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Hybanthus enneaspermus is a rare medicinal plant. We defined a protocol for micropropagation, exvitro rooting of cloned shoots and their acclimatization. Surface-sterilized nodal segments were cultured on Murashige and Skoog (MS) medium with different concentrations of 6-benzylaminopurine (BAP) and kinetin (Kin). Medium supplemented with 1.5 mg L–1 BAP was found optimum for shoot induction from the explants and 6.4±0.69 shoots were regenerated from each node with 97% response. Shoots were further proliferated maximally (228±10.3 shoots per culture bottle with 7.5±0.43 cm length) on MS medium augmented with 1.0 mg L–1 each of BAP and Kin within 4–5 weeks. The shoots were rooted in vitro on half strength MS medium containing 2.0 mg L–1 indole-3 butyric acid (IBA). The cloned shoots were pulse-treated with 300 mg L–1 of IBA and cultured on soilrite® in a greenhouse. About 96% of the IBA-pulsed shoots rooted ex vitro in soilrite®, each shoot producing 12.5±0.54 roots with 5.1±0.62 cm length. The ex vitro rooted plantlets showed a better rate of survival (92%) in a field study than in vitro rooted plantlets (86%). A comparative foliar micromorphological study of H. enneaspermus was conducted to understand the micromorphological changes during plant developmental processes from in vitro to in vivo conditions in terms of variations in stomata, vein structures and spacing, and trichomes. This is the first report on ex vitro rooting in H. enneaspermus and the protocol can be exploited for conservation and large-scale propagation of this rare and medicinally important plant.