The Factors Influencing Wheat Genetic Transformation And The Productions Of Transgenic Plant Mediated By Agrobacterium Tumefaciens

In this study, the foreign gene was transferred into wheat immature embryos by Agrobacterium tumefaciens mediated, and the factors influencing wheat transformation were analyzed. The wheat genotypes with high tissue culture response and good agronomic traits were screened out from 10 potential Chinese wheat cultivars, the wheat immature embryo callus with 4 days preculture were acceptor, using high-toxicity Agrobacterium strains transferred the vector, activate culture for 3 days in YEP solid medium, and the Agrobacterium resuspending in WCC liquid medium was donor, the plasmid vector pZWS101 containing a target gene and marker gene was transferred into wheat. The infected callus were selected several times on selectable medium with 10-50mg/lgeneticin(G418), SOmg/l cefotaxime, 50mg/l vancomyciru 50mg/l ticarcillin. By nptll ELISA, PCR, PCR-Southern analysis, 13 transgenic plants were obtained from 6 experiments for three genotypes, with an average transformation efficiency of 0.62%. The optima! conditions of wheat transformation mediated by Agrobacteriurn was confirmed and a high efficient and stable Agrobacterium-mediated transformation system was estabilished. The main results were as following:1. Wheat genotypes Yangmai 158, Yangmai 10 are excellent transformation materials. Wheat immature embryos with 4 days preculture have high tissue culture response and strong regeneration capacity. Agrobacerium strain C58c1 has high toxicity, with which the recombant plasmid vector was transferred, in favr of the foreign gene was integrated and transformation efficiency was increased.2. The improvement of medium and the optimization of cultivationconditions, they are profitable to form the embryogenic callus and produce the regeneation plants. AS added in medium can increase vir gene expressed; 2,4-D added in medium can promote callus formed. While infecting and co-culturing, the OD650 value of Agrobacterium concentration is 0.6-0.8, after directly infecting 30 minutes, the callus were transferred onto the solid co-cultivation medium with pH5.2, and co-cultured under 21-22癈,dark conditions, the good quality of resistance callus were obtained.3. The selection agent concentration was confirmed. Geneticin (G418) was only used for the selection of npt II, kanamycin or neomycin was not available, its suitable concentration was 10-50 mg/l. At one time three kinds of antibiotics were used in the experiment, which are 50 mg/l cefotaxime, 50mg/l vancomycinx 50mg/l ticarcillin. The transformation effectiveness was good.4. T0 generation resistance plants were identified by molecular analysis. By npt II ELISA analyzing, PCR amplifying, PCR-Southern blotting, the result showed the alien genes were integrated into plant cells, and certified that the transgenic wheat plants were obtained with Agrobacterium tumefaciens-mediated.