An efficient and reproducible protocol has been developed for in vitro propagation and conservation of Hybanthus enneaspermus using stem explants. Stem explant showed high frequency of callus induction potentiality on MS (Murashige and Skoog 1962) medium supplemented with 2.0mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) plus 0.8mg/l 6-Benzylamino purine (BAP) followed by 2.0mg/l α- naphthaleneacetic acid (NAA) plus 0.8 mg/l Kinetin (Kn). The highest frequency of shoot regeneration (88%) and number of shoots per explant (28.2) were obtained on medium supplemented with 2.5mg/l BAP plus0.6mg/l NAA. Rooting was best achieved on half-strength MS medium augmented with 1.5mg/l Indole-3-butyric acid (IBA) and optimum number of 19.7 roots per explants with average 9.3cm root length. The plantlets regenerated in vitro with well developed shoot and roots were successfully established in pots containing garden soil and grown in a greenhouse with 90% survival rate. The regenerated plants did not show any immediate detectable phenotypic variation. The described method can be successfully employed for large-scale multiplication and long term in vitro conservation of H. enneaspermus.
