ABSTRACT
Grain legumes are important crop plants for their protein-rich seeds as a major source of dietary protein for human and livestock consumption. Chickpea is the second most important pulse crop in the world, particularly in India and the African subcontinent, although it is grown in more than 40 countries. Despite large acreage of these crops, the total productivity remains low and consistent for the last few decades worldwide owing to physiological and biological constraints. Traditional breeding practices have been exhausted for further genetic improvement including agronomic characters and incompatibility barriers in the existing available germplasm (Ranalli and Cubero, 1997). Genetic transformation of grain legumes may provide solutions to certain constraints in widening their gene pool and genetic improvement for crop productivity (Popelka et al., 2004). However, grain legumes including chickpea are recalcitrant to tissue culture propagation and incompatible to DNA-mediated genetic transformation (Sharma et al., 2002; Somers et al., 2003). Stable transgenic plants of different largeseeded grain legumes have been produced using various plant tissues and DNA delivery methods including microprojectile bombardment of shoot meristems (McCabe et al., 1988), embryogenic suspension culture (Finer and McMullen, 1991) and Agrobacterium tumefaciens-mediated T-DNA delivery into immature cotyledons (Parrott et al., 1989; Yan et al., 2000), embryogenic suspension cultures (Trick and Finer, 1998) and cotyledonary nodes in several grain legumes (Hinchee et al., 1988; Meurer et al., 1998;
Muthukumar et al., 1996; Olhoft et al., 2003; Sanyal et al., 2003, 2005). Each system in the absence of efficient somatic embryogenesis in grain legumes has resulted in relatively inefficient production of transgenic plants owing to inefficient T-DNA delivery, selection of transformed cells, and plant regeneration. Among different explants available in grain legumes, the cotyledonary node is the preferred choice for direct organogenesis and genetic transformation in a number of grain legumes (Somers et al., 2003). Cotyledonary nodes of large-seeded grain legumes possess sectors of preexisting axillary meristematic cells and also potentially regenerative cells in the L2 layer that are competent to differentiate into new meristematic cells or direct organogenesis subject to the application of a suitable combination of growth regulators and culture conditions. Considering the significance and versatile nature of cotyledonary node explants, their mode of regeneration, and interaction of Agrobacterium with target cells for T-DNA transfer along with screening and selection of putative transformants in major grain legumes. We have developed and optimized a reproducible procedure for preparation of cotyledonary nodes in chickpea and parameters for A. tumefaciens cocultivation that influence efficient genetic transformation in this recalcitrant grain legume with an idea to create optimal conditions for Agrobacterium interaction with excised cells, which are competent for de novo regeneration of shoots via an organogenic pathway.
