Study On Regeneration Systems And Genetic Transformation Via Agrobacterium Tumefaciens In Wheat

Immature embryos and embryogenic callus are regarded as the best explants in genetic transformation of wheat, but it is difficult to obtain immature embryos throughout the year. This problem has restricted the research of transgenic wheat to a great extent. In addition, the potential hazard to the environment and human of antibiotics and herbicide selection agents has caused a negative influence to the application of commercial production. So it is necessary to apply other explants and the selection systems based on safe agents in genetic transformation. In order to set up a effective regeneration system, immature inflorescence and mature embryos of several wheat varieties, which are currently cultivated in the Yangtze River valley and the central region of China, were studied to compare the characteristics of callus induction and plant regeneration. Moreover, a genetic transformation system of wheat immature embryos with manA as a safe selection marker gene mediated by Agrobacterium tumefeciens was studied. Main results are as follows:1. Factors affecting immature inflorescence tissue culture of ten wheat varieties were studied with the different parts under the conditions of different callus induction and differentiation media. The results showed that the middle part was the best one, and the L7V induction medium and the L7Z differentiation medium were the best combination for green plant regeneration. Callus induction, callus rooting and the green plant regeneration have significant differences among the various genotypes, but there was no significant correlation among the three factors. The wheat varieties with higher regeneration capacity, such as Yangmai 158 and Huamai 13, were suitable for genetic transformation.2. Mature embryos of three wheat varieties were studied with different callus induction media and differentiation media. The results showed that there are significant differences in callus induction and the green plant regeneration among the various genotypes; Huamai 9, which has the highest regeneration capacity, was suitable for genetic transformation; ABA and AgNO₃ have positive effect on promoting the frequency of embryogenic callus and differentiation; proper desiccation treatment can improve the differentiation frequency; the optimum media for callus induction and differentiation were MSA and MTA media.3. Immature embryos calli of Zheng9023 were transformed via Agrobacterium tumefeciens strain GV3101 and after selection with PMI/mannose system, the integration of target gene in two transgenic seedlings were identified by PCR. The transformation frequency in this experiment was 0.35%, which demonstrated that we have set up a transformation system and paved the way for further transformation in wheat.