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.