农杆菌介导的大豆高频遗传转化

大豆(Glycine max(L.)Merrill)遗传转化目前常用的两种方法为农杆菌介导的子叶节转化系统和基因枪介导的体细胞胚转化,但这两种转化系统都存在转化频率低、难于重复及依赖于特定的基因型等问题.为了提高农杆菌介导的大豆子叶节的转化频率,采用了一种基于bar基因作为筛选标记基因的固体-液体筛选系统,与农杆菌共培养3d的大豆子叶节在MS添加2 mg/L 6-BA和5 mg/L的glufosinate的筛选培养基培养2周后,再转到含有0.01 mg/L TDZ和2mg/L glufosinate的液体培养基中筛选,并每周更换一次培养液.得到的再生芽首先经GUS分析为阳性后再转入生根培养基得到完整转化植株,然后通过Southern杂交分析证实外源基因整合到大豆基因组,转化植物含有1～2个基因拷贝数.该转化系统具有转化频率高、转化周期短以及不依赖于大豆基因型等优点,对影响该转化系统的一些因子进行了讨论.

High-efficiency Agrobacterium-mediated Transformation of Soybean

Soybean (Glycine max (L.) Merrill) is one of the recalcitrant crops to be manipulated in vitro. To improve Agrobacterium-mediated soybean cotyledonary node transformation efficiency, a new solidliquid medium selection system based on using the bar gene as the selectable marker has been developed. The cotyledonary nodes were first cultured in MS solid medium modified with 2 mg/L 6-BA and 5 mg/L glufosinate for two weeks, and the surviving explants were transferred into a liquid selection medium with 2 mg/L glufosinate and 0.01 mg/L thidanzuron (TDZ) instead of 6-BA for further selection. The liquid medium was changed every week until the transgenic shoots were regenerated from the liquid selection medium. Several factors affecting the transformation efficiency have also been evaluated. GUS assay and Southern blotting analyses confirmed the integration of the transgenes into the soybean genome. This transformation system is genotype-independent and efficient, and has less escape due to the tight selection.