Micropropagation and validation of genetic and biochemical fidelity among regenerants of Nothapodytes nimmoniana (Graham) Mabb. employing ISSR markers and HPLC

An in vitro protocol has been established for clonal propagation of Nothapodytes nimmoniana which is an important source of Camptothecin (CPT). Elite source was identified based on the chemical potency to accumulate the optimum level of CPT. Different types and concentrations of plant growth regulators were used to study their effect on inducing multiple shoots from the explants regenerated from embryos of N. nimmoniana. Of these, a combination of N6-benzyladenine (0.2 mg L-1) and Indole-3-butyric acid (IBA) (0.1 mg L-1) proved optimum for differentiating multiple shoots in 90.6 % of the cultures with an average of 10.24 shoots per explant obtained within 8 weeks of inoculation. Nearly, 92 % of the excised in vitro shoots rooted on half strength Murashige and Skoog (MS) medium containing 0.05 % activated charcoal, supplemented with 1-naphthaleneacetic acid and IBA at 0.1 mg L-1 each. The micropropagated plants were evaluated for their genetic fidelity by employing inter simple sequence repeats (ISSR) markers. Ten individuals, randomly chosen from a population of 145 regenerants, were compared with the donor plant. The regenerated plants were also evaluated for their chemical potency using high-performance liquid chromatography (HPLC) analysis of CPT content. The true-to-type nature of the micropropagated plants was confirmed based on their monomorphic banding profiles with that of the mother plants using ISSR markers. Besides, HPLC evaluation of the CPT content confirmed the existence of chemical uniformity among the regenerated plants and the elite mother plant.