Abstract
Common bean is a crop recalcitrant to in vitro regeneration and therefore it lacks an efficient transformation protocol that can be reproduced using A. tumefaciens. The main goal of this study was to establish a protocol for A. tumefaciens mediated transformation of Phaseolus vulgaris var. Brunca by marker genes (gusA and nptII) together with the gene for trehalose-6-phosphate synthase from Saccharomyces cerevisiae (TPS1) used in other species to increase tolerance to abiotic stress. The β-glucuronidase activity was detected in 45 % of the LBA4404 ElectroMAX® pCAMBIA1301 infected explants. Transformed explants regenerated new shoots after four to five months period in a kanamycin rich media. Surviving plants were evaluated by PCR and presented an 0.5 % efficiency of transformation. The established protocol for genetic transformation of common bean has two additional advantages with respect to previous reports: (1) it allows for obtaining transformed regenerants and (2) the genetic transformation was stable for the selective gene.
Highlights
Phaseolus vulgaris is cultivated and consumed primarily in Latin America, Africa and Asia (Gepts et al, 2008)
Transgenic plants of P. vulgaris have been obtained with resistance to bean golden mosaic virus (BGMV) and with increased tolerance to herbicide ammonium gluphosinate Imazapyt by the biolistic approach (Aragão, Vianna, Albino, & Rech, 2002; Rech, Vianna, & Aragão, 2008; Aragão, Nogueira, Tinoco, & Faria, 2013)
Regenerants with kanamycin resistance were assessed by PCR to determine the presence of the nptII gene (Fig. 2 B). These results indicate a transformation efficiency of 0.5 % for the nptII gene in plants of common bean
Summary
Phaseolus vulgaris is cultivated and consumed primarily in Latin America, Africa and Asia (Gepts et al, 2008). A BGMV (EMBRAPA 5.1) was resistant line made by transforming with a genetic silence system against the viral gene rep (AC1) (Bonfim, Faria, Nogueira, Mendes, & Aragão, 2007; Aragão & Faria, 2009; Aragão et al, 2013). Such transformation has a low efficiency and higher cost compared to the use of Agrobacterium-mediated transformation (Amugune et al, 2011). Brunca via Agrobacterium tumefaciens considering several factors like bacterial concentration, incubation of the pre-culture and co-cultivation, antibiotics used and lethal concentrations of selective agents
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