1 Department of Genetics, Facuty of Sciance, Kasetsart University. 2 Department of Agronomy, Facuty of Agriculture, Kasetsart University

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1 BEEDING FO DISEASE ESISTANCE IN SILKWOM BY BACKCOSS METHOD Natalee. jaiyen,panapa Saksoog 1, Lertluk Ngernsiri 1 and Krisda Samphatharak 2 ABSTACT Breeding silkworm strain for NPV resistance with high cocoon productivity was conducted by the backcross method. The crosses were made between the NPV resistant Thai silkworm variety (Nang Lueng) and NPV susceptible strains (Chinese and Japanese strains). The results of backcrossing showed that, the new inbred Chinese lines and inbred Japanese lines not only had more stability of golden yellow cocoon, non-hibernating eggs but also the performance of the cocoon quality characters such as the shell percentage. All of the inbred lines had the higher percentage of shell than that of Nang Lueng in every hybridized generation. For example, the shell percentages of NL/C 7 and NL/J 7 were in respective order, , in female and , in male, while the shell percentages in female and male of Nang Lueng were and in female and male. On the contrary, the stability of golden yellow cocoons and nonhibernating eggs did not reach until the 6 th generation of backcross. For NPV resistant screening, the logarithmic dose of NPV suspensions were employed viz. 10 6, 10 5, 10 4, 10 3 and 0. Accordingly, the two backcross hybrid lines were resistant and moderately resistant races accorded with decreasing of NPV resistant degree. The two recurrent parents had different degree of resistance, the Chinese strain was moderately susceptible race while Japanese strain was highly susceptible race. Key word; Breeding, Backcross method, silkworm, resistance. N Jaiyen: 1 Department of Genetics, Facuty of Sciance, Kasetsart University 2 Department of Agronomy, Facuty of Agriculture, Kasetsart University

2 INTODUCTION Among the silkworm diseases, those caused by virus are the most serious problem in Thailand especially grasserie, which is caused by an infection by the nuclear polyhedrosis virus NPV. The disease results in failure in obtaining high cocoon yield since the outbreak usually occurs at the fifth (final) instar at which stage the larvae have already consumed almost all the mulberry needed for the whole larval rearing. Some cocoons may be retrieval but often yield low raw silk quality. Therefore, intensive efforts have been consistently investigated to improve the breeding lines for grasserie disease resistance in addition to the high yield character. Yet, the desired lines have not been successfully achieved so far. The backcross (bc) method is a form of recurrent hybridization by which a superior characteristic may be added to an otherwise desirable strain. By this method the breeder has considerable control of the genetic variation in these gregating population in which selections are made. The bc method has been extensively used for transferring qualitative characters, such as disease resistance (Allard et al., 1960). The main objective of the experiment is to synthesize two bivoltine silkworm strains with a NPV resistant character by backcrossing method. MATEIALS AND METHODS Nang Lueng was considered resistant to NPV disease and was thus used as a donor parent. The donor parent was crossed with two recurrent parents, Chinese strain (C), Japanese strain (J) (Nang Lueng x Japanese, Nang Lueng x Chinese) for transferring allured to later NPV disease resistant character. After egg laying on an acid resistant paper, F 1 eggs were hatched and reared and used as a maternal parent for the backcrossing programs. The female moths of F 1 were mated with a recurrent parent and thus producing the first backcrossing generation progeny (BC 1 ). Backcrossing was carried on for 5 generations. The emerged moths with any deformity such as deformed wings, abdomen and antennae were discarded. Peanut shape and golden cocoon in J strain and oval shape and golden cocoon in C strain were maintained in all backcrossing generations. Screening test for the nuclear polyhedrosis virus (NPV) resistance The 4 th instar larvae of parents line, Nang Lueng, C strain, J strain and BC 6 F 1 line were screened for NPV resistance. On the screening test, individual larvae were fed on mulberry leaf of 1 cm 2 which had been dipped with different concentrations of polyhedral suspension 0, 10 3, 10 4, 10 5 and 10 6 polyhedral per ml (PIB/ml). Ten larvae were used in each treatment with three replications. For viruses infection, NPV dipped leaves were fed to each larva in separated paper box until the leaves were all eaten. The number of death larvae caused by NPV disease was recorded daily until cocoon formation.

3 esults and Discussion The larval weight was determined by weighting ten full larvae at the final stage of instars. The larval weight of polyvoltine female parent strain was light but usually heavy in bivotine male parent strain. The ten larval weight were ± 0.507g, ±0.356g in F 1 C and F 1 J generation, respectively. The results of backcrossing program showed that the larval weight generally increased and related with the larval weight of the recurrent parents. (C strain, J strain) as presented in Table 1 For example, the larval weight of BC 1 (C) and BC 1 (J) were ±3.33 g and ±0.12 g, when raised up to ±0.227g and ±0.213g in BC 6 (C) and BC 6 (J)generations. As has been mentioned earlier, the ten fulls grown larval weight usually reflects the successful rearing. The larval weight with a small standard deviation means that the entire silkworm is in good health and hence possibly a good profit from the investment. Nevertheless, silkworm health depends on rearing techniques and mulberry quality. Comparison, between the BC hybrid therefore will confine to those of the controls of individual rearing periods. The weight of the worm has been comparatively increased progressively in each breeding generation up to the BC 2 [BC 2 (C )(15.813±0.202g.), BC 2 (J) (16.711±0.147g.)].Thereafter, the weight increase could not be detected although. There was weight different between generations which weight is due to the quality of mulberry leaves and rearing temperature (Doira, 1978). The latter, although has been controlled by an air -conditioner, there was temperature fluctuation however. During the BC 4 -BC 6, it was cool season in Thailand and hence the differences of temperature during days and might were narrow. This resulted in a relatively less fluctuations of rearing temperature. Unfortunately, temperature was recorded only in the first feeding of the day which could not reveal fluctuation of temperature during the days and might. The overall optimum temperature for larval rearing is between 20 C to 28 C (Agura, 1994; Tiumpitux, 1995) Fresh cocoon weight includes weights of the cocoon shell, pupa and molted skin. The fresh cocoon weight also varies with six of the silkworm. Usually the cocoon of the female larva is heavier than that of the male larva (Aruga, 1994). In accordance with resulted in Table 1. They revealed that F 1 hybrid expressed the higher yield than female parent component, i.e. fresh cocoon weight and single shell weight. In F 1 C and F 1 J fresh cocoon weight were1.008±0.055g, 1.089±0.034g in female and 0.951±0.009g, 0.991±0.126g in male. Single shell weight were 0.143±0.003 g, 0.165±0.003 g in female and 0.136±0.0017g, 0.151±0.002 g. in male respectively. In the backcross generation, the fresh cocoon weight and single shell weight was increased in the BC (C) line and BC (J) line, However some generation the fresh cocoon and shell weight were very low. This was the quality of cocoon depending on the quality, quantity of mulberry and rearing environmen. The silkworm reared in June gave low cocoon and shell weight. Because the mulberry leaves were very poor in quality. Generally, rearing in October it should had hight cocoon and shell weight, on the other hand, the

4 cocoon and shell weight was low because there had been rain this month to bring about quantity mulberry no enough. Thus, the quantity of mulberry affected the fresh cocoon weight and single shell weight. The other characteristic of cocoon (Table 2 and 3) indicated that shell percentage of the new silkworm strain from backcross breeding program yield shell percentage near recurrent parent. For example, recurrent parent, such as the BC 6 (C) and BC 6 (J) had15.437%,16.242% in female shell percentage and15.664%,16.321% in male shell percentage. Comparison of shell percentage between male and female showed that the male shell percentage was higher than that of female shell percentage in all generation from the backcross-breeding program. The high hatching percentage, the young silkworm survival percentage, good cocoon percentage and cocoon yield percentage, which are hardy characters (Nopsenee, 2001).From the Table 2 and 3 indicate the hardy characters of the BC( C) and BC (J) were placed in the Nang Lueng group. Accordance to Table4, resistance or susceptible to NPV among strains of silkworm in the experiment showed that the backcrossing hybrids are higher than the recurrent parent ( C and J strain). While the backcrossing hybrids is nearly the level of resistance than donor parent( Nang Lueng). Because the hardy characters were used as selected in each generations. Hence the high hatching percentage, the young silkworm survival percentage, good cocoon percentage and cocoon yield percentage were used to indicator for selection the hardy characters. In experiment, the LD 50 value were employed in order to determine the mode of inheritance of the nucleare polyhedrosis virus resistant characters of silkworm. The results indicated, the both of BC(C) and BC (J) progeny were moderately resistant and resistance. In the parent strain, C and J strains were susceptibility for NPV and Nang Lueng was resistance to NPV. There were different level of NPV resistance in the backcrossing hybrids and the parental strains. This indicated that the level of resistance to NPV in silkworm was controlled by polygene with additive gene effect. (Uzigawa and Aruga, 1966; Aratake, 1973; Watanabe, 2002; Narayanan, 2003). For the Table 4, found that the LD 50 values in the four lines out of 10 lines in C strain backcross 1, 8,9,10 had x10 5, x10 5, 3.20 x10 5, x 10 5 respectively, were NPV resistance and the two lines in J strain backcrossing, 9 and 8, had x10 5 PIB/ml, 3.13x10 5 respectively, resist to NPV. The lines in BC(C) and BC(J) to NPV resistance chosen to be further maintained. Therefore, the appropriate rearing can eventually control the spread of the disease since these silkworm strains already inherited resistance character in some degree. This good genetic materials can be manipulated through biotechnology and breeding program so that more resistant strain to virus infection can be developed (Sornchattrarak, 1997).

5 Table 1 The ten full grow larval weight and larval duration (day : hr) and Commercial qualities of cocoons produced by various breeds Characters Strain Dec,01 (F 1 ) Feb,02 (BC 1 ) June,02 (BC 2 ) Apr,02 (BC 3 ) Aug,02 (BC 4 ) Oct,02 (BC 4 F 1 ) Dec,02 (BC 5 ) Feb,03 (BC 6 ) 10 full grown BC( C) ± ± ± ± ± ± ± ±0.227 larval wt.(g) BC(J) ± ± ± ± ± ± ± ±0.213 Larval duration BC( C) 21:12 22:14 21:14 21:22 22:21 23:12 22:21 21:20 Day : hr BC(J) 21:17 22:12 21:11 21:18 22:17 23:09 22:14 21:15 Cocoon wt(g) BC( C) 1.008± ± ± ± ± ± ± ±0.088 female BC(J) 1.089± ± ± ± ± ± ± ±1.451 Cocoon wt(g) BC( C) 0.951± ± ± ± ± ± ± ±0.08 male BC(J) 0.991± ± ± ± ± ± ± ±0.073 Shell wt. (g) BC( C) 0.143± ± ± ± ± ± ± ±0.004 female BC(J) 0.165± ± ± ± ± ± ± ±.004 Shell wt. (g) BC( C) 0.136± ± ± ± ± ± ± ±0.005 male BC(J) 0.151± ± ± ± ± ± ± ±.0015 % shell female BC( C) ± ± ± ± ± ± ± ±1.001 BC(J) ± ± ± ± ± ± ± ±0.94 % shell male BC( C) ± ± ± ± ± ± ± ±0.131 BC(J) ± ± ± ± ± ± ± ±0.94 Temperature 25.03± ±1.3 33± ± ± ± ± ±2.0

7 Table 2 Comparative different characters in the backcross hybrids (BC 6 C) against those of recurrent parent ( C) Characters BC 6 C C NL % young survival ±4.21 a ±4.06b ±0.922 a % good cocoon ±4.02 a ±4.59b 92.26±0.469 a 10 full grown larval ±0.227 a ±0.280 a 8.00±0.220b wt.(g) Larval duration 21:20±0.18ab 25:20±0.25 a 19:12±0.189b Day : hr Cocoon wt(g) female 1.179±0.088 a 1.314±0.002 a 0.468±0.022b Cocoon wt(g) (male) 1.047±0.08 a 1.200± a 0.238±0.007b Shell wt. (g) (female) 0.182±0.004 a 0.219±0.004 a 0.046±.0002b Shell wt. (g) (male) 0.164±0.005 a 0.200± a 0.023±.0001b % shell (female) ±1.001 a ±0..23 a 9.736±0.151b % shell (male) ±0.131 a ±0.77 a 9.798±0.141b Different letters stand for significantly different at the 5% level by DMT Table 3 Comparative different characters in the backcross hybrids agains(bc 6 J) t those of recurrent parent (J) Characters BC 6 J J NL % young survival ±1.46a ±3.49b ±0.922a % good cocoon ±2.41a ±2.87b 92.26±0.469a 10 full grown larval ±0.213a ±0.557a 8.00±±0.220b wt.(g) Larval duration Day : 21:15±0.20ab 25:21±0.226a 19:12±0.189b hr Cocoon wt(g) female 1.29±1.451a 1.378±0.040a 0.468±0.022b Cocoon wt(g) male 1.109±0.073a 1.297±0.0025a 0.238±0.007b Shell wt. (g) female 0.210±.004a 0.231±0.007a 0.046±.0002b Shell wt. (g) male 0.181±.0015a 0.218±0.001a 0.023±.0001b % shell female ±0.94a ±0.049a 9.736±0.151b % shell male ±0.14a ±0.147a 9.79±8±0.141b Different letters stand for significantly different at the 5% level by DMT

8 Table 4The LD 50 values of iso-hybrid lines of Chinese and Japanese against NPV innoculum Strains LD 50 (PIB/ml) esistant Scales NL X 10 5 J X 10 3 S C X 10 3 S BC.C X X 10 4 M X 10 4 M X 10 4 M X 10 4 M X 10 4 M X 10 4 M X X X 10 5 BC.J X 10 4 M X 10 4 M X 10 4 M X 10 4 M X 10 4 M X 10 4 M X 10 4 M X X X 10 4 M J= Japanese strain, C = Chinese strain, BC= backcross line, NL= Nang Lueng, esistance() strain : LD 50 > 3.1 x10 5 PIBs/ml, Moderate resistance (M) strain : 3.1 x10 5 >LD x10 4, Susceptibility (S) strain: LD 50 < 1.53 x10 4. lines chosen to be further maintained

9 EFEENCES Allard,. W Principles of Plant Breeding. John Wiley and Sons, New Yord Aratake, Y Seric.Sci 42, pp 440. In Watanabe, H Current science 83(4): Aruga, H Priciples of Sericulture AA Balkemal/otherala,India. Doira, H Genetic Stocks of the Silkworn, pp In Tazima, Y. The Silkworm an Improtant Laboratoy Tool Kodansha Ltd.Tokyo. Hongthongdaaeng, S Study on Silkworm Variety esistance to Nuclear Polyhedrosis Viruses. MS. Thesis Kasetsart University Bangkok. Morohoshi S Development Physiology of silkworm. Published by Science Publishers, Inc., Enfield, NH,USA. Nagaraju, J.,, Urs., and.k.datta.1996.crossbreeding and Heterosis in The Silkworm,Bombyx mori, Sericologia 36(1) Nolsenee, P Breeding of Thai-hybrid silkworm. Department of Agricultural Extension Ministry of Agriculture and Cooperative.. Sornchattrarak N Phylogenetic relationships of different lines of Bombyx mori by andon Anplified polymorphic DNA (APD)and the inheritance mode of BmNPV disease resistance. MS thesis, Kasetsart University Bangkok. Tiumpitux, S Breeding and Selection of Polyvoltine Silkworm(BPY 1 ), Bombyx mori MS Thesis, Kasetsart University Bangkok. Watanabe, H Genetic resistance of the silkworm, Bombyx mori to virul deseasis Current science 83(4): Uzigawa, K. and Aruga, H.(1965) Seric.Sci 35, pp 440 In Watanabe, H Current science 83(4):

SUMMARY AND CONCLUSIONS

SUMMARY AND CONCLUSIONS S SUMMARY ilk, the queen of fibres, is known for its luster and elegance and it is an integral part of Indian rich tradition and culture. In recent years, its utility in various