Physicochemical factors modulate regeneration and Agrobacterium-mediated genetic transformation of recalcitrant indica rice cultivars - ASD16 and IR64

Abstract

Abstract A comprehensive study of various components of rice tissue culture media and physical parameters was carried out to characterize the factors affecting in vitro plantlet regeneration and subsequent transformation from two recalcitrant indica rice cultivars, ASD16 and IR64. The choice and concentrations of carbon sources, light/dark incubation, gelling agents, amino acid supplementation and hormone combinations in the regeneration medium greatly influenced the regeneration potential. Among four different carbon sources tested maltose (3%) was identified as the best for callus and shoot induction. Among different formulations of hormones tested, both cultivars recorded the highest regeneration frequencies when the medium was supplemented with 3.0 mg/L BAP+1.0 mg/L NAA. Partially desiccating the calli for 48 h resulted in a higher frequency of regeneration and among different gelling agents, gelrite induced maximum callus induction while agarose was found to be a better choice in regeneration. Callus induction and embryogenic calli initiation were higher when the calli were incubated in the dark, whereas high shoot numbers and regeneration efficiency in both cultivars were enhanced when incubated in the light. The optimized conditions yielded higher regeneration frequencies during transformation experiments and the transformants were analyzed by PCR, Southern blotting and GUS histochemical staining for asserting successful transformation. The results indicated that manipulation of media supplements and partial desiccation reported here used either individually or in combination can greatly enhance plantlet regeneration which is essential for an efficient genetic transformation system in recalcitrant indica rice cultivars.