Application of lateral branching to overcome the recalcitrance of in vitro regeneration of Agrobacterium-infected pigeonpea (Cajanus cajan L.)

Abstract

Establishment of a compatible and suitable regeneration and gene transformation protocol is of immense significance to achieve the desired improvement in pigeonpea through biotechnological approach. However, no reproducible protocol for large-scale generation of independent primary transformants plant lines through organogenesis virtually exists. Therefore, an attempt was initiated to develop a simple and straightforward protocol for easy generation of transformed lines through the facilitation of the production of multiple shooting and their lateral branching followed by selection of generated axillary buds. The protocol involves infection of longitudinally dissected cotyledonary node and shoot-tips of germinated seedling with Agrobacterium harboring genetic cassette having both the bialaphos-resistant and green fluorescent protein (gfp) genes as a plant selection marker and reporter gene respectively. The infected explants were allowed to grow in medium with 0.25 mg/L BAP and 0.1 mg/L gibberellic acid (GA3) for development of multiple shoots followed by micropropagation of nodal segments and selection under 4 mg/L bialaphos. The selected plants were found to contain integrated genes and carried through successive generations as revealed by dot blot, Southern hybridization, and expression of the reporter gene. In spite of having the scope of the generation of siblings, the resulted transformation frequency was found to be 6.66% irrespective of genotypes used in the present study as revealed by dot blot analysis. Overall, the protocol ensured generation of pigeonpea transgenic plants with considerable easy within a short time irrespective of genotypes/cultivars of the crop.