Agrobacterium tumefaciens mediated transfer of Phaseolus vulgaris alpha-amylase inhibitor-1 gene into mungbean Vigna radiata (L.) Wilczek using bar as selectable marker.
Morphologically normal and fertile transgenic plants of mungbean with two transgenes, bar and alpha-amylase inhibitor, have been developed for the first time. Cotyledonary node explants were transformed by cocultivation with Agrobacterium tumefaciens strain EHA105 harboring a binary vector pKSB that carried bialaphos resistance (bar) gene and Phaseolus vulgaris alpha-amylase inhibitor-1 (alphaAI-1) gene. Green transformed shoots were regenerated and rooted on medium containing phosphinothricin (PPT). Preculture and wounding of the explants, presence of acetosyringone and PPT-based selection of transformants played significant role in enhancing transformation frequency. Presence and expression of the bar gene in primary transformants was evidenced by PCR-Southern analysis and PPT leaf paint assay, respectively. Integration of the Phaseolus vulgaris alpha-amylase inhibitor gene was confirmed by Southern blot analysis. PCR analysis revealed inheritance of both the transgenes in most of the T(1) lines. Tolerance to herbicide was evidenced from seed germination test and chlorophenol red assay in T(1) plants. Transgenic plants could be recovered after 8-10 weeks of cocultivation with Agrobacterium. An overall transformation frequency of 1.51% was achieved.
Sonia
Advanced Centre for Biotechnology, M D University, Rohtak, India.
Saini
Raman
R
Singh
Rana P
RP
Jaiwal
Pawan K
PK
D016960:Agrobacterium tumefaciens / Q000235:genetics*; D001681:Biological Assay; D015139:Blotting, Southern; D007887:Fabaceae / Q000235:genetics* / Q000254:growth & development; D018014:Gene Transfer Techniques; D017343:Genes, Plant; D005819:Genetic Markers; D018525:Germination; D053209:Herbicide Resistance / Q000235:genetics; D009943:Organophosphorus Compounds / Q000378:metabolism; D027805:Phaseolus / Q000235:genetics*; D010637:Phenolsulfonphthalein / Q000031:analogs & derivatives / Q000378:metabolism; D018515:Plant Leaves / Q000166:cytology; D037121:Plant Lectins / Q000235:genetics*; D018520:Plant Shoots / Q000254:growth & development; D030821:Plants, Genetically Modified; D016133:Polymerase Chain Reaction; D014170:Transformation, Genetic
D005819:Genetic Markers; D009943:Organophosphorus Compounds; D037121:Plant Lectins; C061510:alpha-amylase inhibitor, Phaseolus vulgaris; C003122:bialaphos; C007016:chlorophenol red; D010637:Phenolsulfonphthalein