关键词: 大豆, 根癌农杆菌, 转化, 萌动种子, 子叶节

Abstract:

An efficient transformation method for obtaining a high frequency of transformants of soybean [Glycine max (L) Merrill] was developed by inoculation of germinating cotyledonary node with Agrobacterium tumefaciens cells. Soybean seeds were germinated for 12–16 h, and the cotyledonary node cells of half seeds were inoculated with Agrobacterium tumefaciens cells harboring a binary vector pGB that contained the bar and sgfp genes conferring phosphinothricin (PPT)-resistance and green fluorescent protein (GFP) activity, respectively. The inoculated explants were selected on the shoot initiation，shoot elongation, and root induction media containing PPT, respectively. The optimal selection concentrations of PPT for 5-day-old explants were 5 mg/L for shoot initiation, 3 mg/L for shoot elongation, and 3 mg/L for root induction. To determine the effect of the different target tissues on the transformation efficiency, a total of four different cotyledonary nodes germinated for 12–16 h, 2 day, 5 day, and 10 day, respectively, were tested under the optimal selection system. The highest average transformation efficiency (8.9%) of soybean was obtained when the cotyledonary nodes germinated for 12–16 h were used as a target tissue, while in case of using the cotyledonary nodes germinated for 5 and 10 days, respectively, as a target tissue, their transformation efficiencies were remarkably reduced. Stable integration and maintenance of the transgenes in the genome of the PPT-resistant plants were confirmed by Polymerase chain reaction and genomic Southern blot analysis. GFP analysis revealed that the transgenes were highly expressed in the leaves and stems of the transformants. Transgenic plants were resistant to 100 mg/L PPT when applied on the leaves, demonstrating their herbicide-resistance. The transformation strategy described in this study will provide a practical protocol to generate diverse transgenic soybean plants

Key words: Soybean (Glycine max L. Merr.), Agrobacterium tumefaciens, Transformation, Germinating seed, Cotyledonary node