RESUMO
Phaseolus beans are among the major legumes for food consumption, especially in Latin America, Africa, and Asia. Tepary bean (Phaseolus acutifolius L. Gray) is one of the five cultivated species of the genus Phaseolus. This chapter describes an Agrobacterium-mediated transformation protocol for P. acutifolius based on cocultivation of callus, derived from cotyledonary nodes, with Agrobacterium. The selectable marker gene used is neomycin phosphotransferase II (nptII), and the selection agent is geneticin. Selection of transgenic callus material is achieved through four to five passages on geneticin-containing medium, after which shoots are induced on medium without selection agent. The protocol as described here has been applied to transform a cultivated variety of P. acutifolius, TB1, and also with some modifications to a wild genotype, NI576 and another cultivated variety, PI440795.
Assuntos
Agrobacterium tumefaciens/genética , Cotilédone/genética , Técnicas de Transferência de Genes , Phaseolus/genética , Plantas Geneticamente Modificadas/genética , Transformação Genética , Agrobacterium tumefaciens/crescimento & desenvolvimento , Cotilédone/citologia , Cotilédone/microbiologia , Resistência a Medicamentos/genética , Marcadores Genéticos , Phaseolus/citologia , Phaseolus/microbiologia , Plantas Geneticamente Modificadas/citologia , Plantas Geneticamente Modificadas/embriologia , Especificidade da EspécieRESUMO
Light conditions during Agrobacterium-based plant transformation, the most routinely used method in plant genetic engineering, differ widely and, to our knowledge, have not been studied systematically in relation to transformation efficiency. Here, light effects were examined in two already optimized transformation procedures: coculture of Agrobacterium tumefaciens with callus from two genotypes of the crop plant Phaseolus acutifolius (tepary bean) and coculture of root segments from two ecotypes of Arabidopsis thaliana. Except for the light conditions during coculture, all steps followed established procedures. Coculture was done either under continuous darkness, under a commonly used photoperiod of 16 h light/8 h darkness or under continuous light. beta-glucuronidase (GUS) production due to the transient expression of an intron-containing uidA gene in the binary vector was used to evaluate T-DNA transfer. In all situations, uidA expression correlated highly and positively with the light period used during coculture; it was inhibited severely by darkness and enhanced more under continuous light than under a 16 h light/8 h dark photoperiod. The promotive effect of light was observed with Agrobacterium strains harboring either a nopaline-, an octopine- or an agropine/succinamopine-type non-oncogenic helper Ti plasmid. The observed positive effect of light has obvious implications for developing and improving transient and stable transformation protocols, specifically those involving dark coculture conditions.
Assuntos
Agrobacterium tumefaciens/genética , Plantas/genética , Plasmídeos/genética , Transfecção/métodos , Agrobacterium tumefaciens/efeitos da radiação , Arabidopsis/genética , Arabidopsis/efeitos da radiação , DNA Bacteriano/genética , Glucuronidase/genética , Glucuronidase/metabolismo , Luz , Phaseolus/genética , Phaseolus/efeitos da radiação , Plantas/efeitos da radiação , Plantas Geneticamente ModificadasRESUMO
Over the past decade, several high value proteins have been produced in different transgenic plant tissues such as leaves, tubers, and seeds. Despite recent advances, many heterologous proteins accumulate to low concentrations, and the optimization of expression cassettes to make in planta production and purification economically feasible remains critical. Here, the regulatory sequences of the seed storage protein gene arcelin 5-I (arc5-I) of common bean (Phaseolus vulgaris) were evaluated for producing heterologous proteins in dicotyledonous seeds. The murine single chain variable fragment (scFv) G4 (ref. 4) was chosen as model protein because of the current industrial interest in producing antibodies and derived fragments in crops. In transgenic Arabidopsis thaliana seed stocks, the scFv under control of the 35S promoter of the cauliflower mosaic virus (CaMV) accumulated to approximately 1% of total soluble protein (TSP). However, a set of seed storage promoter constructs boosted the scFv accumulation to exceptionally high concentrations, reaching no less than 36.5% of TSP in homozygous seeds. Even at these high concentrations, the scFv proteins had antigen-binding activity and affinity similar to those produced in Escherichia coli. The feasibility of heterologous protein production under control of arc5-I regulatory sequences was also demonstrated in Phaseolus acutifolius, a promising crop for large scale production.
