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Plantlet regeneration in Asteracantha longifolia (L.) Nees (Acanthaceae), a medicinal herb has been achieved from seedling explants on basal MS medium. Three different seedling explants including node, internode and leaf segments were used. Of these three explant, leaf explants gave better response for both callus mediated organogenesis and direct multiple shoot induction. Number of explants showing differentiation of shoot buds was higher on MS media supplemented with BA compared to kinetin. MS medium fortified with BA (2.0 mgl-1) and NAA (0.5 mgl-1) was found to be most suitable for both callus mediated organogenesis and elongation of shoots. The elongated shoots were successfully rooted on MS medium fortified with NAA or IBA. Among them 0.1 mgl-1 NAA or 0.2 mgl-1 IBA provides better response for rhizogenesis. Regenerated plantlets were successfully established in soil where 85.4% of them developed into morphologically normal and fertile plants. RAPD profiling using four decamer primers confirmed the genetic uniformity of the regenerated plantlets and substantiated the efficacy and suitability of this protocol for in vitro propagation of A. longifolia.