Optimization Of Agrobacterium-Mediated Transformation Using Wheat Immature Embryos

Wheat immature embryo is considered to be the best explant material source for calli development and shoot regeneration because of its high cultural characteristics and better regeneration potential. It is be propitious to drive the process of genetic breeding by Agrobacterium tumefacienens-mediated transformation using the immature embryos. Immature embryos of different wheat genotypes were cultured to optimize the system of induction, differentiation and regeneration of wheat immature embryos. Some genotypes with higher culture potential and transformability were screened out. The target genes EeAP2-1.1 and OsDREB2.2 were introduced into wheat by employing suitable culture media and culture condition.The factors influencing the efficiency of transformation mediated by Agrobacterium tumefacienens were researched. The major results showed that:1. The ages of immature embryos, genotypes, induction medium, KT and NAA were significant factors influencing the induction, differentiation and regeneration of wheat immature embryos callus. The best suitable time for immature embryos culture of winter wheat henong827 was 12¹6 d in baoding. The improved induction medium was NO.1ˊ, DIC, W1, W2. The best suitable incretion concentration of differentiation medium was 1.0 mg/L KT+0.2⁰.4 mg/L NAA. Four wheat genotypes with higher efficiency of induction and regeneration of immature embryos callus were selected out of 14 wheat genotypes. The optimal system of wheat tissue culture was established.2. The wheat genotype henong827 with better culture potential was screened out from 14 genotypes after infected by Agrobacterium, whose transient GUS expression frequencie of embryonic calli was 91.91%. With the aid of an orthogonal design, we optimized method for Agrobacterium-mediated transformation of immature embryos of henong 827. Optimal conditions for T-DNA delivery were obtained for immature embryos precultured for 6 days in W2 medium, followed by immersing in inoculation suspension of OD₆₆₀=1 in darkness at 23²5℃for 3 h. The best suitable antibiotic concentration of selected medium was 25 mg/L G418+250 mg/L carb. After co-culture of Agrobacterium and callus, we acquired higher resistant calli survival rate with PCM medium washing callus. The study showed that the environmental temperature at 22℃during 3 d co-culture period was the most suitable condition to decrease callus browning frequency and enhance the efficiency of transformation. In the infection medium surfactant 0.01% F68 treatment was helpful to improve transformation efficiency. we resolved the problem of the regenerated plants taking root with 1 mg/L IBA accession. The system optimized relatively of genetic transformation of wheat immature embryos mediated by Agrobacterium tumefacienens was established.3. The target genes EeAP2-1.1 and OsDREB2.2 were introduced into wheat callus by employing by optimized genetic transformation system, obtaining 47 regenerated plants of T₀ after screening. PCR analysis indicated 7 transgenic plants were obtained from the survival of the regenerated plants.