DEVELOPMENT AND CHARACTERISATION OF PRODUCTIVE BIVOLTINE INBRED LINES OF SILKWORM BOMBYX MORI L.

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1 Indian J. Anim. Res., 41 (3): , 2007 DEVELOPMENT AND CHARACTERISATION OF PRODUCTIVE BIVOLTINE INBRED LINES OF SILKWORM BOMBYX MORI L. Ajay Kumar Goel, Chandrashekharaiah and Y. Sreenivasa Reddy* Andhra Pradesh State Sericulture Research and Development Institute, P.O. Kirikera, Hindupur (Andhra Pradesh), India ABSTRACT Three breeding plans were initiated using three bivoltine commercial hybrids from China with an objective to inbreed productive bivoltine pure lines. A total of 09 inbred lines including two sexlimited larval marking, one each of oval and dumbbell, two plain oval, three plain dumbbell and two marked dumbbell lines were developed. Evaluation and characterisation of all these lines was detailed in the investigation. The lines showed productive merit for pupation rate, cocoon weight, single shell weight, cocoon shell ratio, filament length and reelability besides exhibiting tolerance/resistance to BmIFV, BmDNV and BmNPV. INTRODUCTION In the changing global economy, evolving suitable strategies for increased quality silk production has become major concern (Chandrashekharaiah, 2001). In spite of significant increase in the overall silk production in the country in recent years, India is still far behind to meet the quantitative and qualitative needs of the domestic and international market. Among various factors, improvement of silkworm variety has always been an important measure of increase in production in sericulture. Continuous renewal and change of existing breeds/hybrids with new superior varieties and their extension are the most economic and efficient methods of increasing silk quality and quantity. As being followed in all the sericulturally advanced countries, development of breeds with targeted economic merits is the basic objective to develop more productive hybrids for commercial exploitation. Since exchange of parent genetic materials is highly improbable task on regular basis, pure line isolation from the commercial hybrids followed by re-hybridisation is the potential way to improve the genetic base and the productivity. In India, many productive breeds with good combining ability have been synthesized by pure line isolation from the elite commercial hybrids (Harada 1961, Kalpana * Department of Sericulture, University of Mysore, Mysore (Karnataka), India. et al., 1994, Chandrashekharaiah et al., 2000, Datta et al., 2000). In this direction, attempts are made to develop bivoltine-inbred lines utilizing the Chinese commercial hybrids at Andhra Pradesh State Sericulture Research and Development Institute (APSSRDI), Hindupur. The present investigation carries out the isolation, characterisation and evaluation of the bivoltine-inbred lines evolved. MATERIAL AND METHODS Three Chinese bivoltine commercial hybrids viz., Quju x Xing6, Feng1 x 54A and 871 x 872 constitute the breeding resource material. Three breeding plans were initiated at APSSRDI, Hindupur to isolate the pure lines by following the inbreeding and appropriate selection. The breeding programmes were initiated with the rearing of F1 generation reared as composite (representing broods) of each of the 03 hybrids. The layings for F2 generation were prepared after random mating within each group without making any selection to allow the recombination of genes. Layings were prepared for all the lines by utilizing 300 active, healthy and uniformly grown larvae in each of the three replications selected after 3 rd moult and retained up to spinning. The inbred lines were reared following standard rearing techniques as suggested by Krishnaswamy (1978 and 1990)

2 158 INDIAN JOURNAL OF ANIMAL RESEARCH Quju x Xing6 Chinese Hybrid Quju: Sex limited Oval Xing6 : Plain Dumbbell Composite rearing without selection F 1 - F 2 Composite rearing and line isolation on larval marking and cocoo n shape F 3 All plain larvae: Males All marked larvae: Females Plain oval Mark oval Plain Dumb Mark Dumb F 4 F4 - F1 0 : Cellular rearing and selection Plain Dumb. (NSL) Scre ening fo r BmDNV1, BmNPV and BmIFV Resistance F 10 Fixation of lines with SL character and improved metric traits Quju SLO Quju SLD Xing6 PD BREEDING PLAN OF QUJU SLD, QUJU SLO AND XING6 PD

3 Vol. 41, No. 3, x 872 Chinese Hybrid 871: Short Oval 872: Slight Dumb. Composite rearing without selection F 1 - F 2 Composite rearing and line isolation on larval marking and cocoon shape F 3 Plain oval Mark oval Plain Dumb Mark Dumb F4-F10: Cellular rearing and selection F 4 Not selected Screening for BmDNV1, BmNPV and BmIFV Resistance Fixation of lines with respect to cocoon shape and desired metric traits 871 PO 872 PD 872 MD BREEDING PLAN O71 PO, 872 PD AND 872 MD

4 160 INDIAN JOURNAL OF ANIMAL RESEARCH Feng1 x 5 4 A Chinese Hybrid Feng1: Plain Oval 54 A: Dumbbell Composite rearing without selection F 1 - F 2 F 3 Composite rearing and line isolation on larval marking and cocoon shape Plain Plain Mark Dumb. F4 - F10: Cellular rearing and selection Screening for BmDNV1, BmNPV and BmIFV Resistance Fixation of lines with improved metric traits Feng1 PO 54A PD 54A MD BREEDING PLAN OF FENG1 PO, 54A PD AND 54A MD

5 Vol. 41, No. 3, and Datta (1992). Mulberry leaves of V1 variety were given as feed. Temperature and Relative Humidity varied from C and 60-95% respectively owing to fluctuating tropical environmental conditions in different seasons of the year. The recombinant inbred lines were identified and separated based on egg shell colour, larval markings and cocoon shape. At F2 generation, composite layings were reared in three replications each and random mating was allowed without selection for the preparation of F3 layings in order to allow the genes of the desirable characters to recombine. At F3, these composite layings were reared in three replications and the recombinant inbred lines were derived through appropriate selection with regard to larvae markings and cocoon shape targeting the desired economic characters. At F3 generation, plain and marked larvae were also separated in all the three hybrid combinations. Later at the pupal stage, the plain and marked larvae from the hybrid combination Quju x Xing6 were identified as males and females respectively. The sex-limited character of the two lines obtained from Quiju x Xing6 was confirmed through verification of sex for both plain and marked larvae in the subsequent generations also. Besides, a plain dumbbell line was also isolated from Quiju x Xing6. Similarly, six lines, one each of plain oval, plain dumbbell and mark dumbbells were derived from the hybrid Feng1 x 54A and 871 x 872. Besides larval markings, the lines were also segregated on the basis of their cocoon shape as oval and dumbbell. The purification of inbred lines continued through appropriate selection in the following generations. Cellular (batch) rearings, each with three replications for the isolated breeding lines, were conducted from F4 onward for the improvement of economic characters. Thus a total of 09 including 02 sex-limited lines were isolated from the three hybrids. The data pertaining to eight morphological traits viz., cocoons shape, cocoon colour, serosa colour, egg shell colour, hatched larval colour, late age larval colour, larval markings and haemolymph colour along with ten economic traits viz., fecundity (number of eggs per laying), hatching (%), cocoon yield by number per larvae, cocoon yield by weight per larvae, pupation rate (%), single cocoon weight (g), single shell weight (g), cocoon shell ratio (%), filament length (meter) and reelability (%) were recorded as detailed by Thangavelu et al. (2000) and Anonymous (2001). The reelability and filament length of the inbred lines were evaluated from a sample of 300 cocoons, at F10 generation. RESULTS AND DISCUSSION The performance of Chinese hybrids (F1) is presented in Table 1. A perusal of the rearing performance of Chinese hybrids indicates that all the Chinese hybrids performed better than the control hybrid, APS9 x APS8. Among them, Quju x Xing6 with shortest larval duration, showed the best performance with maximum cocoon yield per larvae (8989 No, 16.8 kg), highest pupation rate of 93.64%, maximum cocoon weight (1.81 g), reelability (85.64%) and more robust as compared to others. The hybrid 871 x 872 recorded highest values for the traits cocoon weight (1.81 g), shell weight (0.446 g), cocoon shell ratio (24.64%) and filament length (1216 meters). The Non breakable filament length (NBFL) was recorded maximum in the hybrid Feng1 x 54A (1024 meters). Except neatness, the performance of the Chinese hybrids was better than the control hybrid for all the traits. A total 09 inbred bivoltine lines (Quju SLO, Quju SLD, Xing6 PD, 871 PO, 872 PD, 872 MD, Feng1 PO, 54 A PD and 54A MD) were segregated from the three hybrids. The inbred lines Quju Sex Limited Oval (Quju SLO), Quju Sex Limited Dumbbell (Quju SLD) and Xing6 Plain Dumbbell (Xing6 PD) were isolated

6 162 INDIAN JOURNAL OF ANIMAL RESEARCH Table 1. Rearing performance of chinese hybrids (F1) S. No. Lines V age Total Yield/10000 larvae Pupation Cocoon assessment FL NBFL Denier Relability Neatness larval larval rate (meter) (meter) (%) (%) duration period No. Weight (kg) (%) SC Wt (g) SSWt (g) CSR (%) (D:H) (D:H) 1 Quju x Xing6 5:17 19: x 872 6:03 21: Feng1 x 54A 5:23 20: Cont. APS9 x APS8 6:06 20: SC Wt.: Single cocoon weight; SS Wt.: Single shell weight; CSR%: Shell to cocoon ratio (%); FL: Filament length; NBFL: Non-breakable FL. Table 2. Characteristic features of new inbred lines S.No. Inbred Voltinism, cocoon shape and colour Serosa colour Egg shell Hatched larvae colour Late age Haemolymph Disease line colour larval colour* tolerance** colour 1 Quju SLO Bivoltine, Larval marking sex limited, white-oval cocoons Green Yellow Blackish Brown with bristles Bluish white TY BmDNV resistant 2 Quju SLD Bivoltine, Larval marking sex limited, white-dumbbell cocoons Greyish Brown White Brown with bristles Bluish white T Y BmDNV resistant 3 54A PD Bivoltine, Plain larvae, white-dumbbell cocoons Light Brown White Brown with bristles Bluish white T 4 54A MD Bivoltine, Marked larvae, white-dumbbell cocoons Dark Brown Milky white Blackish brown with bristles Bluish white T BmDNV resistant PO Bivoltine, Plain larvae, white-oval cocoons Brownish Green Light Yellow Blackish brown Bluish white T Y PD Bivoltine, Plain larvae, white-dumbbell cocoons Dark Brown Milky white Blackish brown with bristles Bluish white T MD Bivoltine, Marked larvae, white-dumbbell cocoons Dark Brown Milky white Blackish brown with bristles Bluish white T 8 Xing 6 PD Bivoltine, Plain larvae, white-dumbbell cocoons Brown White Blackish brown Bluish white T BmIFV tolerant 9 Feng1 PO Bivoltine, Plain larvae, white-oval cocoons Yellowish green Yellow Blackish Brown Bluish white TLY BmDNV resistant * T: Transparent; TY: Transparent with yellowish tint; TLY: Transparent with light yellowish tint; ** Sivaprasad et al., Screening of Germplasm Sericologia, 43(2):

7 Vol. 41, No. 3, from the hybrid Quju x Xing 6. Similarly, the breeds, 871 Plain Oval (871 PO), 872 Plain Dumbbell (872 PD) and 872 Marked Dumbbell (872 MD) were segregated from the hybrid 871 x 872. The hybrid Feng1 x 54A was segregated into three inbred lines viz., Feng1 Plain Oval (Feng1 PO), 54A Plain Dumbbell (54A PD) and 54A Marked Dumbbell (54A MD). In India, isolation of pure lines from commercial hybrids was initiated by Harada (1961) resulting in the lines like Kalimpong-A (KA) and Kalimpong-B (KB) evolved from Japanese Single and Double Hybrid. Further, during 60 s many improved polyvoltine breeds viz., Kolar Gold, Kollegal Jawan, Mysore Princess and Hosa Mysore were successfully evolved using Pure Mysore (PM), an exotic race Nan Nung 6D and Japanese hybrid Shungetsu x Hosho (Niramal Kumar and Sreerama Reddy, 1994). To improve the metric traits, an out cross with a suitable breed was also provided during isolation and purification process of some inbred lines. It was observed that the most commonly used breed, PM is also supposed to be derived from Chinese race (Datta, 2003). During 80 s the productive bivoltine breeds namely NB7, NB18 and NB4D2 were derived from commercial hybrids (Kinshu x Shova) and (Kokko x Seihaku) x (N124 x C124) (Krishnaswami, 1983). During 90 s many CSR breeds were derived from commercial hybrids (Mal Reddy et al., 2003). Chandrashekharaiah et. al,.(2000) isolated five promising lines (one multivoltine APM1 and four bivoltine lines; APS4, APS5, APS8 and APS9) from the exotic elite commercial hybrids. Datta et al.,(2000) isolated the inbred lines, CSR2 and CSR5 from the Japanese commercial hybrid Shuneri x Shogetsu and the line CSR4 from a cross of NB4D2 with the Japanese commercial hybrid BN18 x BCS25. Many such efforts were made by various breeders to synthesize inbred lines (Kalpana and Sreerama Reddy 1988; Subramanya et al. 1991; Maribashetty 1992; Maribashetty et al. 1994; Kalpana et al. 1994; Krishna Rao 1994 and Krishna Rao et al., 2001 and 2003; Basavaraja, 1996; Siddique et al., 2003). Out of which some of the lines became popular. The present investigation also carried out on similar pattern of pure line isolation from exotic commercial hybrids, which has resulted in the evolution of 09 productive bivoltine breeds. The characteristic features of all the 09 inbred lines are presented in Table 2. Out of 09 segregated lines two lines namely Quju SLO and Quju SLD were found to be sex limited for larval markings. The rest included were two plain oval (871 PO and Feng1 PO), three plain dumbbell (Xing6 PD, 872 PD and 54A PD) and two marked dumbbell (872 MD and 54A MD) lines. The serosa colour of the egg in all the oval lines of both sex limited and non-sex limited character was observed as green, brownish green or yellowish green with the egg shell ranging from light yellow to yellow colour. The serosa colour of the egg in the dumbbell lines was observed to be light brown, brown, greyish brown or dark brown having white, dull white or milky white egg shell colour. No differentiation is observed in the oval and dumbbell lines for newly hatched larval colour, which ranges from brown to blackish brown. The newly hatched larvae of Quju SLO, Quju SLD, 54A PD, 54A MD, 872 PD and 872 MD were found to have small bristles on their body. The grown up larval colour was bluish white for all the isolated lines. In addition, all the inbred lines were screened for their tolerance/resistance to BmIFV, BmDNV and BmNPV. The line Xing6 PD exhibited higher degree of tolerance to BmIFV. The lines Quju SLO, Quju SLD, 54A MD and Feng1 PO showed resistance to BmDNV. None of the lines was found tolerant to BmNPV. The average rearing performance of four crops (F7 - F10) of all the 09 newly evolved

8 164 INDIAN JOURNAL OF ANIMAL RESEARCH Table 3. Average rearing performance of inbred lines (F7-F10) S.No. Line Fecundity Hatching Yield/10000 larvae Pupation rate Cocoon assessment FL (meter) Relability (No.) (%) (%) (%) No. Weight (kg) SC Wt (g) SS Wt (g) CSR (%) 1 QUJU SLO 426 ± ± ± ± ± ± ± ± QUJU SLD 412 ± ± ± ± ± ± ± ± XING6 PD 418 ± ± ± ± ± ± ± ± PO 428 ± ± ± ± ± ± ± ± MD 434 ± ± ± ± ± ± ± ± PD 426 ± ± ± ± ± ± ± ± FENG1 PO 428 ± ± ± ± ± ± ± ± A MD 439 ± ± ± ± ± ± ± ± A PD 408 ± ± ± ± ± ± ± ± APS8 (Control) 450± ± ± ± ± ± ± ± ± ±2 APS9 (Control) 460± ± ± ± ± ± ± ± ±25 75±3 Minimum Maximum SC Wt.: Single cocoon weight; SS Wt.: Single shell weight; CSR%: Cocoon shell ratio (%); FL: Filament length.

9 lines for 08 commercial characters is presented in Table 3. The fecundity of the new lines was recorded within the range from 408 eggs/ laying (54A PD) to 439 eggs/laying (54A MD) while the hatching percentage ranged from a minimum of 87.08%(Feng1 PO) to % (54A PD). The cocoon yield per larvae was recorded maximum for Xing6 PD (15.02 kg) followed by Quju SLD (14.17 kg), 871 PO (13.64 kg) and 54A MD (13.52 kg). The line 871 PO recorded maximum pupation rate with 94.84% followed by Xing6 PD (91.59%) and 54A MD (90.35). The pupation rate in other lines was recorded in the range of 85% to 89%. In case of cocoon weight, Quju SLD recorded highest (1.794 g) followed by Xing6 PD (1.648 g) and 872 PD (1.526 g). The highest cocoon shell weight of g was recorded in Quju SLD followed by Xing6 PD (0.336 g) and Quju SLO (0.320). Shell Ratio (SR%) ranged from a minimum of to The line Quju SLO recorded highest cocoon shell ratio (SR%) of 21.32% followed by 872 MD (21.29%) and 871 PO (21.13%). Among all the lines developed, 871 PO recoded with highest filament length (1017 meters) along with highest reelability (81.5%). The line Xing6 PD Vol. 41, No. 3, was observed with minimum filament length (870 meters) and reelability (72.3 %). Better consistency in expression of characters of economic importance besides the compactness, shape and grain scheme etc. in all the isolated lines indicate about their potential to perform under Indian tropical conditions. The newly evolved inbred lines possessing productive merits are expected to serve as potential parents for developing viable and productive hybrids for commercial exploitation. Further, the developed lines exhibiting disease tolerance/resistance for various viral and bacterial diseases make them suitable potential parental material for hybridisation and commercial exploitation and the sustainable hybrid(s) thus developed ensure crop stability. ACKNOWLEDGEMENT The authors are highly thankful to Shri Y. Umamaheshwar Rao, Assistant Sericulture Officer, who has been associated with all the rearings and data compilation. The authors are also thankful to Dr.V. Siva Prasad, Scientist for screening the inbred lines for BmIFV, BmDNV and BmNPV tolerance/resistance. REFERENCES Anonymous (2001). Descriptors for Silkworm (Bombyx Mori L).- Guide for recording morphological character data (Part-A) and Economic characters (Part-B), CSGRC, Hosur, pp Basavaraja, H.K. (1996). Ph D Thesis, University of Mysore, Mysore, India, pp Chandrashekharaiah et al. (2000). In: 4 th China International Silk Conference, Yinchuan, Ningxia, PRC, 4 th May 2000, pp Chandrashekharaiah (2001). In: Proc. of the National Seminar on Mulberry Sericulture Research in India, KSSRDI, Nov. 2001, pp Datta, R.K. (1992). Guidelines for Bivoltine Rearing, Central Silk Board, Bangalore, India, pp. 24. Datta, R.K. et al. (2000). Sericologia., 40(1): Datta, R.K. (2003). In: National Conference on Tropical Sericulture for Global Competitiveness, 5-7 Nov. 2003, CSRTI, Mysore, pp Harada, C. (1961). Bull. Sericult. Exp. Sta., 17: Kalpana, G.V. et al. (1988). Proceedings of the International Congress on Tropical Sericulture Practices, Bangalore, India, February, 1988, Part. 4: Kalpana, G.V. et al. (1994). In: Silkworm Breeding. (Ramireddy, G.S. ed.) Oxford and IBH Publishing Co. Pvt. Ltd., 1998, pp Krishna Rao, S. (1994). In: Silkworm Breeding. (Ramireddy, G.S. ed.) Oxford and IBH Publishing Co. Pvt. Ltd., 1998, pp Krishna Rao, S. et al. (2001). In: Proc. of the National Seminar on Mulberry sericulture Research in India, Nov. 2001, pp

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