Influence of Meta-Topolin on in Vitro Propagation and Foliar Micro-Morpho-anatomical Developments of Oxystelma Esculentum (L.f.)sm

Oxystelma esculentum (L. f.) Sm. (Apocynaceae) is a perennial medicinal climber, enormously explored in traditional and modern systems of medicines. Due to over-harvesting from the wild, this species is categorized as ‘rare’. Hence, this study was aimed to develop an in vitro regeneration system for O. esculentum as a conservation measure. Nodal explants were cultured on Murashige and Skoog (MS) medium supplemented with cytokinins [benzyladenine (BA) and meta-Topolin (mT)] for the establishment of cultures. The growth of shoots from the pre-existing meristems of explants was obtained on MS medium containing 4.14 µM mT with the highest mean number (7.25) of shoots. The maximum shoot proliferation frequency (124.8 shoots) was achieved on MS medium containing 2.07 µM mT and 1.42 µM indole-3-acetic acid (IAA). The foliar micro-morpho-anatomical stability of in vitro raised shoots (from BA and mT) was analyzed using light microscopy. Although the in vitro raised leaves possessed microscopic anomalies, the BA-derived leaves showed a higher degree of abnormalities, such as underdeveloped stomata with single guard cells, reduced vein and trichome density, poor deposition of cutin, and less differentiation of ground and vascular tissue systems. Comparatively, mT-derived in vitro leaves had functional stomata, a higher amount of cutin deposition, and remarkable developments in vascular and ground tissue systems. The regenerated shoots were rooted following pulse treating with 976.08 µM indole-3-butyric acid (IBA) under greenhouse (ex vitro) conditions. All the mT raised plantlets (100%) were survived in the field conditions. This study could suggest a highly efficient cytokinin (mT) for large-scale propagation and conservation of O. esculentum, which could ultimately help in the production of the quality shoots in terms of essential structural developments for successful rooting and hardening processes. A reproducible in vitro propagation protocol for Oxystelma esculentum was developed usingmeta-Topolin. The influence of meta-Topolin on micro-morpho-anatomical developments hasbeen proved through the foliar micro-morpho-anatomical analysis for the first time.