Agrobacterium-mediated transformation of Phaseolus vulgaris L. using indirect organogenesis

Abstract

A protocol for genetic transformation of Phaseolus vulgaris L. cv. CIAP7247F via Agrobacterium tumefaciens was established. Primary green nodular calli and proliferative calli were used as target explants. Several factors such as Agrobacterium strain, plasmid, light conditions, bacterial concentration, co-cultivation period and type of callus were studied for optimization of the Agrobacterium-mediated transformation. The highest DNA transfer occurred when proliferative calli were inoculated with strain EHA105 harbouring pCAMBIA3301 plasmid at a density of OD600 = 0.5, and co-cultivated under 16 h light/8 h dark photoperiod for 6 days. The transformation system integrates Agrobacterium-mediated DNA transfer with efficient regeneration via indirect organogenesis, and using the bar gene as selectable marker and glufosinate ammonium (herbicide finale) for callus selection. The proposed system allowed obtaining transgenic lines with Mendelian inheritance of the transgenes, as demonstrated by PCR analyses. These results also validated the effectiveness of a regeneration protocol via indirect organogenesis for regeneration of transformed bean cells. This system constitutes a major initial step for common bean transformation using A. tumefaciens.