Optimization of tissue culture protocol for rapid regeneration of traditional therapeutic rice genotype ‘Kavuni’

Abstract

Kavuni, one of the traditional rice genotypes possessing blackish purple grain is known for its nutritional and therapeutic properties. Genetic improvement using biotechnological tools and generating variability through genome editing necessitates development of a rapid, reproducible and efficient regeneration protocol. Callus induction from immature embryo explants of Kavuni was found at its maximum in NB media containing 2 mg l-1 2,4-D, 1 mg l-1 NAA, 1 mg l-1 6-BA, 2% sucrose, 1% glucose. After 3 weeks, embryogenic calli were transferred to pre-regeneration media (NB-PR) containing 2 mg l-1 2,4-D, 1 mg l-1 NAA, 1 mg l-1 6-BA, 3% maltose and 0.3 g l-1 glutamine which increased its regeneration frequency. Embryogenic calli failed to regenerate in a commonly used regeneration media (RNM) containing1 mg l-1 NAA, 3 mg l-1 6-BA, 3% maltose and 0.3 g l-1 glutamine. Out of several combinations of regeneration media tried, regeneration was found to be maximum (80%) in the media combination # IV containing 3 mg l-16-BA, 0.5 mg l-1 kinetin, 3% maltose and 0.3 g l-1 glutamine which produced an average of 12 shoots per calli. Regenerated shoots were transferred to half MS basal media for rooting and finally hardened under greenhouse conditions.