[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.