Agrobacterium–mediated transformation of chickpea using shoot meristem

Abstract

78-84Agrobacterium-mediated gene transfer to pre-organized meristematic tissue combined with axillary regeneration was standardized for transformation and regeneration of chickpea, which otherwise was difficult to achieve from other explants. Different Agrobacterium strains harbouring binary vectors pCGP1258, containing the GUS as a reporter and bar [gene for resistance to phosphinothricin (PPT)—the active ingredient of the herbicide Basta] as the selectable marker, were used for the transformation experiments. After co-cultivation, the shoot apex explants were transferred onto a PPT-free regeneration medium and their tops (2 mm) were thoroughly wetted with PPT solution (2 mg/mL). The multiple axillary shoots developing from the shoot apices were excised and placed onto a medium containing 10 mg/L PPT. The surviving shoots were subcultured every 2nd wk onto fresh medium containing 20 mg/L PPT. After each subculture, the number of surviving shoots decreased until it stabilized. Some of the chimeric shoots surviving the PPT selection eventually developed new healthier axillary shoots, which could be rooted or grafted on in vitro grown seedling. This whole process took 6-9 months. Average transformation frequency was found between 1.29-3.33%. Transmission of the transgenes into progeny was also studied following the inheritance of uid A gene in T₁ and T₂ progenies. The overall segregation ratio among progenies of plants derived from T₀ plants appeared to be close to 3:1 Mendelian ratio, indicating integration of the transgene at single locus