根癌农杆菌对结球白菜的转化

Transformation of Chinese Cabbage (B.campestris L.ssp.Pekinensis) by Using Agrobacterium

为了给外源有用基因导入结球白菜并高效稳定地表达提供依据 ,以结球白菜的子叶—子叶柄为外植体 ,采用农杆菌共培养法把外源基因 C5 8/ p V11导入结球白菜 .在选择最优的转化条件——根癌农杆菌侵染外植体 15min,农杆菌培养和共培养时在培养基中均加入 AS,培养基 p H为 5 .2 ,共培养 3d的条件下 ,获得转基因植株 .其中抗性植株的抗草丁膦试验、转基因植株 DNA的 PCR及 PCR- Southern斑点杂交鉴定等结果表明 ,5 0 %的抗性植株的基因组中有外源基因的整合 .

To provide basis for introducing LMV-CP into the Chinese cabbage genome and its high effective and permanent expression,experiments were conducted under the optimum conditions including soaking the outplant by Agrobacterium for 15 min,putting AS into the media during the process of Agrobacterium cultivation and co-cultivation,keeping the pH of media at 5.2.After precultivation for 3 days, the cotyledon petioles were co-cultivated for 3 days with Agrobacterium C58 harhouring binary vectors carrying Bar gene for Basta resistance and LMV-CP gene, then the cotyledon petioles were placed in the selective medium containing 5 mg/L Basta. Shoot induction continued for 4～6 weeks. The shoots were transferred to the root induction medium. Resistant plants frequency was 6%. Through polymerase chain reaction (PCR) analyses and PCR-Southern dot blot hybridization analyses, 3 of 6 resistant plants, which were randomly selected, confirmed the introduction of the LMV-CP into the Chinese cabbage genome.