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  • Author or Editor: Zhanyuan Zhang x
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Optimization of parameters influencing biolistic transformation is a crucial stage towards repeatable transformation of common beans. However, there has been no published study on such optimization of this crop species in a helium particle delivery system (BioRad). Using an intron-containing β-glucuronidase (GUS) gene as a reporter, we optimized several critical parameters of biolistic PDS-1000/He delivery system for common bean transformation. The target explant tissues included cotyledons, zygotic embryos, and meristemic shoot tips suitable for organogenesis. Thus, pretreatment of target tissues with osmotic medium containing 0.15–0.25 m mannitol and 0.15–0.25 m sorbitol, positioning of target tissues in 4 cm microcarrier flying distance, the use of 1.6-μm gold particle and high concentration of coating DNA, and bombardment of young immature tissues twice at 2000 psi, etc., significantly increased transformation rate and achieved the best coverage and penetration of the meristemic areas involved in direct shoot organogenesis.

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Factors influencing Agrobacterium–mediated DNA transfer of P. vulgaris were examined using an intron-containing β-glucuronidase (GUS) gene as a reporter system. Tissue culture procedures used were based on direct shoot organogenesis. Two A. tumefaciens strains, A2760 and EHA105, were used with more emphasis on the former due to its overall higher transformation rate. Ten bean entries including breeding lines and cultivars from both Meso-American and Andean origins were compared for compatibility with the two bacterial strains under different pre- and coculture conditions. Pinto `Othello' was extensively used in testing different transformation conditions. Factors found to have significant effects on transformation rate included Agrobacterium-host interactions, explant maturity, preculture and cocultivation conditions, as well as selection schemes, based on transient expression. Some factors, such as the effect of explant maturity and dark preconditioning of explants on gene transfer, have not been reported before. The best transformation conditions included the use of susceptible genotypes and mature explants, preconditioning of explants in darkness, followed by a maximum cocultivation period in the presence of cytokinin, and the use of high selection pressure.

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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 × 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 = 0.8-1.0) 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.

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