybrid rice breeding efficiency, developing high-yielding hybrids, and optimizing technological packages for hybrid rice cultivation and hybrid rice seed production, training, and awareness are the major areas covered under this network. Recent developments in hybrid rice R&D in Sri Lanka are presented in this paper. Improvement of parental lines Promising CMS maintainer and restorer lines For the development and use of hybrid rice in Sri Lanka, the three-line system has been identified as the most feasible system since it has been widely adopted in countries such as China and India, where hybrid rice R&D has been very successful. In 342 Abeysekera et al

323 Table 1. Performance of IRRI and Indian cytoplasmic male sterile (CMS) lines at RRDI during the minor season of 2001 in Sri Lanka. All lines showed uniform adaptability. CMS line Panicle Spikelets Duration of Stigma Pollen sterility Outcrossing length panicle 1 floret opening exsertion (%) b rate (%) c (cm) (min) (%) a IR58025A* IR62829A* IR64608A IR68275A IR68280A IR68281A IR68887A* IR68890A IR68895A* IR68897A IR69616A* IR69623A IR69625A* IR69628A* IR68896A IR68899A IR69626A IR67684A IR66707A PMS11A* Ref. Bg Bg a Stigma exsertion rate = no. of spikelets with exserted stigma/total number of spikelets 100. b Ratio of the number of sterile pollens to the total number of pollens averaged over three fields. In each field, two separate slides were prepared using 10 plants each. c OCR% = number of fertile spikelets/total number of spikelets 100.* = promising CMS lines. the three-line system, CMS lines and their maintainer and restorer lines are important. IRRI and Indian-developed CMS lines were tested for stability, sterility, and other desirable traits. The CMS line PMS 11A from India and seven CMS lines IR58025A, IR62829A, IR68281A, IR68887A, IR69623A, IR69625A, and IR68889A from IRRI have been identified as adaptable and stable under local conditions (Table 1). The outcrossing rates of the IRRI CMS lines ranged from 30.2% to 43.0%, while showing 100% pollen sterility. During the major season, some Indian and locally developed CMS lines were evaluated at RRDI. The CMS line Krishna from India, two CMS lines (BgCMS 1 and BgCMS 2) developed in Sri Lanka, and 28 CMS lines from IRRI have been identified as stable for both pollen and spikelet sterility and adaptable under Sri Lankan conditions. The performance of these CMS lines is presented in Table 2. Hybrid rice research and development in Sri Lanka 343

324 Table 2. Performance of IRRI, Indian, and locally developed CMS lines at RRDI during the major season in Sri Lanka. All lines showed uniform adaptability. CMS line Panicle Spikelets Duration of Stigma Pollen sterility Outcrossing length panicle 1 floret opening exsertion (%) b rate (%) c (cm) (min) (%) a IR73318A DZ 97A G 46A IR68885A IR68899A IR69622A IR70368A IR70368A IR70759A IR71563A IR71564A IR73323A IR73327A IR73328A IR73794A IR75595A IR75601A IR75602A IR75605A IR75607A IR76765A IR76766A IR76767A IR76768A IR76769A IR77289A IR77801A IR77803A IR77804A IR77807A IR77808A IR77811A IR78353A IR78354A IR78359A IR78364A IR78365A Krishna A V 20A Bg CMS 1A Bg CMS 2A Ref. Bg Bg a Stigma exsertion rate (%) = no. of spikelets with exserted stigma/total number of spikelets 100. b Ratio of the number of sterile pollens to the total number of pollens averaged over three fields. In each field, two separate slides were prepared using 10 plants each. c OCR% = number of fertile spikelets/total number of spikelets 100. * = promising CMS lines. 344 Abeysekera et al

325 Table 3. Characteristic features of new CMS lines developed at RRDI. CMS line DFF a HT PN PL PE% TS OCR GT (days) (cm) (cm) % BgCMS Small grain (samba) BgCMS Small grain (samba) a DFF = days to 50% flowering, HT = plant height, PN = panicle number, PL = panicle length, PE = panicle exsertion, GT = grain type, TS = total no. of spikelets panicle 1, OCR% = outcrossing rate. Two CMS lines (BgCMS 1A and BgCMS 2A) developed at RRDI have small round (samba) grain type with 100% sterility and panicle exsertion rate (PER%). BgCMS 1A and BgCMS 2A are producing 14 and 18 panicles plant 1 and 203 and 243 spikelets panicle 1, respectively, with an outcrossing rate of 25 27% (Tables 2 and 3). These new CMS lines are now being used to develop experimental hybrids. Several selected local varieties/lines were identified as maintainer lines for some of the adaptable CMS lines and were backcrossed to their CMS sources. The most important of those are Bg , Bg , Bg , Bg , Bg , Bg , Bg , Bg , At , IR68281B/Bg 1528, IR68275B/At 354, Ob 2552/Bg 12-1//Bg 379-2, and IR69628B/Bg CMS lines are being developed in the genetic background of these maintainers. Many high-yielding locally developed elite lines have been tested for restoring ability along with some good restorers from IRRI and BRRI. The identified restorers (IR R, IR R, IR R, IR R, IR R, IR R, IR R, BR168-2B-23R, and H4) are being purified and multiplied for producing hybrids. Promising rice hybrids Cytoplasmic genic male sterility is the most effective and stable system being used to develop hybrids. The F 1 rice hybrids tested during were early maturing but failed to outyield local high-yielding inbred varieties. Some new hybrids were tested during Fifty experimental hybrids were tested in preliminary yield trials at RRDI and other test locations. Some promising hybrids showed a considerable grain yield increase over the local inbred check varieties under experimental conditions (Table 4). Three locally developed hybrids BgHR1 (IR69616A/H4), BgHR12 (IR58025A/BR R), and BgHR6 (PMS 11A/IR R) have consistently shown high standard heterosis for grain yield (31 45%) in yield trials conducted at two locations (Batalagoda and Girandurukotte) in the target regions. BgHR1 has also shown high standard heterosis for grain yield (45%) at Mahailiuppallama. The CMS lines IR69616A, IR58025A, and PMS 11A used to develop BgHR1, BgHR6, and BgHR12, respectively, have consistently shown 100% sterility over seasons under local conditions. Hybrid rice research and development in Sri Lanka 345

326 Table 4. Promising hybrids identified in different locations during and major seasons in Sri Lanka. Hybrid combination Year Days Yield ( t ha 1 ) Yield increase over (location) a to the check maturity Hybrid Check (t ha 1 ) (%) IR69616A/H4 (BgHR1) 1999 (MI) (Bg 403) PMS 11A/IR R (Bg 357) PMS 8A/H (Bg 357) PMS 8A/At (Bg 357) PMS 11A/IR R (Bg 357) IR69616A/H (Bg) (Bg 403) PMS 11A/IR R (BgHR6) (Bg 357) IR58025A/BR R (BgHR12) (Bg 357) IR62829A/IR R (Bg 357) IR58025A/IR R 1999 (Bg) (Bg 357) IR62829A/IR R (Bg 357) PMS 11A/At (Bg) (Bg 357) IR69616A/H (Gk) (Bg 403) PMS 11A/IR R (Bg 357) IR58025A/BR R (Bg 357) a MI = Mahailluppallama, Bg = Batalagoda, Gk = Girandurukotte. The promising hybrids were further evaluated in the International Coordinated Hybrid Rice Trial (ICHRT) during the major and 2001 minor seasons. Twenty-seven promising hybrids along with one international check (IR72), one hybrid check (PSBRc2), and two national checks (Bg 300 and Bg 357) were included in the trials. The early hybrids IR69676H and IR69690H, which involve locally adaptable CMS line IR58025A as the female parent, have been identified as the most promising and adaptable rice hybrids under Sri Lankan conditions. These two hybrids have shown consistently high standard heterosis for grain yield over locations and seasons, ranging from 12% to 84% (Table 5). In addition, two hybrids IR69680H and IR75217H also outyielded the local and international check varieties at RRDI. 346 Abeysekera et al

327 Table 5. Performance of selected rice hybrids in the international trial at different locations during the major and 2001 minor seasons in Sri Lanka. Designation Year Days to Yield (t ha 1 ) Yield increase (location) a maturity over the check Hybrid Check t ha 1 % IR69689H (M) PSD (M) APHR (M) IR72064H (M) IR68284H (M) IR76901H (M) IR69676H (M) IR64618H (M) IR72073H (M) IR69690H (M) IR67265H (M) IR65487H (M) KRH (M) NSD (M) COHR (M) PSBRc (M) IR73863H 2001 (Bg) IR73854H 2001 (Bg) IR73870H 2001 (Bg) IR75217H 2001 (Bg) IR69686H 2001 (Bg) IR73871H 2001 (Bg) IR73409H 2001 (Bg) IR74615H 2001 (Bg) IR65489H 2001 (Bg) IR69672H 2001 (Bg) IR69672H 2001 (Gk) IR73860H 2001 (Gk) a Bg = Batalagoda, Gk = Girandurukotte, M = Malwanegama. These two hybrids showed a standard heterosis for grain yield of 30 40% and 23 50% over the best local checks, respectively. Evaluation of hybrids in Observational Yield Trials (OYT) During the 2001 minor season, 21 RRDI-developed experimental hybrids were evaluated at RRDI in Observational Yield Trials (OYT). Among these, eight showed superior performance compared with the inbred check varieties. The promising hybrids and their standard heterosis for grain yield over the check varieties are presented in Table 6. It is interesting to note that the new hybrids in the pipeline are developed using new CMS lines rather than using IR58025A, which has a mild aroma. However, entries with standard heterosis above 15% yielding more than 1 t ha 1 when Hybrid rice research and development in Sri Lanka 347

328 Table 6. Standard heterosis for grain yield and other important characteristics of promising RRDI-developed experimental hybrids at RRDI in Observational Yield Trials in the minor season of Promising hybrid combination Standard heterosis (%) DF a No. of Panicles Plant Root PL S/P FG/P 1,000 Grain tillers plant 1 height volume GW yield *IR68275A/Bg *IR68280A/IR R *IR68275A/IR46R **PMS11A/IR46R **IR58025A/BR 168-2B-23R **IR69616A/BR 168-2B-23R **PMS 11A/IR R **IR68902A/IR R Bg 359 std (3½ mo) Bg 403 std (4 mo) a DF = date of flowering, PL = panicle length standard heterosis, S/P = spikelets panicle 1, FG/P = filled grains panicle 1, GW = grain weight, * = standard heterosis compared with Bg 359, ** = standard heterosis compared with Bg Abeysekera et al

329 Table 7. Performance of locally developed hybrids in on-farm trials and in trials at the research farm at RRDI. Hybrid Year Days to Yield (t ha 1 ) Yield Reaction to variety (location) a maturity increase pests b Hybrid Check over the check (t ha 1 ) BPH RGM BgHR1 2001(Ta) (Bg 403) 1.1 R/MR MR (MI) (Bg 403) 1.3 (Bg) (Bg 403) 1.0 BgHR6 2001(Ta) (Bg 358) 1.3 R MR (MI) (Bg 358) 1.7 (Bg) (Bg 357) 3.7 BgHR (Ta) (Bg 358) 2.0 R/MR R/MR (MI) (Bg 358) 1.4 (Bg) (Bg 357) 1.4 a Ta = Talawa, MI = Mahailluppallama, Bg = Batalagoda. b BPH = brown planthopper, RGM = rice gall midge, R = resistant, MR = moderately resistant. compared with the checks are selected and advanced for further yield testing. The rice hybrids found consistently superior to the checks with desirable agronomic and quality traits are tested in on-farm trials. Evaluation of hybrids in on-farm trials On-farm trials of locally developed hybrids BgHR1, BgHR6, and BgHR12 were carried out at three locations in the target area with the collaboration of the extension staff of the Sri Lanka Mahaweli economic agency at Thambuththegama (dry zone of Sri Lanka). All three hybrids outyielded the inbred checks Bg 357, Bg 358, and Bg 403 by more than 1 t ha 1 at different locations (Table 7). Ten on-farm trials in farmers fields will be carried out during the coming season before the hybrids are recommended to the National Seed Releasing Committee for commercial cultivation. Evaluation for grain quality characteristics of hybrids Quality is one of the key factors that determine the consumer acceptance of hybrids. Added emphasis is being given to this aspect and adequate care is taken to include the elite breeding lines with better quality characteristics in the source nursery. The hybrids evaluated in the large-scale on-farm trials were tested for grain quality characteristics (Table 8). The main characteristics related to grain quality are appearance and milling, cooking, and eating quality. BgHR6 was the best in appearance, having low translucent grains and high total milled rice recovery (more than 60%). However, in cooking quality, it tends to have high amylose content, high gelatinization temperature (GT), and hard gel consistency (GC). BgHR12 was the best in cooking quality. It tends to have high amylose content and high GT, and medium GC. BgHR1 is a red pericarp variety. It also has a good grain appearance. But the head grain recovery is 45%. However, it is the best in cooking and eating quality, with soft GC Hybrid rice research and development in Sri Lanka 349

330 Table 8. Grain quality characteristics of promising locally developed hybrids at RRDI, Batalagoda, major season Hybrid variety/check Head Broken Length/ Shape a Pericarp b Chalkiness c Translucency d Amylose e GC f GT g grain wt grain wt width color content (g) (g) BgHR1 (IR69616A/H4) L/S R WB-3 I H Soft I BgHR6 (PMS 11A/IR R) L/M W WB-3 I H Hard H BgHR12 (IR58025A/BR R) L/M W WB-4 I H Medium H Bg I/B W WB-3 I H Hard L Bg L/M W WB-2 I H Hard I a L/S = long short, L/M = long medium, I/B = intermediate bold. b R = red, W = white. c WB = white belly. d I = intermediate. e H = high. f GC = gel consistency. g GT = gelatinization temperature, L = low. 350 Abeysekera et al

331 Table 9. Seed yield (t ha 1 ) of three promising locally developed hybrids during the major and 2001 minor seasons in largescale seed production plots. Hybrid combination Major 2001 Minor (t ha 1 ) BgHR1 (IR696116/H4) BgHR6 (PMS 11A/IR R) BgHR12 (IR58025A/BR R) Table 10. Seed yield of large-scale CMS multiplication (A/B) plots during the major and 2001 minor seasons. A/B combination Major Minor (t ha 1 ) IR58025A IR69616A PMS 11A and intermediate GT, although amylose content is high. All three entries were superior in milling quality to the local standard checks. Hybrid rice seed production The success and sustenance of hybrid rice technology depend on two main factors: economic and efficient hybrid seed production and an economically viable and high level of heterosis. Results of the past two years show the possibility of raising the hybrid seed yield in Sri Lanka. Seed yield varied, however, with hybrid, season, and the optimum seed production package. The seed yield of both A lines and F 1 hybrids was generally higher during the minor season than in the major season. Small-scale seed production (18-m 2 plots) is necessary to provide seed for yield trials. Seed yield of t ha 1 could be produced in small-scale seed production plots at RRDI using the isolation-free method. Large-scale seed production (plot size 300 m 2 ) was tried out in the target area using three locally developed promising hybrid combinations BgHR1, BgHR6, and BgHR12 and three locally adaptable CMS lines. All three promising locally developed hybrids have given a seed yield of more than 1 t ha 1 in both the major and minor seasons (Table 9), whereas seed yield of the large-scale CMS multiplication plots varied around 1 t ha 1 (Table 10). Hybrid rice research and development in Sri Lanka 351

332 Agronomic management of hybrids To realize the full yield potential of hybrids, an appropriate agronomic package is needed. The high grain yield of hybrid rice is attributed to its high vegetative biomass production, high leaf area, and increased number of spikelets and filled grains per panicle. This shows the importance of an optimum agronomic package for hybrids. Extensive studies on aspects such as nursery management, seedling age, number of seedlings hill 1, planting geometry, time of planting, and nutrient management were carried out at RRDI and the CIC seed farm in Palwehera. To date, a broad agronomic package for the cultivation of hybrid rice has been developed. However, there is a need to fine-tune the package for each region, specific hybrid combination, and growing season. The response of hybrids to N was higher than that of inbreds at all levels of N. The N requirement of hybrids varied from 120 to 150 kg N ha 1. The best spacing for hybrids should be one plant hill 1 with the spacing of and cm (seed rate of kg ha 1 ). Training and awareness National and international linkages are useful for developing human resources and improving infrastructure. Therefore, RRDI maintains close collaboration with IRRI and receives technical support, including seed material and training for an effective hybrid rice R&D program in Sri Lanka. During the first phase of the hybrid rice project, several hybrids have been developed and the human resource base to take up hybrid rice seed production has been strengthened. To take the fruits of research to the end-users rice farmers aggressive efforts are needed. This can be achieved by imparting awareness and training on hybrid rice cultivation and seed production. Training programs 1. Ten training programs were conducted at RRDI. About 250 participants were trained in various aspects of hybrid rice cultivation and seed production. 2. As a special program, 10 officers from the Mahaweli Economic Agency and 30 progressive farmers were made aware of the hybrid rice R&D program using hybrid rice seed production and cultivation plots in and around RRDI. The farmers were very much impressed by seeing the hybrid rice crop at RRDI. 3. Five informal training courses and technical briefings were conducted during the 2001 minor season for the private- and public-sector officers from different organizations. This program increased the awareness of hybrid rice technology among the target group. 352 Abeysekera et al

333 Constraints and problems identified during Phase I of the project period The main constraints and problems related to hybrid rice R&D that emerged during Phase I of the project period can be identified under two areas: technical and institutional. Technical problems were 1. Limited grain yield heterosis in experimental hybrids. One of the reasons for this may be the low level of genetic diversity among a few CMS sources that have been identified as adaptable under local conditions. Another reason may be the narrow genetic base of the locally available varieties that can be used as restorers. 2. In Sri Lanka, direct seeding is practiced over 85% of the rice land area so that promotion of transplanting with hybrid rice would be difficult. 3. The habit of rice farmers of using farm-saved seeds. 4. The inadequate supply of pure seeds of parental lines and hybrids. 5. Inconsistent seed yield in the seed production program and the high cost of GA The lack of uniformity in hybrids probably because of the low level of genetic purity in parental lines. Institutional problems were 1. Inadequacy of infrastructure and transportation facilities. A separate small laboratory unit with laboratory equipment (e.g., light microscopes, portable moisture meters, seed processing unit, etc.) within RRDI is necessary for an efficient and effective hybrid rice R&D program. Conducting on-farm trials is extremely difficult without adequate transportation facilities. 2. A shortage of skilled labor is still a problem for the hybrid rice R&D program. Future plans involve the following: 1. Hybrid rice R&D activities in Sri Lanka will be strengthened. 2. We hope to release the first hybrid in Sri Lanka by the end of The hybrid rice program for the next three years has already been prepared. Future emphasis will be placed on the following areas and the development of hybrid rice technology in Sri Lanka will be further enhanced by Enhancing the level of heterosis through indica/tropical japonica hybrids. Developing better agronomic and pest management strategies to maximize the yield of rice hybrids. Improving grain quality and disease/insect resistance of hybrids. Overcoming the problem of direct seeding in hybrid rice cultivation Gradually introduce transplanting with hybrid rice varieties to enhance spread Hybrid rice research and development in Sri Lanka 353

334 Increase hybrid rice seed yield as much as possible Improve and introduce low-density seedling broadcasting Beginning production of nuclear seed. Establishing large-scale CMS and F 1 seed production plots on public- and private-sector farms. Getting NGOs and interested farmers involved in hybrid rice seed production. Organizing training classes on hybrid rice seed production. Training extension officers of public semigovernment institutions and NGOs on hybrid rice cultivation and promoting it through them. Making policy favorable for hybrid rice R&D. For the policy for hybrid rice R&D, The policy is to promote the cultivation of hybrid rice in Sri Lanka. Initially, promote hybrid rice cultivation in high-potential areas with farmers who adopt transplanting. Next, change farmers gradually from broadcasting to transplanting wherever possible in the high-potential areas with the promotion of hybrid rice as a package. Seek foreign funding to firmly establish the hybrid rice R&D system in Sri Lanka and develop it to a sustainable level with adequate human resources and infrastructure. Not import hybrid rice seed but produce it within the country in collaboration with the private sector, NGOs, and interested farmers. Conclusions The hybrid rice R&D program in Sri Lanka will be continued and expanded because remarkable progress has been made during the past few years. The activities of the hybrid rice R&D program in Sri Lanka for the next three years (under Phase II of the hybrid rice project) have already been prepared to make good progress by solving the problems and constraints identified during the past few years. References Abeysiriwardana DS de Z, Sandanayake CA Future rice research as directed by trends in cultivated extent and yield of rice during the recent past. In: Proceedings of Annual Symposium of the Department of Agriculture, Sri Lanka. 2: Abeysekera SW, Abeysiriwardana DS de Z Recent developments in hybrid rice research in Sri Lanka. Proceedings of the Annual Symposium of the Department of Agriculture, Sri Lanka. 2: Abeysekera et al

335 Notes Authors address: Rice Research and Development Institute, Batalagoda, Ibbagamuwa, Sri Lanka. Citation: Virmani SS, Mao CX, Hardy B, editors Hybrid rice for food security, poverty alleviation, and environmental protection. Proceedings of the 4th International Symposium on Hybrid Rice, Hanoi, Vietnam, May Los Baños (Philippines): International Rice Research Institute. 407 p. Hybrid rice research and development in Sri Lanka 355

336 Hybrid rice development and use in Vietnam Nguyen Tri Hoan and Nguyen Huu Nghia Rice is the number-one agricultural product and it accounts for 90% of national food production. It supplies 80% of the carbohydrate requirement of the 75 million Vietnamese. Before 1998, Vietnam had to import 0.8 to 0.9 million tons of milled rice annually to meet the country s food requirements. Because of the shift to a marketoriented economy from a centrally planned economy complete with technology innovations, rice production in the country increased from 15.9 million t in 1985 to 32.7 million t in 2000, for an average increase of more than 1 million t of rice each year. Vietnam can now export 3 to 4 million t of milled rice each year to the world market. Vietnam s rice area occupies 4.2 to 4.5 million ha. The annual harvested rice area is 7.2 million ha. In general, two rice crops can be grown in the northern provinces, whereas two to three crops can be grown in the southern provinces. For productivity, average rice yield in Vietnam increased from 2.13 t ha 1 in 1975 to 2.9 t ha 1 in 1997, 3.9 t ha 1 in 1998, and 4.2 t ha 1 in In some provinces, such as Nam Dinh, yield can reach 11 t ha 1 year 1. Among the technology innovations bringing about the increase in rice production in Vietnam, the development and use of hybrid rice have been major contributors. Establishing multidisciplinary and multiagency/institutional research and seed production programs for hybrid rice technology Hybrid rice research in Vietnam started on a limited scale in 1979 at the Vietnam Agricultural Science Institute (VASI). In 1992, a National Hybrid Rice Research Program was formulated and, in 1994, the Hybrid Rice Research and Development Center (HRRDC) was established. Currently, the HRRDC has 34 staff (2 with a Ph.D., 4 with an M.Sc., 18 with a B.Sc., and 10 technicians). With increased human resources involved in hybrid rice R&D and improved facilities, Vietnam has been able to develop and implement multidisciplinary programs involving breeding, genetics, seed production, plant pathology, entomology, rice chemistry, and biotechnology. The primary breeding objectives have become 357

337 clear and comprehensive, including various traits such as earliness ( d duration) to fit into Vietnam s cropping system, longer grains vis-à-vis the bold-grain Chinese hybrids, resistance to major insects such as brown planthopper and major diseases such as bacterial leaf blight and sheath blight, in addition to high yield and good hybrid seed productivity. Short-duration hybrids also allow two to three crops per year in the south and escape from natural calamities such as floods, storms, and disease and insect damage. In addition, with the hybrid rice research network, multiagency collaborative projects have been carried out such as those involving two-line hybrid and parental line development and yield evaluation. For yield trials, the most promising hybrids are entered in the National Hybrid Rice Yield Trial conducted at four locations, with three replications, two seasons year 1, and 8-m 2 plots. Entries in national trials have come from different institutions that have developed capacity to breed hybrids and parental lines, such as HRRDC, the Agricultural Genetics Institute, Hanoi Agricultural University, and Food Crop Research Institute. Hybrids must exhibit 15 20% yield superiority over inbred check varieties before they are released for commercial cultivation. Developing national capacity to undertake large-scale parental and hybrid seed production as well as hybrid rice cultivation From 100 ha in 1991, hybrid rice area increased substantially to 480,000 ha in Hybrid rice cultivation is now spreading to the South-Central Coast and Central Highlands in addition to the 31 northern provinces. In general, hybrid rice yields 6.5 ha 1 compared with the national average yield of about 4.0 t ha 1 (Table 1). Seed production area increased from 267 ha in 1996 to 620 ha in The yield and production of hybrid seed also increased from 1.75 to 2.3 t ha 1 and from to 1,426 t in the same period, respectively. In 2001, about 1,450 ha were used for hybrid seed production, a 250% increase over the 2000 level. In some areas, more than 2.0 t ha 1 seed yields could be obtained, but, because of unfavorable climatic conditions in 2001, average yields dropped to 1.7 t ha 1. Hybrid seed production in 2001 reached 2,400 t. For two-line hybrids, a 100-ha seed production area had average yields of 1 2 t ha 1 (Table 2). Over the short time that Vietnam has engaged in hybrid rice R&D, it has been able to develop a technology package for large-scale seed production for hybrids such as AMS24A/IR and AMS24A/Que99. Large-scale seed production of hybrids was done by farmers in the provinces under the close supervision of HRRDC technical personnel or extension specialists. Furthermore, the country has also been able to develop a system for purifying parental-line seeds and support local hybrid rice seed production activities, thus solving the foremost constraint encountered in local hybrid rice seed production the low purity of CMS lines. The development of seed certification standards for hybrid rice in the country has been helpful in providing seed quality control. 358 Nguyen Tri Hoan and Nguyen Huu Nghia

338 Table 1. Current status of hybrid rice commercial production in Vietnam, Year Area (ha) Yield (t ha 1 ) All year Spring Summer All year Spring Summer ,094 1,156 9, ,648 17,025 17, ,077 45,430 4, ,503 39,598 33, ,713 60,416 67, , ,802 77, , ,000 80, , , , , , , , , ,000 Table 2. Yield of hybrid rice seed production in Vietnam, Year Spring crop Summer crop Total Area Yield Area Yield Area Yield (ha) (kg ha 1 ) (ha) (kg ha 1 ) (ha) (kg ha 1 ) , , , , , , , , , , , , , , , , , ,450 1, ,600 2,500 a a Estimated. For seed supply, in 2001, 14,400 t of hybrid seed were used in Vietnam; of this, 2,400 t were produced locally, whereas 12,000 t were imported from China. Vietnam has high potential for hybrid rice development and use because farm size is small and there is excess labor in rural areas, the area suitable for hybrid rice is large, particularly in the South-Central Coast, the mountainous highlands, and the Central Highlands, and government support nationally and provincially is strong. According to the hybrid rice development plan of the Ministry of Agriculture and Rural Development, the hybrid rice target area for 2005 will be 500,000 ha, while that for 2010 will be 1 million ha. Hybrid rice development and use in Vietnam 359

339 Table 3. Characteristics of CMS lines in the summer of Name Flowering Plant height Panicle Panicle exsertion Stigma Outcrossing (50%) (cm) length (cm) exsertion seed set of a line (%) A B A B A B A B (%) IR58025A IR62829A IR68897A IR69628A IR70369A AMS30A Zhenshan 97A AMS24A AMS39A Developing and evaluating newly developed hybrids Evaluation of CMS lines for developing three-line hybrids CMS lines are introduced or extracted from the F 1 population. They are studied for stability of sterility and adaptability to Vietnamese conditions. CMS lines with the most adaptability and good floral characteristics were selected for use in the breeding program. The characteristics of the selected CMS lines appear in Table Nguyen Tri Hoan and Nguyen Huu Nghia

340 Table 4. Some major characteristics of promising TGMS lines in Vietnam. Name of Name of Plant Duration Panicles Spikelets 1,000- TGMS institute a height in d (10% hill 1 panicle 1 grain weight line (cm) heading) (g) 7S HRRDC S HRRDC S HRRDC S HRRDC S HRRDC CL64S HRRDC VN01 AGI VN05S AGI T2TS HAU T29S HAU a HRRDC = Hybrid Rice Research and Development Center, AGI = Agricultural Genetics Institute, HAU = Hanoi Agricultural University. Evaluation of TGMS lines for use in the breeding program The characteristics of promising TGMS lines selected from the hybrid rice breeding network in Vietnam appear in Table 4. Results from genetic combining ability of these TGMS lines with 15 good male parents (testers) indicated that, among 10 TGMS lines, 7S (VNTGMS7S), 21S, CL64S, and T29S have a good general combining ability (GCA) value for yield. The remaining TGMS lines have a negative value for GCA of yield characters. The male parents RDT, RQ838, RQ5, R8, R130, R24, and R117 were found to have good GCA for yield. Results of breeding work and evaluations The adapted CMS lines were used for crossing with available restorers as well as elite lines in Vietnam. In total, 2,000 crosses were made in and every season. For two-line hybrid breeding, 1,000 1,500 testcrosses were made between TGMS lines with high yield and inbred lines. The yield potential of experimental hybrids was tested in observation yield trials and primary yield trials. The best hybrids selected in the summer and spring crops for each regional area from the national hybrid rice yield trial or multilocation yield trials for 1998, 1999, 2000, and 2001 are presented in Tables 5, 6, 7, 8, 9, and 10. Among these hybrids, HYT57, VN01/D212, AMS24A/Que 99, and TN15 have been released for large-scale hybrid rice commercial production. The remaining promising hybrids have been studied for F 1 seed production and further evaluation before releasing them for commercial exploitation. In addition to the crossing program, the four hybrids with high yield and good quality were selected after conducting the International Hybrid Rice Yield Trials for two years at four locations in Vietnam (Table 8). The newly developed or selected hybrids in Vietnam have yield equal to or Hybrid rice development and use in Vietnam 361

341 Table 5. Characteristics of promising hybrids selected in the National Hybrid Rice Yield Trials in the summer of Hybrid Duration Panicles Filled grains 1,000- Yield (d) m 2 panicle 1 grain weight (t ha 1 ) (g) Summer 1998 HYT * a VN01/D ** (2-line hybrid) TG4 (check) TG1 (hybrid check) CV (%) 5.2 LSD (5%) Summer 1999 AMS24A/Que ns AMS24A/M ns AMS24A/IR ns TG4 (hybrid check) ns CV (%) 6.2 LSD (5%) a * = significant at the 5% level, ** = significant at the 15% level. higher than that of the hybrid check. However, all selected hybrids have better quality than the commercial hybrids or inbred checks. Refining seed production technology through adaptive research The seed production technology of hybrids such as AMS24A/IR and AMS24A/ Que99 has been well developed in Vietnam. Considerable success has been achieved in the production of these hybrids primarily because of the high outcrossing rate of the female parent AMS24A. By the end of 1999, seed yields of t ha 1 were being obtained in the northern provinces of Vietnam. The Vietnamese government, because of the high cost of seed that is imported from China, has encouraged indigenous seed production activities. Experiments with different aspects of seed production technology such as synchronization parameters, dosage and stage of GA 3 application, row ratios, and plant population were carried out. The development of seed production technology specific for most of the released and promising hybrids also began. Results of these experiments are summarized as follows: 1. To achieve flowering synchrony in the parental lines of commercially released hybrids AMS24A/IR and AMS24A/Que99, the A line must be seeded when the R1 line reaches the 3.2- and 4.7-leaf stage, respectively. For IR58025A/BR827, the A line should be sown when the R1 line reaches the 5.5- and leaf stage in the spring and summer season, respectively. 362 Nguyen Tri Hoan and Nguyen Huu Nghia

342 Table 6. Characteristics of promising hybrids identified for different ecological locations in the summer of 2000, Vietnam. Hybrid Duration Panicles Filled % 1,000- Actual (d) m 2 grains sterility grain yield panicle 1 weight (t ha 1 ) (g) Ha Tay, summer A/IR (HYT91) HYT AMS24A/M HYT (2-line hybrid) 25A/IR (HYT88) AMS24A/Gui (hybrid check) Hungyen, summer A/IR (HYT88) HYT AMS24A/M HYT AMS24A/R Khangdan (inbred check) Binh Dinh, summer 2000 TN HYT DH (inbred check) 2. Prolonged storage of parental lines affected flowering synchrony in hybrid seed production. Storage of the male parents BR827, Que99, R242, and IR for one season resulted in flowering occurring 1 day earlier. On the other hand, seed storage shortened days to flowering by 2 3 days for the CMS lines IR58025A and AMS24A and by 1 2 days for Zhenshan 97A. 3. For seed production of the hybrid AMS24A/IR , an R:A ratio of 2:16 and A line transplanting space of cm resulted in the highest seed yield (2.5 t ha 1 ), which was confirmed in large-scale seed production for this hybrid during 1998 to 2001, in which yields of up to 4 t ha 1 were obtained. Similar results were obtained for the hybrid AMS24A/Que99 at different locations during the same period (average yields of 1.6 to 2.7 t ha 1 and maximum yield of up to 3.3 t ha 1 ) (Table 11). Hybrid rice development and use in Vietnam 363

343 Table 7. Characteristics of promising hybrids identified for different ecological locations in the spring of 2000, Vietnam. Hybrid Duration Panicles Filled % 1,000- Actual (d) m 2 grains sterility grain yield panicle 1 weight (t ha 1 ) (g) Ha Tay, spring 2000 HYT HYT84 (2-line hybrid) HYT TN TH2 (2-line hybrid) TG1 (check) DH85 (inbred check) Binh Dinh, spring S/R8 (2-line hybrid) HYT AMS24A/Que OM576 (inbred check) Nghe An, spring 2000 HYT S/R130 (2-line hybrid) HYT S/RO5 (2-line hybrid) HYT TG1 (hybrid check) CR203 (inbred check) Another problem in HYT57 (IR58025A/BR827) seed production is the low degree of stigma exsertion and less capacity of the stigma of IR58025A to receive pollen from the male parent. It was found that gibberellic acid (GA 3 ) application at 250 g ha 1 increased the outcrossing rate to 39% compared with 30% in the 180 g ha 1 control treatment. Furthermore, spraying of boric acid (H 3 BO 3 ) at 0.5% after the first GA 3 application increased seed set to 30% compared with 14% when GA 3 alone was applied (Table 12). 5. At Ha Tay, in the summer season, for HYT57 (IR58025A/BR827), a 2R:12A ratio and transplanting space of cm resulted in high yield (1.2 t ha 1 ), but, at Binh Dinh in south-central Vietnam, a 2R:10A ratio and transplanting space of cm gave the highest yield ( t ha 1 ) during the summer season (Table 13). 6. Hybrid rice seed production technology in Vietnam currently uses 3,000 4,000 m 2 for the seedbed or seedling nursery for each ha of seed production. Because of cold temperature in early spring, farmers need to cover the seedling nursery with nylon or plastic, thus raising seed production costs. A sim- 364 Nguyen Tri Hoan and Nguyen Huu Nghia

344 Table 8. Results of hybrid rice multilocation yield trials in Vietnam, spring crop season, Hybrid Grain yield (t ha 1 ) by location Mean Maturity (d) Ha Thai Nam Vinh Nghe Tay Binh Dinh Phuc An HYT90 (2-line) HYT HYT HYT A/IR A/IR A/R A/IR AMS24/R HYT HYT85 (2-line) HYT HYT HYT84 (2-line) S/R Boitap 77 (check) HYT CV (%) LSD (5%) Table 9. Results of hybrid rice multilocation yield trials in Vietnam (wet crop season, 2001). Hybrid Grain yield (t ha 1 ) by location Mean Maturity (d) Nam Tuyen Hai Ha Ha Quang Phong Tay HYT HYT HYT84 (2-line) HYT85 (2-line) HYT HYT TH2 (2-line) TH1 (2-line) II-32A/838 (check) Boyou 903 (check) HYT AMS24A/AYT AMS24A/ AMS24A/ AMS24A/ AMS24A/ AMS24A/ CV (%) LSD (5%) Hybrid rice development and use in Vietnam 365

345 Table 10. Yield of promising hybrids selected from the International Hybrid Rice Yield Trials in Vietnam in 1999 and Hybrid Ha Tay, Nghe An, Thai Binh, Binh Dinh, Mean spring 1999, spring 1999, spring 1999, spring 1999, (t ha 1 ) spring 2000 spring 2000 spring 2000 spring 2000 IR67693H IR68877H TG1, TN (checks) IR (inbred check) CV (%) LSD (5%) IR76901H IR69689H Local check (hybrids) National check CV (%) LSD (5%) plification of this technology was developed in which, instead of sowing 1 kg A line 50 m 2 seedling nursery and transplanting at the 5.8- to 6.0-leaf stage, 1 kg A line seed was sown on a 3-m 2 seedbed (high-density seeding method) and transplanting was done at the 3-leaf stage. Thus, instead of a 2,500-m 2 female-line seedling nursery requirement per ha of seed production, only a 210-m 2 seedling area was needed. Using this method, for hybrid AMS24A/Que99, about US$230 could be saved per ha (15% of the seed production cost). This technology has been gradually adopted in commercial hybrid seed production in several provinces. During , the available thermosensitive genic male sterile (TGMS) lines that were used as female parents in two-line hybrid rice breeding were characterized to determine their respective critical sterility points (CSP) and critical fertility points (CFP). Selected TGMS lines were further evaluated at Sapa and in the Red River Delta area. During 2000, six promising TGMS lines were studied for their CSP and CFP under artificial (growth chamber) and natural conditions. Results showed that these promising TGMS lines selected were all sterile at 25.5 C (Table 12). Work on specific locations and seasons for TGMS multiplication and two-line hybrid seed production was done. To determine the appropriate timing of TGMS seed multiplication and two-line hybrid seed production in Hanoi, for example, average temperatures from 1960 to 1980 were analyzed. It was found that the average temperature for May to September ranged from 27.4 to 29 C, suggesting that this period was suitable for two-line hybrid seed production. However, there were three 366 Nguyen Tri Hoan and Nguyen Huu Nghia

346 Table 11. Yield and yield components of male and female parents in seed production of AMS24A/Que99 at different locations ( ). Location/ No. of hills No. of No. of Ratio of F 1 seed Theoretical Av Max. season ha 1 (000) panicles spikelets R:A set yield yield yield ha 1 (000) ha 1 (million) (%) (t ha 1 ) (t ha 1 ) (t ha 1 ) R A R A R A R A Ank Hanh, Hoai Duc, , Ha Tay, spring 1996 Vu Thu, Thai Binh, , spring 1999 Vinh Bao, Hai Phong, , spring 1999 Ank Hanh, Hoai Duc, , Ha Tay, summer 1999 Co Do, Can Tho, ,153 4, spring 2000 Vinh Bao, Hai Phong, , spring 2000 Tien Lang, Hai Phong, , spring 2000 Hybrid rice development and use in Vietnam 367

347 Table 12. Effectiveness of different chemicals for fertilization capacity (outcrossing seed set) of IR58025A. Days after H 3 BO 3 DHHN a GA 3 Check flowering Seed Seed Seed Seed spikelets set spikelets set spikelets set spikelets set pollinated (%) pollinated (%) pollinated (%) pollinated (%) Total or av 2, , , , a DHHN = chemical components used in Vietnam. Table 13. Yield and yield components for different treatments of F 1 seed production experiment with HYT57 at Binh Dinh, summer Character Spacing Ratio of R and A (treatment) Mean of A line (cm cm) 2R:10A 2R:12A 2R:12A 2R:14A a 1.8 a 1.7 a 1.7 a 1.8 Actual a 1.4 b 1.4 b 1.3 b 1.5 yield b 1.3 c 1.2 c 1.2 c 1.3 (t ha 1 ) c 0.8 d 0.7 d 0.8 c 0.8 Av of treatment consecutive days in May when the temperature dropped to C, suggesting that it was unsafe to produce two-line hybrid seed in the Red River Delta during the spring season. On the other hand, there were no consecutive days with average temperature below 26 C during August, suggesting that it was safe to use TGMS lines with CSP of <26 C for two-line hybrid seed production during the summer season, with the flowering period arranged to occur in early September. The season for using TGMS lines for hybrid rice seed production in northern provinces was determined in Figure 1. These seasons have been exploited successfully for limited commercial seed production of two-line hybrids in Vietnam for the past 2 3 years. 368 Nguyen Tri Hoan and Nguyen Huu Nghia

348 Month Red River Delta Multiplication of TGMS for high yield F 1 seed production for TGMS lines having CTP 26 C Multiplication of TGMS in mountain areas (Sapa) Fig. 1. Seasons determined for using TGMS lines for hybrid rice seed production in northern provinces. CTP = critical temperature point. Challenges for hybrid rice technology in Vietnam The constraints and challenges that Vietnam faces and needs to overcome to further enhance the development and use of hybrid rice technology in the country are summarized as follows: Lack of hybrid rice combinations with good grain quality, tolerance of pests and diseases, and short duration ( d) that meet the requirements of the various agroecological zones of the country. Hybrid rice development and use in Vietnam 369

349 Only a few genetic materials are needed to develop hybrid rice suited to South Vietnam and with resistance to pests and diseases during the summer crop in the North. Currently, hybrids are blast-resistant but susceptible to major diseases such as bacterial leaf blight and sheath blight. Hence, they perform well only in the spring season because of lower disease pressure. Lack of standardization of seed production packages for different agroecological zones targeted for hybrid seed production for each season, and for particular hybrids. Lack of a strong local seed production system involving both the public and private sectors. The private seed industry and companies in Vietnam, at the national and provincial level, are still too weak for efficient and competitive hybrid rice seed production and distribution. The poor seed supply or difficulty in buying hybrid seed has been noted among farmers. Seed production led by the public sector, on the other hand, usually involves too many farmers even for small seed production targets, thus leading to poor quality control and related problems. Furthermore, both public and private seed companies are not linked closely with public hybrid rice R&D institutions. Low seed yields. With the exception of seed yield for AMS24A/IR and AMS24A/Que99, seed yield for other hybrids used in commercial production is still low ( t ha 1 ) and must be increased to t ha 1. Vietnam could produce parental and hybrid seeds during the spring season for summer-season cultivation but low seed yields are obtained in the summer season because of unfavorable climate and high disease pressure. Hence, purified seed of parental lines for spring-season hybrid rice is still inadequate. Yields in seed production of newly developed hybrids HYT56 (IR58025A/ 242R) and HYT57 (IR58025A/BR827), among others, have been lower than that of AMS24A-derived hybrids, partly because of the poor to moderate outcrossing potential of the CMS lines. Except for HRRDC, R&D institutes are not well equipped (with manpower, facilities, and equipment) to shoulder responsibility for nucleus and breeder seed production of locally developed and imported hybrids. Furthermore, isolated areas lack foundation seed production. Nonsuitability of hybrid rice seed production in the north, where hybrid rice is mostly cultivated to date, because of erratic climate and late harvesting time; hence, the need to identify and develop alternative seed production sites. Reluctance of small farmers to locally produce hybrid rice seeds because of greater risk; the very high financial requirement; lack of proper warehouse space, storage space, and cold storage for unsold seed; unavailability of pure CMS lines; and farmer preferences for imported seeds. Lack of an explicit policy to support domestic hybrid rice R&D in terms of manpower training and provision of physical infrastructure, facilities, and equipment. Funds for hybrid rice research and technology transfer, human resources in hybrid rice research (scattered in various institutions and with 370 Nguyen Tri Hoan and Nguyen Huu Nghia

350 Notes limited training on hybrid rice technology), and information sharing are still perceived to be inadequate. Inconsistent government policy of allowing the importation of Chinese hybrids for the summer season without regard to local F 1 seed production of the selected hybrids such as AMS24A/Que99, AMS24A/IR , and HYT57 (IR58025A/BR827) that can compete with imported hybrids in yield and grain quality. Authors addresses: Nguyen Tri Hoan, director, Hybrid Rice Research and Development Center, VASI; and Nguyen Huu Nghia, director general, Vietnam Agricultural Science Institute (VASI), Vietnam. Citation: Virmani SS, Mao CX, Hardy B, editors Hybrid rice for food security, poverty alleviation, and environmental protection. Proceedings of the 4th International Symposium on Hybrid Rice, Hanoi, Vietnam, May Los Baños (Philippines): International Rice Research Institute. 407 p. Hybrid rice development and use in Vietnam 371

351 Hybrid rice technology and achievements in Iran G.A. Nematzadeh, M. Sattari, A. Valizadeh, A. Alinejad, and M.Z. Nori Rice is a second main staple crop after wheat in Iran. More than 600,000 hectares of cultivated land are allocated to rice cultivation. Rice cultivation has had three distinguished periods in Iran: (1) before 1979, when all local rice varieties (tall, low-yielding, and excellent quality) were used; (2) , when high-yielding semidwarf varieties; either local improved ones such as Dasht, Nemat, Neda, and Sepeadroad or introduced varieties such as Amo13, Khazar, and others, were used, besides local varieties; the contribution of improved semidwarf high-yielding varieties to total rice production varies highly annually and it has depended on the quantity of imported rice; and (3) a period that actually began in 1991 with the introduction of some CMS lines from IRRI, when hybrid rice technology began in Iran. V20A, W32A, IR58025A, and IR28298A were the first CMS lines introduced from IRRI. Our primary attempt with hybrid rice technology started in 1991 and so far we have improved at least six new CMS lines from welladapted high-yielding improved varieties such as Kazar A, Nemat A, Neda A, Dasht A, and Champa A. The development of restorer lines such as IR24R, IR60969R, IR56R, and Amol 1R for Neda A, including a study of allogamyassociated traits, for all new improved CMS lines was carried out. This is our starting point for hybrid seed production. Two books on hybrid rice on hybrid seed production and a hybrid rice breeding manual were also translated into Persian to explain hybrid rice technology in Iran. The heterosis phenomenon and commercial F 1 hybrid seed have a significant role in yield production per unit area. Hybrid seed production technology caused real changes in rice production in China (Virmani and Edwards 1983). Cytoplasmic male sterility (CMS) or environmental genetic male sterility (EGMS) are essential in hybrid rice seed production because of the structure of the rice flower (perfect flower) (Virmani et al 1997). In spite of the study of cytoplasmic male sterility of rice since the 1930s, its application and hybrid seed production began in the 1960s in China (Yuan 1977). China was the pioneer in commercial hybrid seed production (Virmani et la 1981) 373

352 and several new CMS lines (A lines) and B and R lines were improved and released (Yuan 1994). The development of hybrid rice technology in China began in the 1970s. Hybrid rice varieties demonstrated 15 20% more yield than the recombinant inbred lines (Ikehashi et al 1998, Yuan 1977, 1994, Yuan et al 1989). Regular hybrid rice seed was produced in 1975 and 1976 in China (Virmani et al 1997). During , total rice production increased to 300 million tons because of the application of hybrid vigor in China (Ahmad et al 1996). The first attempts with hybrid rice in Iran started at the Sari Science of Agriculture College and Amol Rice Research Center through the introduction of two CMS lines (V20A and W32A) from IRRI in 1987 and later in 1990 (Nematzadeh et al 1996). Two other CMS lines, IR58025A and IR29298A, were introduced from IRRI and the study and breeding of new CMS lines from well-adapted and high-yielding improved varieties started in 1991 (Nematzadeh et al 1996). To harmonize all the activities on hybrid rice technology in Iran, a national hybrid rice project was prepared and approved by the AREEO research council in The goals of the project were to study putative maintainer lines, develop new CMS lines suitable to the region, particularly based on consumers preferences, and identify appropriate restorer lines and develop hybrid rice technology in Iran. Materials and methods Several local high-quality, tall, and low-yielding varieties (Tarom, Domsia, and Rashti Sadri) including high-yielding semidwarf varieties with acceptable quality (Nemat, Neda, and Dasht) were evaluated for type of nuclear gene controlling grain fertility. This evaluation was done using IRRI-source CMS lines such as IR58025A and IR28298A as female parents. The IRRI methods for screening F 1 pollen grain between CMS female parents and a new unknown nuclear gene for controlling fertility were studied (staining of pollen grain with 1% IKI) (Virmani et al 1982, 1997) and pollen grain was classified into four categories: complete sterility (0% fertility), partial sterility (1 30% fertility), partial fertility (30 60% fertility), and complete fertility (>60% fertility) (Govindaraj and Virmani 2000). The putative suitable maintainers were identified and, through several backcrosses, the new nuclear genome was transferred to sterile cytoplasm. After fixation of new CMS lines, the allogamy-associated traits were also studied according to the methods developed by IRRI (Virmani et al 1997). Results and discussion Primary results showed that local rice varieties, in spite of their excellent grain quality, were not suitable for hybrid rice technology because of their very tall plant height, narrow culm, and high susceptibility to lodging. So, all local tall varieties were eliminated from further study. Some high-yielding semidwarf improved varieties such as Nemat, Neda, Dasht, Amol 3, and Champa showed the recessive nuclear gene (rfrf) 374 Nematzadeh et al

353 Fig. 1. Pollen grain of F 1 hybrid of IR58025A/Nemat stained by 1% IKI solution. Nemat B Nemat A Fig. 2. Newly developed CMS lines (Nemat A and B) and their maintainer lines using IR58025A sterile cytoplasm via backcrosses. when crossed to IR58025A because their F 1 hybrid indicated complete sterility. Then, several backcrosses were carefully made and a single plant of each backcross was precisely checked for 100% sterility. Finally, after seven backcross generations, new Nemat A, Neda A, Dasht A, Amol 3A, and Champa A CMS lines with complete sterility were developed. New Nemat A and Nemat B, including their natural CMS seed production, appear in Figures 1 and 2. Hybrid rice technology and achievements in Iran 375

354 Some agronomically important traits of new CMS lines are shown in Table 1 and their allogamy-associated traits in Table 2. Besides this breeding program, some more varieties were also checked for restorer lines. In this regard, restorer lines such as IR24R, Amol 1R, and IR56R were identified for Neda A variety (Table 3). As was mentioned earlier, this is the first attempt at and achievement of hybrid rice breeding technology in Iran. Nemat and Neda recombinant inbred lines developed from Sang Tarom (local variety as a female parent) crossed to Amol 3 (semidwarf improved variety as a male parent) have excellent performance and are completely resistant to blast disease and show tolerance for stem borer (the most common disease and insect in Iran), with more than 8 t ha 1 of average yield and long grain and good quality. Definitely, their F 1 hybrid with another high-yielding semidwarf variety such as IR24R or IR56R will give a better performance (Nematzadeh et al 1996, 2002). Our next programs are to evaluate combining ability, heterosis ratio, and the feasibility of mass hybrid seed production (Fig. 3). References Ahmad MI, Vigaya CHM, Kumar MS, Ramesha, Krishnaiah K Hybrid rice technology, final report Govindaraj K, Virmani SS Causes of incomplete fertility-restoring ability of some established restorer lines of WA cytosterility system in rice. Manila (Philippines): International Rice Research Institute. Ikehashi H, Wan J The wide compatibility system: current knowledge of its genetics and use for enhanced yield heterosis. In: Virmani SS, Siddiq EA, Muralidharan K, editors. Advances in hybrid rice technology. Manila (Philippines): International Rice Research Institute. p Nematzadeh GhA Planning and programming of national hybrid rice project research. Iran Rice Research Institute. Nematzadeh GhA, Arefi H, Amani R, Mani R Release of new high-yield rice variety with good quality, Nemat. J. Agric. Tehran Univ. Nematzadeh GhA, Arefi H, Amani R, Mani R Development of new high-yield rice Neda with good physicochemical characteristics. J. Seed Plant Improve. Virmani SS, Edwards IB Current status and future prospects for breeding hybrid rice and wheat. Adv. Agron. 36: Virmani SS, Chaudhary RC, Khush GS Current outlook on hybrid rice. Oryza 18: Virmani SS, Viraktamath BC, Casal CL, Toledo RS, Lopez MT, Manalo GO Hybrid rice breeding manual. Los Baños (Philippines): International Rice Research Institute. Virmani SS, Aquino RC, Khush GS Heterosis breeding in rice (Oryza sativa L.). Theor. Appl. Genet. 63: Yuan LP, Virmani SS, Mao CX Hybrid rice achievements and future outlook. In: Progress in irrigated rice research. Manila (Philippines): International Rice Research Institute. p Nematzadeh et al

355 Table 1. Agronomically important traits of new CMS lines with their cytoplasm donors (Nemat A, Neda A, Dasht A, Amol 3A, Champa A, and IR58025A). Variety Tillers Plant Panicle Flag-leaf Flag-leaf Flag-leaf 50% Grains 1,000- (no.) height length length width angle flowering panicle 1 grain (cm) (cm) (mm) (mm) ( ) (d) (no.) weight (g) Nemat A Neda A Dasht A Amol 3A Champa A IR58025A Table 2. Allogamy-associated traits of new CMS lines with their cytoplasm donors (Nemat A, Neda A, Dasht A, Amol 3A, Champa A, and IR58025A). Variety Pistil Style Stigma Filament Anther Lemma Opening Period Stigma length length length length length and palea time of color a (mm) (mm) (mm) (mm) (mm) angle ( ) opening (mm) Nemat A Neda A Dasht A Amol 3A Champa A IR58025A a On a scale of 1 5, where 1 = white, 2 = light green, 3 = yellow, 4 = light purple, and 5 = purple. Hybrid rice technology and achievements in Iran 377

356 Table 3. Results of F 1 hybrid pollen grain and spikelet fertility of some putative lines with newly developed CMS line Neda A. Crosses Pollen grain Panicle fertility (%) fertility (%) Neda A/IR Neda A/Amol Neda A/IR Neda A/IR Neda A/IR Neda A/IR Neda A/IR Neda A/Amol Fig. 3. Natural mass CMS seed production of newly developed CMS line Nemat A at Sari Science of Agriculture College experimental field. 378 Nematzadeh et al

357 Yuan LP Increasing yield potential in rice by exploitation of heterosis. Paper presented at the Symposium on Hybrid Rice, Manila, Philippines, April International Rice Research Institute. Yuan LP The execution and theory of developing hybrid rice. Chinese Agric. Sci. 1: (In Chinese.) Notes Authors address: Amol Rice Research Center and Sari Science of Agriculture College. Acknowledgments: We are thankful to Dr. S.S. Virmani for kindly providing us with the CMS lines. The authors also thank the director of AREEO and deputy minister of Agriculture, Mazandaran University, Iran Rice Research Institute, Amol Rice Research Center, and Sari Science of Agriculture College for providing all facilities for this study. Citation: Virmani SS, Mao CX, Hardy B, editors Hybrid rice for food security, poverty alleviation, and environmental protection. Proceedings of the 4th International Symposium on Hybrid Rice, Hanoi, Vietnam, May Los Baños (Philippines): International Rice Research Institute. 407 p. Hybrid rice technology and achievements in Iran 379

358 Hybrid rice development and use in the Philippines, E.D. Redoña, F.M. Malabanan, M.G. Gaspar, J.C. de Leon, and L.S. Sebastian The development and use of hybrid rice technology as a major approach for further increasing rice productivity gained national prominence in the Philippines with the launching of a national hybrid rice program under the Department of Agriculture (DA) in January With the Philippine Rice Research Institute (PhilRice) at the helm, activities on research and development (R&D), seed production, training, technology demonstrations, and information dissemination on hybrid rice were undertaken nationwide. The hybrid rice research program at PhilRice, with 18 multidisciplinary projects conducted by more than 80 scientists, was strengthened with the establishment of a hybrid rice center and collaboration with IRRI and other national programs, the private sector, and Chinese hybrid rice R&D institutions. Season-long training activities for prospective hybrid seed growers in strategic areas led to the organization of cooperatives that began producing hybrid seed on a commercial scale starting in The more than 1,000 trainers, agricultural technicians, seed inspectors, and local government officials that were trained on hybrid rice seed production and cultivation techniques extended this new knowledge to hybrid rice seed growers and cultivators. On large-scale technology demonstration farms, the hybrid rice Mestizo showed more than a 1.2 t ha 1 yield advantage over the best inbred varieties during and 1.3 t ha 1 in commercial cultivation areas during the 2001 wet season. Privatesector participation also increased in the areas of breeding, seed production, and technology promotion. In 2002, the hybrid rice program was embraced as the banner program for agricultural modernization by the Philippine government. Rice in the Philippines is cultivated on 2.3 million hectares (physical area) or 41% of the country s total arable land of 5.6 million ha. It contributes 2.3% to the gross domestic product and 2.2% to the gross national product. In 2000, 16.32% of the real value of agricultural production was accounted for by rice. An average rice farmer in the Philippines owns and tills about 1.5 ha of irrigated rice land (Tolentino et al 381

359 2001). Three-fourths of the country s rice farms are exclusively devoted to rice production, that is, they follow a rice-rice cropping pattern. Rice is the staple food for more than 90% of the country s population, accounting for 41% of average calorie intake to as high as 60 65% for the poorest households (David and Balisacan 1995). Average annual per capita rice consumption in the country has hovered between 90 and 100 kg (Redoña et al 1997). On average, a Filipino consumes 95 kg of rice per year. Eighty percent of Filipino households (10 million out of 12 million) devote at least half of their expenditures to food and about a quarter of the total food budget of Filipino families is used for rice (Tolentino et al 2001). Employment in the rice industry is highest among agricultural subsectors, involving 10.6 million farmers and family members, of which 2.1 million are rice farmers. Close to three-fourths of farm household income is derived from rice farming and related activities. Meanwhile, 80% of the income of the average rice farmer is primarily derived from rice (Tolentino et al 2001). Of the 4.0 million ha of harvested rice area, some 2.66 million ha (67%) are irrigated, 1.28 million ha (32%) are rainfed lowlands, and 50,000 ha (1%) are rainfed uplands. The national average yield is 2.95 t ha 1, with the highest yields in irrigated areas at 3.4 t ha 1, followed by rainfed areas at 2.1 t ha 1 and upland areas at 1.5 t ha 1 (BAS 1999). Rice production in the Philippines increased from 5 million t in 1970 to a record million t in 2001, for an average annual increment of 3%. Traditionally, production volume during the wet season (WS) is higher by about 10%, as both rainfed and irrigated areas are cultivated, compared with the dry season (DS). The harvested rice area also increased from 3.1 million ha in 1970 to 4.0 million ha in The three major rice-producing provinces in the country are Isabela, Nueva Ecija, and Iloilo. In 2000, Isabela posted the highest harvested rice area and total rice production volume of 260,140 ha and 1 million t, respectively. In 1998, both rice production and area harvested drastically decreased because of the El Niño phenomenon. As a consequence, rice importation reached its peak at 2.2 million t in The role of hybrid rice technology Significant strides have been made in increasing rice production in the country over the years, with a 4% growth in 2001, mostly because of the high adoption of highyielding varieties (HYVs), improved irrigation facilities, and intensive use of fertilizers. However, the high and steady growth of the Philippine population (2.3% per annum), the decreasing area devoted to rice production because of urbanization and industrialization, the relatively high per capita rice consumption, and trade liberalization issues resulting from the Philippines participation in the emerging world economic order have become the major forces fueling the country s vigorous pursuance of rice self-sufficiency and food security-related goals. Over the next 25 years, at least 65% more rice, relative to 2000 production volume, would be needed to adequately feed the Philippine population. The Philippine government has realized that a major strategy that can be used to meet this 382 Redoña et al

360 enormous challenge is to increase the yield of rice per unit area per unit time. Recognizing the potential of rice hybrids to increase rice production over and above currently achievable levels, as amply demonstrated in China, and the prospects that the technology offers for increasing the profitability and competitiveness of rice farming, the Philippine government launched a hybrid rice program in 1998 under the Department of Agriculture s (DA s) rice production program. Besides investing in hybrid rice technology using its own resources, the Philippine government obtained two Technical Cooperation Project (TCP) grants from the Food and Agriculture Organization of the United Nations (FAO). Implemented by the Philippine Rice Research Institute (PhilRice) from July 1998 to November 2000, the projects successfully assisted the Philippine government in strengthening its national capacity for hybrid rice development and use (Virmani 2001). In addition to the FAO TCP project, the government also entered into two bilateral agreements with the People s Republic of China (PROC) on hybrid rice technology, one through the Chinese Ministry of Agriculture and the other through the RP-PROC Protocol on Science and Technology Cooperation. Under the former, construction of a US$5 million Philippine-Sino Agricultural Technology Promotion Center will be based at Central Luzon State University in Muñoz, Nueva Ecija. This will focus on hybrid rice technology use and agricultural machinery promotion, which started in Hybrid rice technology has also been looked upon as a major means for creating new employment opportunities in the rice sector. It has become a banner project for agriculture in support of the government s thrust to create a million new jobs nationwide by In December 2001, Administrative Order 25 was signed by Philippine President Gloria Macapagal-Arroyo, formally declaring the hybrid rice program as a banner program of the government, and identifying PhilRice as the program s lead implementing agency. Executive Order 76 was subsequently enacted in March 2002, transferring PhilRice from the DA to the Office of the President. The role of PhilRice At the forefront of the Philippine hybrid rice program is PhilRice, located in the country s erstwhile premier rice-producing province of Nueva Ecija, 143 km north of Manila. PhilRice is mandated to carry out a national rice research and development program, to sustain the gains made in rice production, and to solve locationspecific problems of the rice industry. PhilRice s mandate empowers it to direct and coordinate rice R&D activities of all agencies working on rice in the Philippines. To support the national hybrid rice program (HRP), PhilRice created an R&D program for hybrid rice in July The hybrid rice program at PhilRice aimed at developing and using hybrid rice technology to increase average rice yields by at least 15%. Specifically, the program (1) plans and implements multidisciplinary R&D activities on breeding; seed production; nutrient, water, and pest management; socioeconomics and policy; and other relevant areas of concern; (2) develops and promotes resource- and cost-efficient seed production technologies for parental and Hybrid rice development and use in the Philippines,

361 hybrid seed production and organizes a national hybrid rice seed production network; (3) creates awareness on hybrid rice technology among the general public and promotes its adoption to Filipino farmers; (4) coordinates public-sector activities on hybrid rice R&D, seed production, and technology promotion at the national level, and explores collaboration with the private sector; and (5) links up and collaborates with hybrid rice programs of other countries and other international hybrid rice institutions. Also in 1998, it converted its San Mateo, Isabela, branch station, with 29 staff members, into its Hybrid Rice Center to serve as the main base for applied hybrid rice R&D activities. The program has drawn financial support from the DA, the Asian Development Bank, the Rockefeller Foundation, FAO, and the International Rice Research Institute (IRRI). It has collaborative R&D agreements with several institutions in China, notably the Yunnan Agricultural University, Jiangxi Academy of Agricultural Sciences (JAAS), Guangxi Academy of Agricultural Sciences, and the Fujian Agricultural and Forestry University (Redoña et al 1998). During its first full year (crop year 1999) of implementation, the HRP put into operation 18 hybrid rice research-development and extension (RD&E) projects, with 91 studies implemented by 85 lead scientists and technical personnel. The activities encompassed the areas of germplasm conservation and characterization; breeding; molecular marker technology applications and genetic engineering for improved pest resistance; seed production research; grain quality evaluation; development of water, nutrient, and planting management practices; pest resistance screening; ecology of pests and beneficial organisms; development of IPM protocols; socioeconomics and policy; engineering and mechanization; training; and technology demonstration. The results of the various activities undertaken in the various disciplines engaged in hybrid rice R&D have been summarized in a proceedings that was published as an activity of the FAO TCP project (Redoña and Gaspar 2001). Technology generation Breeding From 1998 to 2001, seven breeding institutions PhilRice, the International Rice Research Institute (IRRI), the University of the Philippines Los Baños (UPLB), and four private seed companies, AgroSeed (a Monsanto subsidiary), HyRice (a local seed company), Aventis, and SL Agritech (local seed company backed up by Chinese experts) were actively involved in breeding or testing rice hybrids using both the three-line and two-line methods in the Philippines. Aventis and SL Agritech began their activities only during this period. The three-line breeding system using the wildabortive or WA cytoplasmic male sterility (CMS) system remained as the main focus of breeding work, but other CMS systems were also being used (Redoña et al 2000). Using the CMS system, three hybrid combinations were commercially released in the Philippines from 1998 to These were PSB Rc26H (Magat, IR62829A/ IR R), released in 1994, which matured in days and showed a yield advantage of 14.6% over the inbred check PSB Rc4; PSB Rc72H (Mestizo, IR58025A/IR R), released in 1997, which showed a yield advan- 384 Redoña et al

362 Table 1. Hybrid combinations being considered for commercial release in Season Hybrid yield % Yield % Yield (kg ha 1 ) advantage over advantage over PSB Rc28 Mestizo IR73860H (IR6888A/IR R) Across season 6, Dry season 5, Wet season 6, IR75207H (IR68888A/IR R) Across season 5, Dry season 5, Wet season 6, IR75217H (IR68897A/IR R) Across season 5, Dry season 5, Wet season 6, tage of 16.4% over the inbred check varieties IR68 and IR74 across seasons (dry and wet) and 26.9% during the dry season in national trials and matured in days; and the early maturing (100 days) PSB Rc76H (Panay, developed by AgroSeed), which was released in 1999 to specific regions of the country, where it satisfied the 15% higher yield requirement vis-à-vis the best inbred varieties. By 1998, improved protocols for the multilocation National Cooperative Tests (NCT), the penultimate testing stage prior to varietal release, were being implemented. Salient features of these new guidelines were the use of two fertilizer levels to identify superior hybrids for low- and high-input cultivation, and the testing of parental lines along with the hybrids to produce vital information for commercial hybrid seed production. In 2001, promising experimental hybrids from IRRI, PhilRice, SL Agritech, and Aventis were entered in the NCT. Strict commercial release requirements notwithstanding, three new hybrids that met the minimum testing requirements in the NCT were identified in 2001 as candidates for commercial release (Table 1). Seed production evaluations for these IRRIdeveloped hybrids (IR73862H, IR75207H, and IR75217H) were undertaken at four locations across the country during the DS as a final release requirement. Following a National Seed Industry Council (NSIC) guideline enacted in 2001 for the naming of rice hybrids, released hybrids would carry the name Mestizo, followed by a number. Naming all future hybrids Mestizo, which literally means hybrid in local parlance, is expected to facilitate and simplify technology promotion/information campaigns on the usage of hybrid rice technology by farmers. Aside from conventional methods, new tools in biotechnology began to be used for hybrid rice breeding in the country for CMS line diversification, for improv- Hybrid rice development and use in the Philippines,

363 ing heterotic potential, and for improving disease resistance (Redoña et al 2000). At PhilRice, some biotechnology-assisted breeding work included marker-aided breeding for two-line hybrids using the thermosensitive genetic male sterility (TGMS) system, pyramiding of bacterial blight resistance genes in promising hybrid rice maintainer (B) lines, and Agrobacterium-mediated transformation of B lines with disease and insect resistance-related genes. Seed production The seed production technology developed at IRRI was initially used by PhilRice for seed production activities nationwide for the released hybrids, particularly Magat and Mestizo. Refinements in the technology package have since been incorporated and PhilRice s collaborative undertakings with Chinese hybrid rice institutions have been useful in increasing seed yields. In the 1999 DS, for example, a record 3.0 t ha 1 F 1 seed yield was obtained in seed production demonstration plots supervised by experts from JAAS (Shen et al 2000). To fine-tune existing technologies, research aimed at optimizing flowering synchronization of parental lines, determining the optimum practices for male (R line):female (CMS or A line) row-ratio, timing and rates of gibberellic acid (GA 3 ) applications, procedures for flag-leaf clipping, and developing planting, nutrient, water, and pest management protocols for hybrid seed production was conducted by PhilRice and the rice R&D network. During 1998 to 1999, for example, the effect of planting in May and June on the number of days to 50% flowering was investigated in the parental lines of Mestizo-IR58025A and IR48686R. Results indicated that seeding in May required a 14-day seeding interval between the parents, with the male parent to be planted first. However, when seeding was done in succeeding months (June and July), a differential seeding in which the male parent is planted 20 days ahead of the female parent was required. Studies on different nitrogen rates applied to the seed production plots, on the other hand, found a dose of 90 kg ha 1 to be optimum. Studies on flag-leaf clipping and optimum dosage of GA 3 application indicated that, with flag-leaf clipping, a dose of 50 g ha 1 is optimum, whereas without flag-leaf clipping a dosage of 80 g ha 1 should be applied to obtain high seed yields. Multieffect triazole (MET) applied at 1,000 g ha 1 produced the most vigorous seedlings. In 1998 to 1999, nucleus and breeder seed production of the parental lines of the released hybrids Magat and Mestizo was undertaken. To strengthen national capability for nucleus and breeder seed production, a tripartite collaborative project among PhilRice, IRRI, and the University of the Philippines Los Baños (UPLB) was established in 2000 to provide an adequate supply of nucleus, breeder, and foundation seeds of hybrid parental lines used in the national hybrid rice production program. About 500 paired crosses produced each season by PhilRice were provided to UPLB and IRRI, which then produced the breeder and foundation seeds in a joint field activity at the IRRI farm. Breeder and foundation seeds from this project were then used to produce certified seeds of the A line by PhilRice and private seed growers, which were authorized to undertake A B seed production. To further improve 386 Redoña et al

364 the system, SL Agritech was authorized in 2001 to produce IR58025A line seeds at a guaranteed purity of 95%. These seeds were then distributed to groups/individuals undertaking hybrid rice seed production of Mestizo in different parts of the country. In 1999, the NSIC s Technical Working Group on Seed Certification and Seed Standards finalized the guidelines for hybrid rice seed testing, seed production, and seed certification. These guidelines were revised and subsequently enacted by the DA in 2001 to include seed certification for parental lines in addition to hybrid seed certification. These guidelines form the basis for field and laboratory inspection/testing/ certification activities undertaken by the Seed Quality Control Services of the Bureau of Plant Industry (BPI), the government agency mandated to enforce seed quality standards for rice and other crops. Highlights of the PhilRice hybrid rice program in 1999 to 2001 are listed in Table 2. Table 2. Research & development highlights from the PhilRice hybrid rice program, Item Germplasm characterization and conservation Varietal development Biotechnology Cultural management Comments 821 accessions characterized for morpho-agronomic traits and pest and disease resistance. 3,865 testcrosses developed from 9 to 22 CMS lines and 1,157 tester inbreds; only 12% and 1% of testcross F 1 s were observed as potential A R and A B combinations, respectively; experimental hybrid yields exceeding 8.5 t ha 1 consistently observed across trials. Low correlation between simple sequence repeat marker heterozygosity and per se hybrid performance; conditions for Agrobacterium-mediated transformation of B lines optimized and putative transgenic calli containing diseaseresistance genes being confirmed; B line-specific polymerase chain reaction products obtained from mtdna of IR69625A and B lines. Relationship between fertilizer and headrice recovery significant in Mestizo hybrid; 8.89 t ha 1 yield of Mestizo observed when 50% of 180 kg N ha 1 was substituted with rice straw incorporated 3 weeks before transplanting; 8.99 t ha 1 yield of Mestizo observed when 90 kg N applied in 3 splits at mid-tillering, panicle initiation, continued on next page Hybrid rice development and use in the Philippines,

365 Table 2 continued. Item Pest management Economics Engineering and mechanization Hybrid rice seed production Comments and flowering; seedlings of Mestizo should be planted at days to increase yield. Green leafhopper, brown planthopper, rice earhead bugs, and stem borers are dominant pests in major hybrid ricegrowing areas; spiders and Cyrthurhinus among the prevalent natural enemies in Region 2 and Cordillera Autonomous Region; no differences in diversity of pests and beneficial organisms of inbred and hybrid rice in Isabela, Mindanao, Nueva Ecija, and Iloilo; natural enemy population increased significantly with chicken manure application while increase in herbivore densities was associated with synthetic fertilizer application. Break-even yield for hybrid rice with inbred seed production found to be 600 kg ha 1 at F 1 seed price of P100 kg 1 ; hybrid rice cultivation relatively more profitable (13% higher net income) than inbred paddy production because of higher F 1 yield; the net incremental benefit from hybrid cultivation estimated as P7,850 per hectare. A three-stage hybrid grain cleaner (model HC13) performed best at 1/16 hopper opening and 1,090-rpm blower speed with an air velocity of 6.2 m s 1 ; efficiency, loss, and purity values were 98.5%, 1.2%, and 98.7%, respectively; seeding rate of the prototype four-row precision seeder still above the 20 kg ha 1 target rate. GA 3 application at 50 g ha 1 optimum with flag-leaf clipping during panicle initiation, g ha 1 optimum with no clipping; 2.04 t ha 1 F 1 seed yield obtained with 200 g ha 1 GA 3 application; GA 3 application did not affect the germination of hybrid and A line seeds; a 2:8 row ratio of Mestizo A and R lines optimum at Maligaya and San Mateo; highest yields obtained with 2:10 and 2:12 ratios; multieffect triazole (MET) growth enhancer improved seedling vigor. 388 Redoña et al

366 Table 3. Number of participants, provinces, and regions represented in hybrid rice seed production training course conducted by the PhilRice TPD Program. Year Participants Provinces 1 Regions a and 1998 b c d Total Agusan del Norte ac, Agusan del Sur c, Albay b, Aurora d, Basilan a, Bataan a, Biliran c, Bohol abcd, Bulacan b, Bukidnon bcd, Cagayan abcd, Camarines Sur abcd, Camiguin c, Cavite d, Cebu abc, Davao del Norte bc, Davao del Sur abc, Davao Oriental ab, Eastern Samar bc, Ilocos Norte abcd, Iloilo abc, Isabela abcd, Laguna abcd, Lanao del Norte cd, Lanao del Sur ac, La Union bcd, Leyte bcd, Maguindanao abcd, Metro Manila bcd, Misamis Occidental c, Misamis Oriental acd, Mt. Province a, Negros Occidental bc, Negros Oriental bc, North Cotabato abcd, Northern Samar bc, Nueva Ecija abcd, Occidental Mindoro bc, Oriental Mindoro bc, Palawan d, Pampanga bcd, Pangasinan abcd, Quezon d, Quezon City bd, Quirino abc, Rizal bd, Sarangani acd, Sorsogon ab, South Cotabato bcd, Sulu a, Sultan Kudarat bcd, Surigao del Sur c, Tarlac bcd, Tawi-Tawi a, Western Samar c, Zambales abc, Zamboanga del Norte bcd, Zamboanga del Sur bd. 2 CAR acd, 1 abcd, 2 abc, 3 abcd, 4 abcd, 5 abcd, 6 abcd, 7 abc, 8 bcd, 9 bcd, 10 abcd, 11 abcd, 12 abcd, ARMM acd, Caraga abc, NCR bcd. Capacity building Training courses Two major types of training courses on hybrid rice technology were conducted by PhilRice beginning in These were the Season-Long Rice Specialists Training Course on Hybrid Rice Seed Production (RSTC-Hybrid) and the 4-day Specialized Training Course on Hybrid Rice Seed Production (STCHRSP). The participants in RSTC-Hybrid were mostly trainers selected from hybrid rice target areas across the country. They were expected to become specialists in hybrid rice cultivation and seed production and to train potential hybrid rice seed growers and farmers in their respective areas of operation. The 17-week course was initially conducted during the 1998 WS with 21 prospective trainers from 16 provinces in 9 regions. Of these, 18 came from DA agencies, 2 from a nongovernment organization (NGO), and 1 from a local government unit (LGU). The STCHRSP, in contrast, was offered more frequently and was designed for farmers/seed growers, seed inspectors, seed analysts, seed coordinators, extension workers, and other R&D implementers. This course discussed the principles of hybrid seed production and cultivation through lectures and practical hands-on exercises. From March 1997 to 2000, more than 20 batches of STCHRSP were conducted in Luzon, Visayas, and Mindanao, drawing a total of 973 participants (Table 3, Yabes et al 2001). Participants included potential seed growers (60%), RD&E personnel (30%), seed inspectors (8%), and seed coordinators (2%). After the training, seed growers were provided seeds of the parental lines of Mestizo for use on their own farms the following season. Hybrid rice development and use in the Philippines,

367 In 2000, the PhilRice Hybrid Rice Center developed a season-long training program on hybrid rice seed production for seed growers the University Without Walls (UWW). Under the UWW, which consisted of an 80:20 practicum:lecture format and once-a-week meetings held throughout the cropping season, a practice field that served as the classroom was planted in advance of actual seed production in seed growers fields. This allowed participants to directly apply new-found knowledge and skills on their own farms. Five batches of UWWs were conducted in different towns of Isabela, Quirino, and Cagayan provinces in the 2000 WS, which eventually triggered the establishment of hybrid rice seed growers associations/cooperatives in these areas the following year. As technical capacity on hybrid rice seed production began to expand, training offerings became decentralized, with training courses initiated in different parts of the country by the ATI and PhilRice Branch Stations. Beginning in 2001, for example, PhilRice-Batac conducted training courses on hybrid rice seed production for northwestern Luzon, while courses were held at ATI-Aklan and Aklan State College of Agriculture for the Visayas, and at ATI-Midsayap and PhilRice-Midsayap for Mindanao. Furthermore, two specialized training courses designed solely for R&D implementers with more than 100 participants were conducted using resource speakers drawn from collaborating institutions in China and through the FAO TCP grant. A course on parental line (A B) seed production was also organized in the 1999 DS for selected seed growers and SeedNet members. On-farm training on hybrid rice cultivation practices was also incorporated in the farmers field school (FFS) training module that was implemented at technology demonstration (techno-demo) sites nationwide. Workshops In 1998 to 1999, workshops were organized in eight provinces targeted for hybrid rice seed production to discuss strategies and work out the modalities for developing and using hybrid rice technology, in general, and for undertaking large-scale hybrid rice seed production, in particular. Participants included seed growers, seed inspectors, provincial seed coordinators, provincial agriculturists, agricultural technicians, and regional seed coordinators. In each province, the number of seed growers was identified and targets for seed production were fixed. Necessary arrangements were made to supply the seeds of the parental lines and technical guidance was given for undertaking hybrid rice seed production. In 2000, preseason workshops held in parts of the country planned the hybrid rice cultivation and seed production targets for the WS and beyond, and discussed various strategies for using hybrid rice technology in these regions. A national policy workshop on hybrid rice was also held in collaboration with the Asia Pacific Seed Association (APSA), IRRI, FAO, and the DA. Moreover, the 2nd National Workshop on Strengthening National Capacity of Hybrid Rice Development and Use was held in collaboration with IRRI and the FAO and a Dialogue for Establishing Partnerships Between Public and Private Sectors for Production and Marketing of Hy- 390 Redoña et al

368 brid Rice Seed was held at IRRI to encourage the participation of large seed companies in hybrid rice seed production. Aside from private seed companies, NGOs, cooperatives, and government personnel from key agencies such as the DA, BPI, UPLB, and PhilRice participated. Issues affecting hybrid rice technology commercialization were discussed and various recommendations were forwarded to increase privatesector participation. Among suggested provisions and support to be given by the public sector were technical support to industry, free access to public hybrid rice germplasm, a favorable investment climate through tax breaks and seed procurement programs, improved purity of parental lines, an effective hybrid rice seed certification process, and the development of more productive and less costly hybrid rice seed production technologies. In 2001, a National Workshop on Hybrid Rice for Policy Formulation was held to critically assess the progress made in the country since 1998 in hybrid rice technology generation, seed production, and technology promotion and to produce policy recommendations for fast-tracking the commercialization of hybrid rice technology in the Philippines. Participants were key public- and private-sector movers in hybrid rice technology. A work plan was prepared to achieve hybrid rice cultivation targets and a resolution was drafted on policy issues for submission to top government policymakers. Briefings Numerous technical briefings and field days, primarily aimed at convincing farmers to use hybrid rice, were held from 1998 to Some briefings were also aimed at farmers interested in becoming seed growers. Organized by PhilRice, ATI, LGUs, and private-sector players in different parts of the country that were targeted for hybrid rice cultivation, the briefings also increased public awareness on hybrid rice technology. For example, in 1999 to 2000, 3,389 farmers were briefed on hybrid rice technology in Region 2 alone 2,602 in Isabela, 509 in Cagayan, 178 in Quirino, and 100 in Kalinga Province. In addition, 8,300 farmers attended hybrid rice field days organized in the same region, including three field days with 2,200 participants and 14 field days with 6,100 participants in 1999 and 2000, respectively. Some of these capacity-building measures are shown in Figure 1. Technology demonstrations To showcase the advantage of hybrid rice cultivation in target areas across the country, large-scale (20 ha) on-farm technology demonstration (TD) activities began in the 1998 DS in the provinces of Isabela, Camarines Sur, Iloilo, Davao del Sur, and Maguindanao. These front-line demonstrations were located in strategic areas and each farm was divided into inbred and hybrid rice demonstration plots to allow easy visual evaluation of crop stand and the yield advantage of the hybrids over the best inbred varieties preferred by farmers in the area. Large billboards were used to draw the attention of passersby and field days and harvest festivals were held toward the end of the season for farmers, technicians, seed growers, millers, local officials, and Hybrid rice development and use in the Philippines,

369 A B C D E F Fig. 1. Selected capacity enhancement activities conducted in the Philippines. (A) Season-long UWW training on hybrid rice, 2001 dry season. (B) Hands-on exercises for hybrid rice training participants, (C) A compact 27-ha hybrid rice seed production area in Kalinga under the UWW concept. (D) Public- and private-sector hybrid rice workshop at IRRI, May (E) Policy workshop on hybrid rice. (F) National hybrid rice seed production workshop, March Redoña et al

370 key administrators (Fig. 2). The TD activities were expanded to cover 16 and 15 provinces during the 1998 WS and 1999 DS, respectively. Results of these technodemo trials clearly demonstrated the yield superiority of hybrids vis-à-vis the best inbred varieties. Weighted average yield superiority of the Magat and Mestizo hybrids (adjusted to reflect the actual production area at each site) was 825 kg (16%) and 1,350 kg (27%), respectively. The highest yield of 12 t ha 1 was obtained from Mestizo in the provinces of Cagayan and Bohol during the 1999 DS (Redoña et al 2000). The 2001 WS data from the DA, produced from various locations with large hybrid rice areas. again demonstrated the superiority of hybrid rice technology over ordinary or farmer-saved and certified seeds used in the national rice program (Table 4). Across the country, the use of hybrid rice resulted in a 1.3 t ha 1 or 31% yield advantage over the use of certified seeds. Information dessimination Awareness of hybrid rice technology among the general public and especially among rice farmers was very limited in the Philippines before Hence, to promote awareness and in support of national capacity-building efforts on hybrid rice technology generation, seed production, and cultivation, a massive information campaign using television, radio, and print media began in 1998 (Fig. 3). Promotional materials translated into local dialects were printed in popular magazines with regional circulation. Informative and educational materials were also produced, including the Hybrid Rice Question and Answer booklet, the Hybrid Rice Production Technology booklet written in five dialects, the Hybrid Rice Program Document, and news features in the PhilRice Newsletter. Posters, calendars, and other information materials on hybrid rice technology were displayed and distributed during planting demonstrations, field days, and training activities. The technology was also promoted during spots acquired by PhilRice on agricultural radio and TV programs. Other activities included the translation of the hybrid rice seed production manual, originally published by IRRI, into the Filipino language. The manual was then used as a reference material in seed production training courses, with more than 3,000 copies distributed nationwide. Also, a proceedings titled Advances and Challenges in Hybrid Rice in the Philippines was published based on the presentations and recommendations of the first national hybrid rice workshop conducted under the FAO TCP grant. Sets of a six-panel Plexiglas-laminated hybrid rice poster display, each containing the aims, targets, and methodologies of the national hybrid rice program, and prospects of hybrid rice seed production and cultivation, were distributed to key stations. Displayed in strategic areas, these attractive poster exhibits drew the attention of farmer visitors while serving as a reference material for technicians and trainers. PhilRice also regularly issued press releases on various hybrid rice topics and hybrid rice-related activities conducted in the country for publication in major newspapers of national circulation. Some press materials were also published in regional Hybrid rice development and use in the Philippines,

371 Fig. 2. Technology demonstration activities for farmers and key officials. (A) For hybrid rice cultivation (with former DA Secretary Salvador Escudero). (B) For hybrid rice seed production (with former DA Secretary Leonardo Montemayor). dailies, usually in local dialects. Moreover, instructional videos on hybrid rice seed production (one in English and one in Tagalog) in VHS and VCD formats were produced and disseminated. During 2001, information materials distributed came in the form of posters on hybrid rice technology as a means for increasing yield; leaflets such as Hybrid Rice Technology, Mag Hybrid Rice Na! (Support Hybrid Rice), and 10 Things We Should Know about Hybrid Rice; billboards titled Dagdag Ani at Kita, Hybrid Rice (Increased Yield and Income in Hybrid Rice) and Sa kita at Anihan, Hybrid Rice ang Number 1 (Hybrid Rice No. 1 in Yield and Income); and a hybrid rice jingle and hybrid rice dramatized plug aired over radio stations. Moreover, PhilRice gave out tarpaulins (tricycle banners) advertising the benefits of the use of hybrid rice technology. With the launching of the PhilRice Web site ( hybrid rice-related information was made available to those with Internet access. Some of the materials produced and information dissemination activities are shown in Figure Redoña et al

372 Table 4. Hybrid rice technology demonstration results (2001 wet season). Region Certified seeds Hybrid seeds % Yield advantage Area Production Average Area Production Average (hybrid vs harvested (t) yield harvested (t) yield check) (ha) (t ha 1 ) (ha) (t ha 1 ) Philippines 458,014 1,956, , , Cordillera Autnomous Region 7,133 37, , I 14,571 68, , II 103, , , , III 56, , , IV 81, , V 19,889 85, VI 13,428 51, VII 4,619 19, VIII 41, , , IX 7,348 32, X 24,598 95, XI 25, , , XII 29, , , CARAGA 22,797 31, Autonomous Region of 4,860 16, Middle Mindanao Source: DA rice program. Hybrid rice development and use in the Philippines,

373 Fig. 3. Promotional materials for hybrid rice. (A) Bulletins/books/VHS tapes/vcd. (B) A joint public- and private-sector poster ( In hybrid rice you win through yield and profit ). (C) A display booth in an agricultural fair. (D) Newspaper features on hybrid rice technology. (E) Hybrid rice discussion on an agricultural TV talk show broadcast nationwide. 396 Redoña et al

374 Policy advocacy Hybrid rice technology advocacy by the DA, PhilRice, and the private sector also reached the highest levels during 1998 to No less than three Philippine presidents (Fidel V. Ramos, Joseph E. Estrada, and Gloria Macapagal-Arroyo) were made aware of the potential role that hybrid rice technology could play in their respective government agricultural program s quest for rice self-sufficiency and food security (Fig. 4). Among the activities that contributed to this successful sensitization of policymakers were study tours for key policymakers, field days attended by top officials, consultancy missions by foreign hybrid rice experts, and a mass media campaign including hybrid rice spots on popular TV programs. The exposure of top government officials to hybrid rice initiatives outside the country, particularly in China and India, was particularly important in their having a positive view on the role that hybrid rice technology could play in Philippine agriculture. As a result, investments of the Philippine government in hybrid rice increased dramatically. From minimal DA funding for hybrid rice technology development and use prior to 1998, the budgetary allocation for the hybrid rice program grew to about P7 million (Philippine pesos) in 1998 and to P121 million in The budgetary outlays were used for hybrid rice R&D, seed production, training, technology demonstrations, an information campaign, and commercialization. The latter included the procurement of hybrid and parental seeds from organized seed growers and private companies. Funding for hybrid rice R&D activities at PhilRice, derived from both government and external sources, correspondingly increased during the period. Support was obtained from the FAO, the Rockefeller Foundation, IRRI, and the ADB. Using these funds, equipment was acquired for hybrid rice breeding, seed production and processing, and seed certification. Moreover, a P9.6 million hybrid rice building with an office, laboratory, and seed processing space was constructed at PhilRice using DA funds. Greenhouse and hybrid seed storage facilities were constructed at the PhilRice Hybrid Rice Center and the two PhilRice branch stations in Mindanao. Seed industry development The Philippines has no organized hybrid rice seed industry. Initial commercial hybrid rice seed production was mainly undertaken by PhilRice along with members of the National Rice Seed Production Network (SeedNet) and two private seed companies. In 1999, a national workshop was organized for key personnel to develop a mechanism for large-scale seed production. Seed production targets were prepared, the specific roles for various institutions involved were identified, and a calendar of training programs was prepared. A consequence of this activity was the submission in 1999 of a proposal to develop a model mechanism for seed production in the Philippines, which may also be used in other countries without a strong seed industry. Partially supported by the IRRI-ADB project and implemented in 2000, the strat- Hybrid rice development and use in the Philippines,

375 398 Redoña et al Fig. 4. Philippine presidents promoting/briefed on hybrid rice. (A) Former President Fidel V. Ramos launching the hybrid rice program, (B) Former President Joseph E. Estrada receiving briefing on hybrid rice technology, (C) President Gloria Macapagal-Arroyo lauding Mestizo hybrid rice, 2002.