Optimization Of Wheat Agrobacterium-mediated Transformation System

Agrobacterium-mediated transformation method is one of the main methods for wheat genetic transformation. In order to optimize the Agrobacterium-mediated genetic transformation efficiency of wheat, we selected four factors that affect what genetic transformation efficiency. The Agrobacterium strain AGL1 containing the binary pAL154/pAL156 vector system and wheat immature embryos were used for transformation. After PCR analysis, a total of 213 independent transgenic lines were obtained. GUS staining test showed that 163 transgenic lines expressed gusA gene. Southern bolt analysis showed that the single copy and multiple copies of exogenous gene both exist in transgenic progeny. Analysis of T1 showed that about 30% had the segregation ratio of 3:1 which accorded with separation ratio of single copy transgene or single expression, and the rest lines had ratios typical of two or three independently segregating loci.The result showed that rate of Agrobacterium infection of explants was increased by amending the liquid and solid co-culture medium with Acetosyringone and L-cysteine, and callus browning was reduced. The frequency of embryogenesis was increased by adding Picloram in solid co-culture and induction medium. The frequency of regeneration was increased by adding CuSO₄ in induction and regeneration medium. The average final transformation efficiency was improved from 2.8% to 6.3%, and the highest frequency of 12.3% was reached in one particular experiment. The most significant effect was observed when the concentration of Acetosyringone was 400 ?M, the concentration of L-cysteine was 400 mg/L, the concentration of Picloram was 10 mg/L, the concentration of CuSO₄ was 50 mM. This study constructed a efficient Agrobacterium-mediated transformation of wheat system which was powerful technique for wheat genetic transformation.