Optimization Research On The Regeneration And Transformation System Of Tomato

Using tomato variety "Zhong Shu No.4" as the materials, the paper optimized the regeneration system of adventitious bud by tissue culture. The appropriate transformation conditions were selected by infecting tomato cotyledons with the Agrobacterium strain EHA105carrying the binary plasmid pBI121(containing the GUS gene and the npt II gene). It laid the foundation for improving tomato traits through genetic engineering technology in the future. The detail results are presented as follows.1. Using the cotyledon of aseptic seedling in tomato as the explants, the experiment studied the effects of different organic additives and different combinations of ZT and IAA on callus and buds induction of the tomato. The results showed that the best medium for the regeneration of tomato cotyledons is MS (added0.07%MES)+ZT2mg/L+IAA0.1mg/L. Comparing the regeneration of different parts of the cotyledon, leaf and hypocotyls, the middle section of the cotyledon was the best explants, the frequency of buds induction was as high as98.3%, followed by leaf, hypocotyls was the lowest. The most optical medium for shoot elongation was MS+ZT1mg/L+GA1mg/L in subculture. Optimum medium for adventitious buds rooting was1/2MS+IAA0.3mg/L, the roots were much and sturdy.2. The resistance of The Agrobacterium was determined by streaking out the activated Agrobacterium on the YEB solid medium with different concentration cefotaxime, streptomycin, ampicillin, kanamycin and rifampicin. The results showed that the EHA105strain is resistant to kanamycin, rifampicin and streptomycin, but cefotaxime and ampicillin inhibit its growth; The activated bacterium was added to the YEB liquid medium and shaken for culture to measure its growth curve, the results showed that the bacteria shaking for20h enter into logarithmic growth phase, and the stable growth period after28h. The Agrobacterium was of high activity when cultured between24h to28h and suitable to infect the explants.3. Optimal concentration of antibacterial antibiotic cefotaxime was400mg/L, while antibiotic kanamycin concentration was50mg/L in the research on the antibiotic sensitivity of tomato. Using the cotyledon of tomato as explants, the paper studied the factors which influence Agrobacterium-mediated transformation efficiency of the tomato by infecting tomato with Agrobacterium strain EHA105 containing pBI121plasmid. The results showed that GUS staining was best and the induction rate of the resistant buds was the highest (22.75%) when the explant were pre-cultured for2d; When200～300μmol/L acetosyringone were added to induction and co-culture media, both Gus staining and induction rate of resistant buds were better than other combinations; The explants grew well if they were infected by Agrobacterium(OD600≈0.6) and GUS staining was the be st and the induction rate of the resistant buds was the highest (25.16%); Infection with Agrobacterium and co-culture time that are suitable to induction of the resistant buds was5min and2d respectively. PCR test results to the resistant plants have indicated that the target gene have been integrated into the genome of tomato. This provides some references for the transformation of tomato in the future.