农杆菌介导小麦遗传转化方法的优化与应用

Improvement and application of agrobacterium mediated genetic transformation of wheat

为了优化农杆菌介导小麦遗传转化方法,以郑麦9023幼胚愈伤组织为材料,研究了60Coγ射线辐照对农杆菌介导法遗传转化的影响。结果表明,在辐照剂量为4 Gy,辐照处理后以继代培养24 h时β-葡萄糖苷酸酶(gus)基因瞬时表达率最高。负压抽拉处理结果表明,抽拉10次可明显提高农杆菌介导的遗传转化效率。转基因抗性再生植株bar基因分子标记检测结果表明,利用改良的遗传转化处理方法(4 Gy辐照+辐照后培养24 h+负压抽拉处理10次),可将遗传转化效率由2.2%提高至3.5%,优化后的小麦农杆菌转化方法可应用于小麦遗传转化。

In order to optimize the method of wheat agrobacterium mediated genetic transformation,the embryonic calli derived from the immature embryos of a wheat cultivar "Zhengmai 9023"were used as materials,to study the effects of gamma irradiation on agrobacterium-mediated genetic transformation.The results indicated that 4 Gy is the optimal dose for agrobacterium-mediated genetic transformation of Zhengmai 9023,using GUS transient expression rates as the indicator. Based on the survival rate and GUS transient expression rate,further observation found that 24 hours reculture after gamma irradiation is the most appropriate for agrobacterium infection. Another negative pressure treatment results showed that pulling 10 times in enclosed injector was the optimal manner for transformation. Bar gene PCR assay results of positive regeneration plantlets showed that transformation efficiency was enhanced from2. 2% to 3. 5% by using the improved method(4 Gy irradiation + 24 h reculture + 10 times negative pressure treatment) described above. The optimied agrobacterium-mediated transformation method can be used in the genetic transformation of wheat.