Shoot organogenesis from leaf callus and ISSR assessment for their identification of clonal fidelity in Rhinacanthus nasutus (L.) Kurz., a potent anticancerous ethnomedicinal plant

An efficient plant regeneration protocol via shoot regeneration from callus derived from immature leaf explants was established for Rhinacanthus nasutus (L.) Kurz., a potent ethnomedicinal plant for various ailments including cancer. 5-day old leaf explants were cultured on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BA; 1.0–5.0 mg/l) or Kinetin (Kn; 1.0–5.0 mg/l) in combination with 0.5 mg/l IBA for callus induction. Optimum morphogenic callus induction (98.8%) was observed on MS medium supplemented with 4.0 mg/l Kn and 0.5 mg/l IBA. The excised callus subcultured on MS medium supplemented with BA (1.0–5.0 mg/l) or Kn (1.0–5.0 mg/l) with NAA (0.2 and 0.5 mg/l). The highest frequency of shoot regeneration (96.3%) and shoot number (36.5 shoots per 1.0 gm calli) was observed on MS medium supplemented with Kn (3.0 mg/l) in combination with NAA (0.5 mg/l). Half MS medium supplemented with IAA (2.0 mg/l) produced an optimum rooting response of 96.23% with an average number of 6.1 roots per shoots. Out of 50 rooted shoots transferred to soil 47 survived after acclimatization. Clonal fidelity of the regenerated plants with mother plant was evaluated using 12 inter simple sequence repeats (ISSR) primers and genetic uniformity among all the regenerants and mother plant is confirmed.