影响农杆菌介导的大豆子叶节遗传转化的因素

利用携带pCAMBIA1301质粒（含hpt和gus基因）的超毒根癌农杆菌菌株EHA105对大豆子叶节外植体进行遗传转化，研究了影响农杆菌介导的大豆子叶节遗传转化的因素。研究结果表明．农杆菌侵染液和共培养培养基中添加200μmok/L乙酰丁香酮和50mg／L抗坏血酸可以有效促进农杆菌对大豆子叶节的转化。农杆菌与子叶节共培养后羧苄青霉素（250mr／L）和头孢霉素（100mg／L）结合使用能有效抑制农杆菌过度繁殖并提高转化芽诱导频率；在转化细胞的分化和转化芽伸长过程中，改进的筛选策略可以明显改善对转化芽的筛选效果，从而提高转化频率。应用优化后的转化体系．获得了3个国内大豆主栽品种的转基因植株，PCR阳性植株频率为3．8％～7．6％。转化植株叶片总DNA的PCR和Southern blot实验表明，T-DNA上的外源基因已经整合到大豆基因组中。

FACTORS INFLUENCING AGROBACTERIUM-MEDIATED COTYLEDONARY-NODE TRANSFORMATION OF SOYBEAN (GLYCINE MAX L.)

Using Agrobacterium tumefaciens strain EHA105 carrying binary vector p1301, which containing the gus and hpt genes, a highly efficient transformation system was developed based on the study of factors influencing the Agrobacterium-mediated cotyledonary-node transformation of soybean. The results demonstrated the additions of acetosyringone (200 micromol/L) and ascorbatic acid (50 mg/L) in both infection medium and co-cultivation medium resulted in a significant increase in the transformation efficiency. The induction of the shoots was benefited from the combined utilization of Carbnicillin (250 mg/L) and Cefotaxime (100 mg/L). The inclusion of a 7-d resting step made the selection scheme using hygromycin B as the selective agent more efficient to produce transgenic shoots. Using the optimized transformation procedure, three soybean cultivars widely grown in North China were successfully transformed and the frequency of PCR-positive plant ranged from 3.8%-7.6%. The integration of the transgenes into the soybean nuclear genome was confirmed by PCR analysis using gus- and hpt-specific primers and by Southern blot using hpt-specific probe.