Study On Agrobacterium Tumefaciens-mediated Transformation Of Common Wheat

As one of the major creal crops for human being, the yield of Wheat (Triticum aestivum L.) is related to the survival of mankind and social development. Salt stress influences on the yield and qualities of the wheat obviously. It’s proved to be difficult to obtain a new salt-tolerance wheat species by conventional breeding methods. Genetic engineering offers new ways in wheat genetic improvement. Due to its huge DNA and complex genetic background, genetic modification of wheat falls behind rice and maize significantly. And the establishment of wheat transformation system by Agrobacterium-medi&ted is more complex and difficult because of the strong recalcitrance in its tissue culture.Transcription factors regulate the expression of genes by combing with the cis acting element region on the promoter. Zinc finger protein is one of the important transcription factors in abiotic stress, such as drought, waterlogging, and salinification etc. TaCHP, a salt-tolerance related gene, was cloned from the somatic hybrid introgression line Shanrong No.3(SR3), which has been generated from somatic hybrid of common wheat Jinan177(JN177) and Thinopyrum ponticum, a salt tolerant grass. Previous investigation has shown that the over-expression of TaCHP transgenic Arabidopsis thaliana has more salt tolerance than wild-type.In this study, the factors involved in genetic transformation of wheat via agrobacterium tumefaciens were optimized. And the conditon for plantlets to survive and develop after transplant into soil was also investigatied.The main achievements are as follows:1TaCHP gene was successfully introduced into calli derived from immature embryogenic of Hesheng3, Yangmai15and Aikang58by inserting it into plasmid pGA3626via Agrobacterium-mediated transformation. And21transgenic lines were obtained. Stable integration and inheritance were confirmed by histochemical staining assay, PCR screening, Southern blotting analysis.2Genetic transformation system of the wheat (Hesheng3) through Agrobacterium-medlated was established, and factors involved in the frequency of transformation were optimized.①Calli dried for30minutes before Agrobacterium inoculation would to enhance efficiency of the GUS gene expression, which reached25.39%.②Co-cultivation at25℃would keep Agrobacterium vigorous as well as calli.③After co-cultivation, calli were rinsed by liquid MS medium1-2times, and then dried with sterile filter paper, followed by transferred the calli onto induction medium containing160mg/1of the antibiotic Timentin. Under this optimum condition, higher transformation frequency was observed.3A suitable method for transferring transgenic plantlets into soil was established, and the survival rate was higher than80%.