Review and Prospect on Japonica Hybrid Rice Research in Anhui Province, China

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1 Rice Science, 2005, 12(2): Review and Prospect on Japonica Hybrid Rice Research in Anhui Province, China LI Cheng-quan, WANG Shou-hai, WANG De-zheng, LUO Yan-chang, ZHANG Pei-jiang, WU Shuang, DU Shi-yun, XU Chuan-wan (Rice Research Institute, Anhui Academy of Agricultural Sciences, Hefei , China) Abstract: The breeding history and commercial exploitation of japonica hybrid rice in Anhui Province, China over the last three decades were reviewed. Besides, the bottleneck problems restricting the development of japonica hybrid rice in China were summarized, and corresponding technological countermeasures were proposed. Key words: Anhui Province; japonica hybrid rice; breeding The research on japonica hybrid rice in Anhui initiated in 1977, and in the following 26 years, went through four stages: from initial three-line breeding to the successful development of the first hybrid combination, from breeding new three-line hybrid combinations to initial two-line hybrid breeding, developing two-line and three-line hybrid combinations simultaneously for both middle- and late-season cropping systems, and integration of breeding and industrialization. In the breeding and commercial utilization practice, we confronted various troubles, such as inadequate heterosis, seed purity problem, lack of financial supports and unexpected poor market demands. Nevertheless, we adjusted our technology proposals opportunely by constantly following the consumer s market demand, solved the bottleneck problems encountered, and thus ensured the steady improvement in breeding and production of japonica hybrid rice in Anhui [1, 2]. Up to 2003, the annual area planted with japonica hybrid rice in middle- and late-season cropping in Anhui reached more than ha, accounting for 18.2% of the total japonica rice planted area ( ha). Furthermore, the japonica hybrid combinations bred in Anhui have been applied extensively in Yunnan, Hubei, Jiangsu, Henan and some other provinces, and have received increasing popularity in the above regions. PROGRESS OF JAPONICA HYBRID RICE BREEDING IN ANHUI From initial three-line breeding to the development of the first hybrid combination ( ) Due to the advanced evolution in japonica rice, Received: 9 December 2004; Accepted: 2 March 2005 Corresponding author: LI Cheng-quan restoring genes were rarely found in the conventional japonica rice varieties. Because of the incompatibility of the cross between indica and japonica, it was difficult to directly utilize the restoring genes in indica rice. Yang [3] and his colleagues at Liaoning Academy of Agricultural Sciences, first developed the so called indica-japonica bridging technology to transfer the indica restoring gene into japonica rice. C57, a representative of japonica restorers, was bred and tested by crossing with Liming A, a BT type cytoplasm male sterile (CMS) line. This combination exhibited strong heterosis in 1975, and thus announced the successful establishment of three-line system for japonica hybrid rice in Northern China [3]. In 1977, this combination was introduced to Anhui. During the planting test period, we found that the combination was affected by photo-thermo-reaction, therefore unsuitable for planting in late season in Yangtze River Valley. Thereafter, CMS lines and restorer lines introduced from Liaoning and Yunnan, were crossed and backcrossed with local elite ecotype japonica cultivars to develop new, early-maturing japonica three lines for developing hybrids suitable in single or double-cropping system in Anhui. After three years of efforts, six CMS lines, Dangxuanwan 2A and 105A (BT type), Nanjing 8A (Dian-I type), Fengwo A, Xuanshan A and Yinli A, were developed, and five restorers, C-Bao, Wanhui 2, Wanhui 3, Wanhui 15 and Wanhui 65 adaptable in southern China, were also bred by means of cross, multiple-way-cross and backcross. Several breeding methods, such as selecting by testcross, restorer/restorer, CMS/restorer, restorer/ resistance or good quality, or high yield with wide compatibility, were applied, indicating the establishment of three-line system for japonica hybrids in Anhui. Using the newly developed three-line system, followed the theory of estimating heterosis by genetic distances, the new CMS lines were crossed with each

2 138 Rice Science, Vol. 12, No. 2, 2005 restorer for estimating the general combining ability (GCA) for each parent and the special combining ability (SCA) for each combination, and for checking the productivity for each combination. Combinations such as Dangyou-C-bao, Liuyou-C-Bao, Dangyou 3, Liuyou 3 were developed and entered the provincial yielding potential trials. Some of the combinations were recommended to enter South China Rice Regional Trial. These achievements provided a foundation for the breeding and production of japonica hybrid rice in Southern China. From breeding new three-line hybrid combinations to initial two-line hybrid breeding ( ) The development of new three-line hybrid combinations was emphasized in this period. In 1984, the CMS multiplication and hybrid seed production as well as production demonstration for japonica hybrid combinations, such as Dangyou-C-bao, started and succeeded at Tongcheng County, a main test site that year. In 1985, along with Dangyou-C-bao, production demonstration for other hybrids, such as hybrid derived from Liuqianxin-A, a CMS line with high yielding potential in its CMS multiplication and hybrid seed production, were enlarged and strengthened. The areas of the production demonstration and seed production for japonica hybrids reached ha and ha, respectively, in Anhui in And Dangyou-C-bao received an award from the Ministry of Agriculture of China for its good grain quality. However, partial selfing was noticed in the CMS lines Dangxuanwan 2A and Liuqianxin-A as applied extensively, when they headed under high temperature condition (higher than 35 ). Most offspring from the selfed seeds were male sterile. However a few, called homogenous restorer(s), showed restoring ability. The homogenous restorer(s) caused the impurity of hybrid seeds, and brought troubles in breeding procedure. In the same year, the similar selfing phenomenon occurred in Liaoning, Zhejiang, Jiangsu and some other provinces, and attracted expert s attentions. To explore new ways for selecting stable male sterile lines, Nongken 58S, a photoperiod sensitive genetic male sterile (PGMS) rice was introduced as a donor of sterile gene(s) in Since then, two-line method breeding was initiated. The selected receptor parents should have some characters, such as the derivative from indica rice (for improving the characters such as outcrossing and combining ability), a moderate photo-sensitiveness (considering a possible correlation between the photo-sensitivity and pollen fertility), ideal growth duration, high-yielding potential, good grain quality and multi-resistances to biotic and abiotic stresses. The conventional japonica varieties, Liuqianxin B, 917, Zao 107 and Pecos, which met the above requirements, were crossed or backcrossed with the available PGMS lines. Some approaches such as multi-generation oriented selection, and generation advancement in Hainan (in winter) or in greenhouse, were employed to accelerate the process of PGMS breeding. Developing two-line and three-line hybrid combinations simultaneously for both middle- and late-season cropping systems ( ) We learned a good lesson from the failure of hybrid seed production in 1986, and therefore improved the conditions for the research and development in three-line breeding. By severely controlled crossing between the pairs of single plants, A-line and B-line to purify the male sterile line, meanwhile we obtained more financial support and increased input in the construction of seed production bases to guarantee the seed purity. According to the market demand, we adjusted our breeding targets, focused on enhancing yield heterosis, good quality, disease resistance, high yield of seed production for both A-line and hybrids. The elite japonica hybrid combinations for the late season, Dangyou 9, 80 You 9, Dangyou and Liuyou were successfully bred and registered in Anhui. The breeding for new japonica hybrid combinations responding to the North Huaihe River area s market demand was also strengthened. The 80 You 121 and Liuyou 121 were bred and released for commercial exploitation, which increased yield by 10% over the conventional japonica rice, and had good grain quality (awarded as national and provincial good quality rice, respectively), favorable growth duration of 140 d and moderate resistance to blast and bacterial blight. Since 1987, the two-line hybrid rice breeding item was listed in the National 863 High-Technology Program, titled as Utilization and study on intra-subspecies heterosis of two-line hybrid rice, the steps of two-line breeding have been speeded up. Against to the characters that the fertility/sterility alterations of PGMS are controlled by both gene(s) and varied environmental factors of day-length/temperature, the breeding methodology for PGMS should differ from that for three-line system hybrid [4]. The following combined methods were used in our PGMS breeding: 1) fertility/sterility alterations were examined both in natural conditions by seeding sequentially at intervals in multi-locations and in phytotron; 2) fertility/sterility alterations were double identified under the conditions of long day-length, high temperature in summer at Hefei, and long day-length, low temperature at high altitude

3 LI Cheng-quan, et al. Review and Prospect on Japonica Hybrid Rice Research in Anhui Province, China 139 mountain area; 3) the sterility selection for PGMS lines was carried out in long day-length of summer, the selection for self-seeding rate was performed through stem-cutting in short day-length of fall; and 4) besides the fertility/sterility alterations, simultaneously considering the examinations on flowering habits, grain quality, resistance, growth duration and yielding potential. By applying the above-mentioned methods, four widely accepted PGMS lines, 7001S, 8087S, 3502S and 3516S were bred to match the standards of sterility. The following characters, i.e. good agronomic traits, wide adaptability, strong resistance, superior quality, suitable growth duration, good combining ability, and high yields of self and hybrid seed production, were pooled together in the lines. The first PGMS line, 7001S, derived from the cross of Nongken 58S/917 (from Huxian 19/IR661//C57), passed the technique evaluation in Using 7001S as male sterile line, the two-line hybrid combinations, 70 You 9, 70 You 04 and 70 Youshuang 9, were developed. The yield advantage of these combinations was % over the conventional japonica check variety in regional trial and demonstration field. They were successively registered in Anhui during Among them, 70 You 9 was registered in Hunan and national wide in So, 7001S has become the most widespread PGMS line in the rice growing area of southern China. During this period, the expert advisory group for hybrid rice, under the Ministry of Agriculture, strengthened the supervision and support on japonica hybrid rice in southern China. In October of 1988, a collaborative network for japonica hybrid rice research and extension in southern China was set up, including 31 organizations, agriculture administrative departments and academies from Jiangsu, Zhejiang, Anhui, Hubei, Hunan and Shanghai. The interdisciplinary cooperation, cross mating, regional trial, demonstration and extension were simultaneously carried out. Annual convention was held to exchange opinions and experiences at some selected regional trial or demonstration site. The extension area of hybrid rice had 5 big jumps in five years, planting area from less than ha in 1986 to ha in 1989, ha in 1990, ha in 1991, ha in 1992 and ha in 1993 [5, 6], making the total planting area of the japonica hybrid rice from ha, during the period of the Seventh Five-Year Plan ( ) to ha, during the period of the Eighth Five-Year Plan ( ) in Anhui, which opened a new prospect for japonica hybrid rice in southern China [6, 7]. In 1994, a program for commercializing the great successes from two-line hybrid rice breeding research under the 863 Program was launched, aiming at hybrid rice industrialization, supported by suitable set of technologies and organizing mainly by scientific institutes and seed companies. Anhui, Hunan and Hubei firstly joined the program. As the stronger financial and administrative support from governments, the commercialization process was effectively accelerated. The total planting area reached ha, being a greatest prosperity period during the Ninth Five-Year Plan ( ) for japonica hybrids in Anhui. During the Tenth Five-Year Plan ( ), rice market weakening, food price lowering and farmers planting activity losing caused the planting area decreased sharply (Table 1). Integration of breeding and industrialization (1997 to the present ) To strengthen the seed production and extension of japonica hybrid rice, in 1995, a cooperative complex was founded by the Rice Research Institute, Anhui Academy of Agricultural Sciences (AAAS), Anhui Seed Company and some counties where the hybrid seed production bases located. Based on the cooperations for our mutual benefits, the seed production bases were kept in stable, the yields of seed productions were increased, the qualities of hybrid seeds were improved, and the steps of extension for japonica hybrid rice were speeded up. So we learned that the seed production should be industrialization. It was not only the link connecting with the market, but also the power for breeding innovation and transforming achievement into productivity. On the previous basis, in 1997, Anhui Huaan Seed Company Ltd. for developing two-line hybrid rice was founded by five shareholders, the Development Center of Biology Engineering of China (DCBEC; a very powerful supporter), Anhui General Seed Company, the Rice Research Institute of AAAS and other two ones. Depending on the technologies provided by the Rice Research Institute of AAAS, an integral service running system coordinated the scientific research, seed production, business management and technical support service for hybrid rice. With the industrialization smoothly progressed (in 2001, followed the government policy, the DCBEC withdrew), a safety technique system for japonica hybrid seed production, stable bases of seed production and teams with skilled technicians were gradually formed, and a series of high-yielding cultivation techniques varied with different hybrids were developed. Under market pressure and intense research competitions, a new breakthrough was achieved in japonica hybrid rice breeding for getting supper high yield, good grain quality and multi-resistance in this period.

4 140 Rice Science, Vol. 12, No. 2, 2005 Table 1. Application of japonica hybird rice developed in Anhui Province. Hybrid rice Type a Year of registration Planting area ( 10 3 ha) Total Remark b Dangyou-C-Bao THLS GN Liuyou-C-Bao THLS GP Dangyou THLS Dangyou 9 THLS Liuyou THLS You 9 THLS GP 80 You 121 THMS GN Liuyou 121 THMS GP 70 You 9 TWLS GP 70 You 04 TWLS Youshuang 9 TWLS GP 40 You 04 TWLS You 98 THMS GP Shuangyou 4183 THLS GP Shuangyou 3402 THLS Total a THMS, Three-line hybrid in middle season; THLS, Three-line hybrid in late season; TWMS, Two-line hybrid in middle season; TWLS, Two-line hybrid in late season. b GN and GP represented the hybrid that was awarded as good grain quality rice at national level and at provincial level, respectively. We participated in super hybrid rice breeding projects funded by the Ministry of Agriculture in 1997, and by National 863 Program in In breeding, a three-line hybrid combination for middle season, III-You 98, in 2003, and two three-line hybrids for late season, Shuang-you 4183 and Shuang-you 3402, in 2004, passed the variety appraisement in Anhui, respectively. All of the three hybrids, having good grain quality and resistance to blast and bacterial blight (BB), increased yield more than 9% over check variety. Here, the III-You 98 (MH2003A/R-18) was bred by the cooperation with Mitsui Chemistry Co. Ltd. Japan, and China Seed Group Company. In Anhui japonica hybrid local trials for middle season, the average yield of III-You 98 was 9.05 t/ha, over 13.75% of the check hybrid of 80 You 121, with moderate resistance to blast and bacterial leaf blight, and its grain quality reached the second grade according to China National Standards [8]. In Anhui local trials for late season cropping, Shuangyou 4183 (Shuangyou 9/ Wanhui 4183, also named as Hua an 2) performed moderate resistance to blast and BB, and its grain quality reached the third grade of China National Standards, average yield was 7.07 t/ha, over 9.0% of the conventional japonica rice check variety. Shuangyou 3402 (Shuangyou A/Wanhui 3402) yielded 7.72 t/ha, over 12.4% of the conventional japonica rice check variety, ranking the first of all cultivars, in two-years regional trials and one year s productivity test at provincial level. Its grain quality reached the third grade of China National Standards. In 2003, its average yield was 9.89 t/ha in a 6.7 ha planting area at Baihu Farm, Anhui. An average yield of t/ha, from a 0.2 ha intensified cultural experiment for late season of double growths was acquired at Anqing Institute of Agricultural Sciences in 2004, showed a very strong yield potential. Some other japonica hybrids, have being evaluated in provincial regional trials for waiting promotion, such as 9201A/R-18, a three-line hybrid for middle season, bred by AAAS, increased yield by 13.3% over the check in In addition, Aiyou 18, a three-line hybrid for middle season, provided by Anhui Seed Company, increased yield by 12.25% over the check hybrid 80 You 121. Based on general judgments on yield increase scale, grain qualities and resistance abilities, all of the above-mentioned hybrids met the standards for super rice. The super hybrid rice and hybrid combinations with more than 66.7 thousand hectare of accumulative planting area were listed in Table 2.

5 LI Cheng-quan, et al. Review and Prospect on Japonica Hybrid Rice Research in Anhui Province, China 141 Table 2. The yield, resistance and grain quality of the super hybrid rice and japonica hybrid combinations with more than 66.7 ha planting area in total. Hybrids Check variety Yield (t/ha) ±CK Demonstration (%) Resistance a Grain milling quality (%) b Grain apparent quality c BB Blast BR MR HR LW CA(%) CR(%) 80 You 9 E-Yi MR MR You D MR MR You 9 E-Yi HR MR You 04 E-Yi MR HR Youshuang 9 D HR HR Liuyou You MR MR III You You MR R Shuangyou 4183 M MR MR Shuangyou 3402 M MR MR A/R You MR MR a R, Resistant; MR, Moderately resistant; HR, High Resistance; BB, Bacterial blight. b BR, MR and HR indicated brown rice recovery, milled rice recovery and head rice rate, respectively. c LW, CA and CR stand for length/width ratio, chalky area and chalky rice rate, respectively; the grain transparency of 80 You 121, Ⅲ You 98, 80 You 9 was grade 2, that of the other hybrids was grade 1. Alki digestion values of all hybrids were low; Gel consistency of all hybrids had high values. The data of grain quality were from Rice Quality Test Center, Ministry of Agriculture, China. d AC, Amylose content. e PC, Protein content. AC (%) d PC (%) e PROSPECTS FOR JAPONICA HYBRID RICE The development of japonica hybrid rice facing the best opportunity Up to 2003, nation-wide productions of cereal crops had decreased in six consecutive years, causing tremendous changes in the relationships between the food provisions and demands, which has drawn great attentions from the topmost policy-makers in China. Therefore, a series of policies to stimulate grain food production have been made and carried out. In Anhui, the grain food production in 2003 reduced by 19.9% over last year, of which, the total amount of rice production were million tons in 2003, seeing a decrease of 23.4% of the last year s rice production, the yield per unit area and total production of middle-season rice decreasing by 28.8% and 30.2%, respectively. The paddy price has been rising since the spring of According to market investigation on March 6, 2004, the prices of japonica paddy and milled rice were 1.9 Yuan RMB/ kg and 2.9 Yuan RMB/kg, respectively, an increase of 81% and 75.6% over those in September last year, respectively, 0.22 Yuan RMB/kg and 0.32 Yuan RMB/kg higher than those of indica paddy and milled rice at the same time (the prices of indica paddy and milled rice were 1.68 Yuan RMB/kg and 2.58 Yuan RMB/kg, respectively). Rice planting farmers rushed to market to looking for japonica hybrid seeds, the market was opened 2 3 months earlier than normal year. The agriculture administration departments estimated that the planting area of japonica rice would have a net increase of more than ha in Anhui. All of the abovementioned offered a best opportunity for the development of japonica hybrid rice. What is the bottleneck for developing japonica hybrid rice? It has been proved that japonica hybrids have heterosis as indica hybrid rice. Japonica hybrids had yield increases of % over the conventional rice check variety or hybrid in regional trials over years, showing that japonica hybrid heterosis have reached a new level. The breeding for elite japonica hybrids aiming at higher heterosis, good grain quality and stress resistance is feasible. However, the ecological adaptability of japonica hybrid rice is not as good as that of indica. We noticed that no japonica hybrid combination developed so far was popularized over ha in a single year. In contrast, the annual planting areas of some indica hybrids, such as Shanyou 63, II You 838 or Gangyou 22, are all over ha in recent years, and that of Liangyoupeijiu is over ha, covering more than 10 southern provinces. One of the reasons is that: the medium and late mature japonica rice has relatively stronger growth photo-sensitiveness, and the parents of japonica hybrids, especially the restorers, have genetic connections with indica rice, so the seed

6 142 Rice Science, Vol. 12, No. 2, 2005 setting rate of japonica F 1 is more sensitive to eco-environment. When japonica hybrid rice meets with low or high temperature at heading stage, the seed-setting rate will be fluctuated and lower. Therefore, the suitable planting regions are restricted. Secondly, although japonica hybrid rice has obvious heterosis for large panicle, but has not so strong heterosis for panicles and yield as indica. And the amount of seeds planting in per unit area is larger. Thirdly, the sterilities, regardless of BT-type CMS or PGMS, are affected by temperature. The high temperature at heading stage can lead to the selfing of BT-type CMS and the low temperature at sterility-sensitive stage can lead to the selfing of PGMS. Therefore, we have to select the suitable eco-area and suitable season for selfed and hybrid seed production, and develop the techniques to obtain high yield and high purity of selfed and hybrid seeds as soon as possible. We also have to develop and intensify the suitable cultural techniques for the specific characters of japonica hybrid rice. If we can solve these bottleneck problems, there will be a new way for japonica hybrid rice development. Strategies for japonica hybrid development Japonica heterosis and breeding objectives Japonica hybrids showed significant heteroses on vegetative growth and panicle, and exhibited the faster growth of seedling, stronger photosynthesis ability and more spikelets per panicle, than conventional japonica varieties. However, compared with indica hybrids, the heteroses of japonica hybrids are weak in tillering ability, productive panicles, and seed-setting rate [9, 10]. The differences between the indica and japonica hybrids were very obvious in 1990s. In recent years, the differences were reduced, and the yield was significantly increased, which was attributed to improving the spikelets per panicle. According to the statistics of Anhui regional trials in 2003, indica hybrid rice in middle season averaged panicles/m 2, 22.7% over conventional indica rice in middle season (211.5 panicles/m 2 ). However, according to the data from regional trials of Southern China and of Anhui for japonica hybrid rice in 1990, hybrid rice had 72.5 panicles/m 2 and 75 panicles/m 2, less than the conventional check, Xiushui 04, decreasing by 18.4% and 19.4%, respectively, number of spikelets per panicle increasing by 51.0% and 42.2%, respectively, number of filled grains per panicle increasing by 39.6% and 33.1%, respectively, seed setting lowering by 6.4% and 5.2%, respectively. According to the result of the Anhui japonica regional trial of middle season in 2003, japonica hybrid rice and conventional rice had 261 panicles/m 2 and panicles/m 2, respectively, the former decreasing by 1.7% over the latter, number of spikelets per panicle of hybrids increasing by 9.0% over that of conventional rice, seed setting rate of hybrids lowering by 6.5% over that of conventional rice. In the Anhui regional trial for japonica rice of late season in 2003, japonica hybrid rice and conventional rice had 303 panicles/m 2 and panicles/m 2, respectively, the former increasing by 3.6% over the latter, total number of spikelets per panicle of hybrids increasing by 34.5% over that of conventional rice, number of filled grains per panicle of hybrids increasing by 12.1% over that of conventional rice (Table 3). Of which, the super hybrid rice, Shuangyou 3402, had productive panicles/m 2 (1.8 panicles/m 2 more than the conventional rice, an increase of 9.2% over the conventional rice), increases of spikelets per panicle by 36.1%, filled grains by 12.1%, yield by 12.4% over the conventional rice (Table 3). From the above analyses, we have concluded that for further increasing the yield heterosis of japonica hybrid rice, in the near future, the breeding objectives for japonica hybrid rice should focus on increasing productive panicles and seed-setting rate, while keeping attentions on the characters of good grain quality, multiple-resistance and easy hybrid seed production. A higher and stable seed-setting rate is closely related to the restorability of male sterile lines, the restorability of restorers, and the tolerances of parents and hybrids to the low and high temperature. Therefore, the improvement for parents should be done first. To solve the problems of wider adaptabilities for japonica hybrid rice, we have to select the parents that are non-sensitive to photo-thermo, and have relative stable basic vegetative growth intervals for finding superior hybrid combinations. By selecting the japonica restorer lines that contained some beneficial genes from indica rice, we can broaden the genetic diversity to increase the yield heterosis. Breeding for new type male sterile line with stable sterility The purities in japonica hybrid seed productions are degraded by multiple ways, such as the seed production fields located within the indica rice planting areas, the isolations were failed and some unexpected hybrid plants occurred. In fact, both BT type CMS lines and PGMS lines could be self-fertilized due to the temperature lower or higher than critical sterile point. The failure of japonica hybrid seed production happened in the rice planting areas of Southern China in Anhui Province was also included. The self-fertilizations of CMS lines (over 20% selfing plants, called homogeneous restorers) occurred in a large area, and the seed productions suffered great loss. The self-fertilizations were more serious in the CMS lines that had later pollen

7 LI Cheng-quan, et al. Review and Prospect on Japonica Hybrid Rice Research in Anhui Province, China 143 Table 3. Comparison of yield components among indica and japonica hybrid rice and inbred varieties. Region trial a Variety type b Productive panicles Spikelets/panicle Filled grains/panicle (ha) ±% no. ±% no. ±% Seed setting rate (%) SJSC SJAH IMA JMA JLA JHR CK (IV)(Xiushui 04) JHR CK (IV)(Xiushui 04) IHR CK (IV) JHR CK (JHR) JHR CK (IV) a SJSC represented for single-season late japonica regional trial in rice area of Southern China in 1990, SJAH for single-season late japonica regional trial in Anhui in 1990, IMA for Indica regional trial of middle season in Anhui in 2003, JMA for Japonica regional trial of middle season in Anhui in 2003, JLA for japonica regional trial of late season in Anhui in b JHR, IHR, IV and CK represented japonica hybrid rice, indica hybrid rice, inbred variety and the check inbred / hybrid, respectively. abortion and high outcrossing rate. Although the degree of the CMS line self-fertilization has being reduced, but this problem has not been completely solved. In 2002, an experiment conducted in Jiangsu Province showed that the selfed seed setting rate of the Liuqianxin-A, a CMS line, was 3.7%, under bagged condition. Now, regardless to three-line or two-line, we firstly have to select the suitable eco-areas and seasons for selfed and hybrid seed production, avoid the effect from higher or lower temperature. Secondly, to guarantee the purity of sterile lines and safety in seed production, we adopted single plant pair mating to purify the BT type CMS lines. For the PGMS lines, we selected ideal individual plants under controlled low temperature to prevent the up-drift of critical sterile point to produce foundation seeds of sterile lines. Thirdly, we bred new type sterile lines with stable sterility. In recent years, we have successfully developed a new breeding method, which can guarantee the purity of male sterile lines. The key point of this method is that using a BT type sterile line as the donor of sterile cytoplasm (A-line), a PGMS line (S-line) with male sterile-maintaining genes as maintainer, by crossing and backcrossing to develop a new type sterile line, which were called SA line. The SA line performed completely male sterility under high or low temperature in summer long day-length, or in winter or spring short day-length in Hainan. If we utilize this advantage in hybrid seed production, we will not only avoid the self-fertilization of BT type sterile lines under high temperature, but also prevent the self-fertilization of PGMS lines under low temperature, and escape from the risk of the failure of hybrid seed production. This newly developed technique, can guarantee the safety in japonica hybrid seed production, and has been patented in China. Combining the technologies of conventional breeding and molecular marker-assisted selection (MAS) Conventional breeding technologies now still play very important roles in japonica hybrid rice breeding, especially in improving quantitative traits such as yield and grain quality traits. Crossing and selection are still used as the basic means to pool the beneficial characters together. However, breeding efficiency can be greatly enhanced for disease/pest resistances and other desired qualitative traits by MAS and transgenic technologies. We have successfully transferred the Bt gene (insect resistance), pepc and ppdk genes (the key isozyme genes, to fix CO 2 in the photosynthesis process of C 4 plants) to the three-line and two-line hybrid parents by MAS and transgenic technologies. Remarkable progresses also have been made in insect-resistance breeding for hybrid rice, in the provinces, Fujian, Hubei, Zhejiang and more. But, in the near future, directly applying these technologies to practical productions are still severely restricted by the law and rule on transgenic plant in China. Currently, the most effective methods for japonica hybrid breeding should be: first, to select the existed near-isogenic lines (NIL) carrying the loci for the insect-resistance, good grain quality, stress-tolerance, low level N and P tolerance, and the quantitative trait loci (QTL) for high yield as well as its component factors; next, to apply the conventional cross and

8 144 Rice Science, Vol. 12, No. 2, 2005 backcross methods to pool them together in existed good parents; and to combine the means, such as antibiotics seedling selection, MAS, field phenotype selection, artificial inoculation and anther culture to smoothly accelerate the breeding progress. Characteristics of japonica hybrid rice and its cultivation techniques The experiments done in Jiangsu and Anhui in 1990s showed that japonica hybrid rice significantly differed from indica hybrid in grain filling. In terms of the dry matter formation of panicle for japonica hybrid, the ratio of the dry matter (transferred from stem and sheath) to the total dry matter of panicle ranged from %, and the ratio of dry matter synthesized after heading to the total dry matter of panicle, from %. However, for indica hybrids, the two ratios were ranged from % and %, respectively. This very significant difference indicated that at late growth stage, the dry matter transferring and grain-filling rate in indica were faster than those in japonica hybrid, which was favorable to seed setting and yield; for japonica hybrid, the net dry matter synthesized at late growth stage contributed much more to yield, but the transferring rate of carbohydrates from stem and sheath to panicles was lower, and the grain filling duration was longer. Compared with indica hybrid, japonica hybrid had more vigorous root system, more active canopy and biological function of leaves at late growth stage. The nitrogen content of leaves during d after heading would decrease by 15% for japonica hybrid and by about 25% for indica hybrid, the content of chlorophyll had the same trend. Therefore, for japonica hybrid cultivation, the transfer of assimilates at late growth stage should be strengthened through the application of fertilizer, especially by potassium topdressing at grain filling stage. And irrigation should be proper at late growth stage to make full use of the advantages of japonica hybrid in biological functions of leaves and root vigor at late growth stage, so that the seed setting and yield could be increased. In addition, selecting fitting parental lines for combination with prior knowledge of genetic information on assimilate translocation, may be one important alternative to solve the problems. ACKNOWLEDGMENTS This work was supported by National 863 Program of China (2004AA211091, 2002AA207001). We thank Dr. CHENG Rong and LI Ze-fu for their revising the manuscript. REFERENCES 1 Li C Q, Sun M, Xu C W, Bai Y S, Zhang P J. The research and development of three-line japonica hybrid rice in Anhui Province. J Anhui Agric Sci, 1994, 22 (suppl): 7 9. (in Chinese) 2 Wang D Z, Wang S H, Li C Q, Luo Y C. The research and utilization of japonica hybrid rice in Anhui Province. J Anhui Agric Sci, 2001, 29 (1): (in Chinese) 3 Yang Z Y. Japonica Hybrid Rice Breeding Researches in Northern China. Beijing: China Agriculture Science and Technology Press (in Chinese) 4 Lu X G, Gu M H, Li C Q, Mou T M, Peng H P, Wang B, Yao K M, Yuan Q H, Zhou C S, Zhu Q S. The Theory and Technique of Two-Line Hybrid Rice. Beijing: Science Press, (in Chinese) 5 Li C Q, Sun M, Xu K N, Zhu X Q, Bai Y S. Yield-increasing heterosis and rational use in southern japonica hybrid rice. Hybrid Rice, 1991, (suppl): The Cooperation Group of japonica Hybrid Rice Research and Extension in Southern China. The prospect of the development and utilization of japonica hybrid rice in southern China. J Anhui Agric Sci, 1994, 22 (suppl): 4 5. (in Chinese) 7 Sun M, Xu C W, Zhang P J, Bai Y S, Yang L S. Breeding of a new three-line medium-maturing japonica hybrid rice 80 You 121. In: Chen J A. Researching on Agricultural Key Projects in Anhui Province. Beijing: China Agricultural Science and Technology Press, , 103. (in Chinese) 8 Bai Y S, Yang L S, Ge W Q. Comprehensive analysis for yield traits to medium-maturing japonica hybrid rice III You 98. J Anhui Agric Sci, 2002, 30(3): , 336. (in Chinese) 9 Yuan L P. Theory of Hybrid Rice. Beijing: China Agriculture Press, , (in Chinese) 10 Tang Y G. Analysis of the heterosis of japonica hybrid rice. Hybrid Rice, 1991, (suppl): (in Chinese)