An efficient direct organogenesis protocol for in vitro clonal propagation of Rubia cordifolia L.

Abstract

This comprehensive study delves into the intricate process of in vitro clonal propagation of R. cordifolia, with a particular focus on optimizing a method of direct organogenesis. The choice of direct organogenesis over indirect organogenesis is driven by the propensity for somaclonal variations associated with the latter. The presented research explores the critical factors influencing direct organogenesis, including the selection of appropriate explants and the optimum use of plant growth regulators. Notably, nodal explants have demonstrated great potential for regeneration, and the choice of cytokinins such as kinetin (KIN) and 2-isopentenyladenine (2-iP) for shoot induction has proven highly effective, with 1.0 mg/L KIN supplementation to MS media emerging as the most favourable choice resulting in 12.14 ± 1.58 shoots and 29.78 ± 1.93 axillary buds. Subsequent adventitious rooting using indole-3 butyric acid (IBA) and indole-3-acetic acid (IAA) resulted in a 100% rooting response. Among the concentrations tested, 1.0 mg/L IBA was found most effective, yielding the best rooting response (22.9 ± 1.06 roots/ micro-shoot). Acclimatization of in vitro cultures was achieved by gradually exposing them to natural conditions, maintaining high humidity and minimizing photorespiration. The morphological transformations observed, including robust leaf development contributed to the high acclimatization success (100% survival). Additionally, the genetic stability was confirmed using DNA-based ISSR markers, with complete monomorphism indicating no variations among the regenerants. The efficient direct organogenesis protocol developed in this study may prove a valuable contribution towards the mass clonal propagation and conservation of R. cordifolia, an industrially and medicinally important plant.