农杆菌介导大豆子叶节转化系统的优化

Optimization of the Agrobacterium-Mediated Transformation Systems of Soybean Cotyledonary Node

【目的】研究影响大豆(Glycine maxL.)子叶节外植体遗传转化的因素。【方法】以农杆菌转化系统为平台,用直接筛选法和延迟筛选法确定卡那霉素的筛选方法和浓度,以GUS基因的瞬时表达计算子叶节区GUS阳性率。【结果】延迟7d进行Km选择、筛选压力为75mg·L-1时选择效果最好;菌株培养阶段和侵染浓度分别在OD600为0.6和0.5时子叶节区GUS阳性率最高;黑农35、绥农14和合丰35为易感的大豆基因型;农杆菌菌株EHA101对大豆的转化能力好于LBA4404和AGL1;共培养培养基中添加的硫醇类化合物对T-DNA的转移有极强的影响效应。【结论】本实验方法可以优化农杆菌介导的大豆子叶节转化系统。

[Objective] The factors influencing Agrobacterium-mediated soybean （Glycine max L.） cotyledonary node transformation were studied so as to improve the transformation frequency of soybean. [Method] Delayed selection and direct selection were conducted to determine selection method and concentration of Km. And percentage of GUS positive in cotyledonary nodes was calculated according to transient expression. [Result] The best selection result was obtained when the explants were cultured by delayed selection of seven days and at 75 ml·L^-1 of Km; the highest percentage of GUS positive in cotyledonary nodes was obtained when strain growth phase and infection concentration were at the level of OD600=0.6 and OD600=0.5, respectively. Heinong 35, Suinong 14 and Hefeng 35 were sensitive genotypes to Agrobacterium. The strain EHA101 had advantages in transformation to soybean over the strains LBA4404 and AGLI. Addition of thiol compounds to co-culture medium had significant effect on T-DNA delivery. [Conclusion] The procedure optimized the systems for Agrobacterium-mediated transformation of soybean cotyledonary node.