Study Of Gna Gene Transfer Of Soybene And Optimization Of The Transformation Systems

To establish the most optimal methods of sterilization on soybean, variety Hefeng No.35 were taken as material to compare effects of seed germination with different disinfectants and different treating time at 28℃ or 4℃ in tissue culture. The different explants, hypocotyl, terminal bud, cotyledonary node, seedling were studied to induce bud or callus with different concentrations of 6-BA and to establish a better system for transformation. Several factors in transformation system of soybean were studied and gna gene was transferred into soybean with seedling explants. The results show:1. In soybean tissue culture , the most optimal methods of sterilization is one-two hour's treatment with saturate bleaching powder solution in 4℃ after 30 seconds' pretreatment with 70% ethanol or two hour's treatment with 0.1% aqueous mercuric chloride in 4℃.2. The different explants need different concentration of 6-BA to induce callus and bud, cotyledonary node is 1.0mg/L, terminal bud 2.0mg/L, seedling 1.0mg/L to bud-inducing, and hypocotyls is 2.0mg/L to induce callus. The effect is better with 6-BA 1.0mg/L supplemented with IBA 0.2mg/L for seedling explant's bud-inducing.3. Hypocotyl, cotyledon, terminal bud are all easy to induce callus, but the inducing rate of bud are lower than those of cotyledonary node and seedling. Regeneration rates of various explants were , in the order from high to low, seedling, cotyledonary node, terminal bud, hypocotyls, cotyledon.4. There exists obvious difference among different genotypes . these results indicates that the stability is uniform between callus and bud inducing with different explants and it affects callus and bud inducing. Dongnong 42 is better than other tested genotypes on its stability. It shows we should pay attention to the difference on the stability of genotypes.5. The sensitivity of soybean cultivars to Agrobacterium tumefaciens is commonly low and there is significant difference among genotypes.6. The preculture, duration of infection and co-culture with Agrobacterium tumefaciens influences soybean transformation evidently. The best combination is preculture 1 day, infection 10 minutes and co-culture 3 days under the experimental conditions.7. gna gene was transferred with regeneration system of soybean seedling, whose regeneration is higher than others. After selected by kanamycin, Among the 15 putative transgenic plants, nine gave positive PCR reaction.