Phaseolus vulgaris L. regeneration systems and their application for Agrobacterium-mediated genetic transformation

Common bean is the most important legume worldwide for direct human consumption. Beans constitute an important source of dietary protein for over half a billion people mainly in developing countries. A great number of bacterial, fungal, and viral diseases occur annually in bean-producing regions all over the world, causing economic losses to bean producers. Major improvements in agronomic traits of cultivated common bean have been achieved through years of conventional breeding. However, traditional breeding methods became limited by low recombination potential as an outcome of self-pollination, low inheritance capacity of some important characteristics, sexual barriers and embryo abortion in interspecific hybrids. Therefore, genetic transformation is needed to allow breeders to introduce novel traits that could contribute to improved performance and quality and to tolerance to abiotic and biotic factors that limit yield and reduce profitability. Consequently, our aim was to establish different regeneration systems for Phaseolus vulgaris L., and their application to develop an Agrobacterium-based transformation method. A reproducible procedure for the regeneration of fertile plants by direct organogenesis from cultures of the economically important Phaseolus vulgaris L. varieties: CIAP7247F, BAT93, BAT304, BAT482 and ICA Pijao using stem sections as initial explant was established. Pre-culture of explants in liquid medium with TDZ, which has not been used for common bean regeneration before, was a critical step for multiple bud induction. Optimization of culture media and parameters influencing explant quality allowed the standardization of several culture phases, including shoot formation, rooting, plantlet acclimatization and growth of fertile plants in the greenhouse. Although the regenerative responses differed among genotypes, this protocol has the advantage of being applicable to five different cultivated varieties. The optimization of several parameters involved in Agrobacterium-mediated DNA-transfer combined with efficient direct organogenesis from stem sections allowed to develop a working scheme to obtain putative transgenic plants. The observation of GUS-staining during the transformation procedure, both transient in epicotyls and stable in multiple buds, clearly indicated T-DNA transfer. However, the Agrobacterium-mediated transformation of Phaseolus vulgaris L., using direct organogenesis established in this study did not yield permanent transgenic plants, only escapes and some chimeras were obtained in this study. In order to solve the difficulties related to transformation based on direct organogenesis, studies on somatic embryogenesis were initiated, in particular the effect of 2,4-D and explant type. The addition of 2,4-D to the culture medium induced somatic embryogenesis in P. vulgaris L., and the response was influenced by the explant orientation on the culture medium. Somatic embryo formation on cotyledons from immature seeds of Phaseolus vulgaris cv. CIAP7247F was achieved. However, the embryos obtained in this work did not regenerate into plants. Therefore, this technique is not yet useful for genetic transformation in P. vulgaris. Consequently our efforts were focused on establishing a protocol for the in vitro regeneration of Phaseolus vulgaris L. cv. CIAP7247F via indirect organogenesis. Several factors such as explant type, seed age, culture media composition and culture conditions were assessed. The use of CN-1 and CN-2 explants (cotyledonary nodes with one or two cotyledons attached), which have not been used as initial explants for common bean regeneration before, was an important factor for the successful formation of morphogenetic green nodular compact calli. Freshly collected seeds and seeds stored for four months are optimal for callus formation without affecting the regeneration capability of the calli. The number of shoots per callus was dependent on both the concentration of TDZ used during callus proliferation and the BAP concentration used for shoot formation. The influence of the genotype was also observed for elongation of rooted plantlets. The protocol established for cv. CIAP7247F was efficiently applied to different commercial P. vulgaris varieties (BAT93, BAT304, BAT482 and ICA Pijao), demonstrating the value of this procedure. Based on the indirect organogenesis regeneration pathway, Agrobacterium-mediated transformation of common bean was conducted. The use of green nodular calli, which have not been used as target explant for common bean transformation before, played an important role to successfully obtain transformed plants. The most efficient T-DNA transfer occurred when proliferative calli were inoculated with Agrobacterium strain EHA105 harbouring pCAMBIA3301 at a concentration of OD600= 0.5, and co-cultivated under 16 h light/8 h dark photoperiod for 6 days. 0.5 mg l-1 of glufosinate ammonium was the minimum inhibitory concentration for selection of transformed tissue. A transformation system integrating Agrobacterium-mediated DNA transfer, efficient regeneration via indirect organogenesis, and the bar gene as selectable marker, was established. This system allowed obtaining putative transgenic lines with Mendelian inheritance of the transgenes based on PCR analysis, demonstrating its value for bean transformation. Further progeny characterization and molecular analysis by Southern blotting are needed to fully validate this approach as efficient and reproducible; this system constitutes a major initial step for common bean transformation using Agrobacterium tumefaciens.