Study On Agrobacterium-mediated Wheat Transformation By Using Its Different Explants

In the research field of wheat Agrobacterium mediated transformation to date, the immature embryo is the only successful explant, and Bobwhite is the mostly used genotype. Because of such situation, the transformation efficiency of wheat has been hampered by some factors such as inconvenient material source, limited available genotypes, and unstable transformation result. Therefore, it is very important to wheat improvement and functional genomic study to establish the transformation systems of different wheat explants including mature embryo, inflorescence, and immature embryo by screening sensitive genotypes to Agrobacterium and modifying co-culture techniques, and to transfer some useful genes related to its characteristics. 83 wheat genotypes from different domestic regions were used in this study, and their mature embryos, inflorescence and immature embryos were infected by Agrobacterium harboring GUS, nptll, WT, 154X and 154Y. Some sensitive genotypes were screened out by the GUS gene transient expression, and the target genes above were introduced into wheat by employing suitable techniques of material preparing and co-culture.The result showed that the inflorescences of CA9924, Beinong 49, Xinong 2611, Jingdong 11 were sensitive to the Agrobacterium infection, and GUS gene transient expression frequency were 66.7%, 75.0%, 80.0% and 82.8% respectively. The immature embryos of 97H2169, Lunxuan208, Lankao 906 and Yangmai 6 were sensitive to the Agrobacterium infection, and GUS gene transient expression frequency were 50.0%, 50.0%, 63.3% and 93.3%. Most wheat genotypes were not sensitive to the Agrobacterium infection. The results also suggested that the co-culture step processing on paper was much better than it on solid medium. Mature embryos and their derived callus from 6 different wheat genotypes as receptor explants were infected by Agrobacterium harboring plasmids containing nptll and GUS or WT, 154X, respectively. Through callus initiation, selection, regeneration and plantlet detection, the foreign genes were successfully transferred into whea. For the first time, wheat transgenic plants were obtained by using its mature embryos as explants, and such success will be contributing to wheat molecular breeding for convenience. It was demonstrated at the same time that the fresh mature embryo tissues of being cut into halves were more frequently infected by Agrobacterium than the tissues prepared with other two methods. Transgenic wheat plants were also obtained from the experiments by using the immature embryos as explants other than from the experiments by using the inflorescences as explants, from which several resistant plants were generated only. All resistant regeneration plants were tested by PCR or ELISA first, and putative transgenic plants were identified by southern next.Segregations of the alien genes were analyzed in T₁ generation.