Regeneration System Optimized And Genetic Transformation Of AhSAD Promoter In Peanut

The study selected the epicotyls of some varieties peanut as explants,such as Yuanza 9307,Jitian 1,Luo Hanguo,Hua 37 and Baisha 1016.We explored the best meida and culture conditions to establish and optimize callus induction and regeneration system.We studied the optimal culture concentration of callus induction by comparing the induce callus differences among preculture age, peanut types and plant hormones concentrations in the medium. And then we explored the best way to epicotyl in vitro regeneration from somatic embryogenesis and organogenesis in peanut, respectively. On this basis,screening for 19 kinds of different genotypes. Agrobacterium mediated genetic transformation was exploed on the base of regeneration system. Different Km gradient screening, different transgenic acceptors,different infection time and different co-culture time were set to improve the process of Agrobacterium mediated genetic transformation.Finally,we transferred AhSAD promoter into the transgenic acceptors from different kinds of peanut. The main results were as follows:(1)There was a significant difference in the preculture time and the callus status was significantly different. The epicotyl from the peanut of precultured 0d was easy to form embryogenic callus, and the callus quality from 7d young epicotyl was generally better than 3d and 10 d.(2)The best medium for the induction of callus was MS+B5+Pic3mg/L. Embryogenic callus induction rate could be up to 60%.This system is suitable for the most of peanut varieties and repetitive somatic embryogenesis can be obtained. The subculture medium was MSB5+Pic3mg/L+Gln1g/L(+)AgNO3(2mg/L).(3)The best culture medium for the somatic embryogenesis was MS+0.4mg/L6-BA +0.1mg/LNAA, the highest seedling rate was 46.77%.It was better to regenerate on MS medium containing 6-BA0.4mg/L+NAA0.1mg/L and MS+6-BA3.0mg/L+NAA 0.5mg/L. The best culture media about adventitious shoot formation was MS+TDZ0.2mg/L+6-BA3.0mg/L+NAA 0.5mg/L, the adventitious shoot differentiation rate as high as 42%.(4)Different genotypes had different induced and regenerative capacity.The percentage of callus induction of 9048 and 9102 was between 70%and 80%.The peanut varieties of 9020,9092 and 9110 were between 60% and 70%.And the lowest percentage of callus induction was 16 % from 9059.The percentage of regeneration was between 40% and 50% from 9099 and 9097.And the regeneration rate from the two peanut varieties was better than 9036 and 9093.(5)When we took the embryonic callus and no vaccine epicotyl materials as transgenic acceptors,Km gradient screening was 80mg/L and 100mg/L.And the browning rate was lowest and the transformation rate was highest when the infection time was 10 min.Best condition of the highest the resistance bud induction rate was the co-culture time in 2d from embryonic callus and the co-culture time in 3d from no vaccine epicotyl materials.(6)After scanning and PCR identification, 34 strains resistant plants were obtained and 10 strains were successfully obtained as positive finally.