Routine and efficient in vitro regeneration system amenable to biolistic particle delivery in chickpea (Cicer arietinum L.)

Genetic engineering can add new capabilities or traits and direct method using biolistic particle delivery holds key for rapid, routine and efficient transformation of chickpea. Regeneration efficiencies of five different explants derived from BAP pre-treated breeders’ chickpea seeds (cv. DCP 92-2) raised in phytohormones combinations (BAP and KIN for shoot primordia induction; GA3 for shoot elongation and NAA for rooting) were compared. Best response was obtained using the embryonic axis (EAX) explants with 86.69% regeneration efficiency followed by epicotyl (EPI) explants (78.69%). Direct genetic transformation were demonstrated in two responding explants by bombarding with pre-treated tungsten, coated with plant expression cassette (harboring Bt and nptII gene) from a distance of 4 cm with 1100 psi helium pressure. Transgenic chickpea lines with multiple and single copy integrations were obtained with transformation frequency of 0.72% for EPI explants and 1.21% for EAX explants, significantly higher than Agrobacterium tumefaciens mediated transformation of same genotype (0.076%). Southern blot based analyses of seven single copy transgenic chickpea lines exhibited presence and transmission to subsequent generations (T1 and T2). Presence of Bt protein were detected in the leaves of transgenic chickpea lines at pre-flowering (6.63–11.95 ng/mg TSP) and post flowering stages (4.85–8.93 ng/mg TSP). Genetic fidelity analysis using genome wide SSR markers of ten independent transgenic lines indicated true to type with original genotype. Taken together, this study describes a protocol that can be adapted for direct genetic transformation of chickpea with high efficiency.