Rapid plant regeneration, analysis of genetic fidelity and camptothecin content of micropropagated plants of Ophiorrhiza mungos Linn. — a potent anticancer Plant

Abstract

An efficient protocol has been established for rapid multiplication of Ophiorrhiza mungos Linn. (Rubiaceae), a potent anticancer plant. Axillary and terminal buds of these in vitro-raised seedlings formed the primary source of explants for direct organogenesis. Explants were inoculated onto MS medium supplemented with different concentrations and combinations of 6benzylaminopurine (BA) and kinetin (KN). The best morphogenic response was observed on MS media supplemented with 0.25 mg L -1 BA and 0.25 mg L -1 KN, which exhibited the highest regeneration frequency (84%), the maximum number of shoots/explants (63.1 ± 1.35) and shoot length (2.8 ± 1.15) within 4 weeks. Fortification of 1.0 mg L -1 GA3 enhanced the shoot elongation by 2.33 fold in 91% of shoot cluster cultures within 3 weeks. A high percent frequency of rooting (92.13%) was achieved within 15 days of shoot implantation on ½ strength MS media fortified with 100 mg L -1 activated charcoal. The rooted plantlets were successfully acclimatized with 95% survival rate. Randomly amplified polymorphic DNA (RAPD) analysis confirmed that all the regenerated plants were genetically identical to their mother plant, suggesting an absence of detectable genetic variation in the regenerated plantlets. High performance liquid chromatography (HPLC) was done to further confirm the existence of qualitative and quantitative differences in the major secondary metabolite (camptothecin) between the mother plant and in vitro-propagated plants. The present results evidently showed comparable chemical profiles. Thus, the present protocol can be used for clonal mass propagation of true-to-type elite O. mungos plants.