Factors affecting Agrobacterium-mediated transformation of common bean

Abstract

Factors influencing Agrobacterium tumefaciens-mediated transformation of common beans (Phaseolus vulgaris L.) were examined using an intron-containing β-glucuronidase (GUS) gene as a reporter system to develop a repeatable transformation protocol. Tissue culture procedures used were based on direct shoot organogenesis. Two A. tumefaciens strains-A2760 and EHA105-were used, with emphasis on the former due to its overall higher infection rate. Eleven common-bean genotypes were compared for susceptibility to strain A2760 or EHA105. The pinto bean 'Othello' was used extensively in testing different transformation conditions. Factors significantly affecting transformation rate were Agrobacterium x host interactions, explant maturity, preculture and cocultivation conditions, and selection schemes, based on transient GUS gene expression. The best transformation conditions were the use of susceptible genotypes and explants derived from mature seeds, preconditioning of explants in a medium containing 20 μmol of benzyladenine (BA) in darkness or on a filter paper, dipping explants in high concentrations of Agrobacterium cell suspension (OD650 = 0.81.0) followed by a long-term (6-day) cocultivation period on a semisolid agar medium in the presence of cytokinin or 3-day cocultivation on a moistened filter paper, and the use of lethal levels of selective agents. About 4% of explants, or 14 % of regenerated shoots or buds, were putatively transgenic, as indicated by GUS blue staining throughout the entire shoot or bud, after explants were transformed with Agrobacterium strain A2760 using an optimized protocol.