Efficient soybean transformation using hygromycin B selection in the cotyledonary-node method.

The efficiency of soybean [Glycine max (L.) Merrill] transformation was significantly increased from an average of 0.7% to 16.4% by combining strategies to enhance Agrobacterium tumefaciens-mediated T-DNA delivery into cotyledonary-node cells with the development of a rapid, efficient selection protocol based on hygromycin B. Wounded cotyledonary-node explants were inoculated with A. tumefaciens carrying either a standard-binary or super-binary plasmid and co-cultivated in the presence of mixtures of the thiol compounds, L-cysteine, dithiothreitol, and sodium thiosulfate. Transformed shoots began elongating only 8 weeks after co-cultivation. Southern analysis confirmed integration of the T-DNA into genomic DNA and revealed no correlation between the complexity of the integration pattern and thiol treatment applied at co-cultivation. All T(0) plants were fertile and the majority of the lines transmitted the beta-glucuronidase (GUS) phenotype in 3:1 or 15:1 ratios to their progenies.