Role of reduced nitrogen for induction of embryogenic callus induction and regeneration of plantlets in Abelmoschus esculentus L.

Abstract

Okra (Abelmoschus esculentus L.) is a difficult species to regenerate in vitro because of its recalcitrant nature. Whitish colored ‘non-embryogenic’ callus formation is a major problem in okra tissue culture. In this study, different levels of inorganic–nitrogen (KNO3 and NH4NO3 with 1:2 ratios, 30–60 mM) in MS media with different PGRs combinations were tested to obtain friable and embryogenic callus masses. Hypocotyls produced the highest callus induction (71%) on media containing 1.0 mgL−1 BAP + 1.5 mgL−1 2, 4-D, whereas cotyledons induced (58%) calli on media containing 1.5 mgL−1 NAA + 0.5mgL−1 BAP. Regarding shooting frequency, media containing 40 mM of total nitrogen and supplemented with 1.5 mgL−1 KIN + 0.5 mgL−1 IBA produced maximum shooting (66.5%) with 3.5 shoots per calli from hypocotyl derived calli, whereas, media having 30 mM of total nitrogen supplemented with 1.5 mgL−1 BAP + 0.5 mgL−1 NAA produced (49.50%) shooting with 2.95 shoots per calli from cotyledons derived calli. In contrasts, calli cultured on media containing 60 mM total nitrogen exhibited very low shooting frequency. Activated charcoal (AC) in rooting media promoted root formation with higher survival rates. Healthy and strong roots (79%) were obtained on media containing 2.0 mgL−1 IBA and 100 mgL−1 AC. Rooted plantlets were successfully acclimatized and grew normally. Hypocotyls have more potential for callus induction than cotyledon explants. The results demonstrated that using a modified MS media with reduced nitrogen reprogrammed the compact non-friable embryogenic callus to friable embryogenic callus with high shoot induction frequency. This study provides a contribution towards the development of efficient regeneration and genetic transformation in okra.