Cotyledonary node pre-wounding with a micro-brush increased frequency of Agrobacterium-mediated transformation in soybean

Abstract

A Japanese soybean variety, Kariyutaka, known to form many adventitious shoots from sections of the cotyledonary node, had a transformation frequency (relative number of T0 transgenic plants, which stably transmitted transgenes to their T1 progenies, to number of explants subjected to Agrobacterium-infection) of about 1.0% in the conventional protocol. We modified this method to obtain the transformation frequency of 4.4%. Modified protocol included mechanical wounding of explants with a micro-brush and application of 0.02% of Silwet L-77 during the infection with Agrobacterium. We obtained T0 plants 8 to 12 weeks after co-cultivation with Agrobacterium. T1 seeds were rapidly obtained from 5 to 6 months after the co-cultivation, for T0 plants flowered precociously. Three T0 plants out of 4, i.e. 75% of all T0 plants, which were generated at 12 weeks after co-cultivation, possessed stable transgene(s) that were transmitted to their T1 progenies, although only 7 T0 plants out of 22, i.e. 32% of all T0 plants, which were generated at 8 weeks after the co-cultivation, stably transmitted the Basta-resistance to their T1 progenies.