Tissue culture studies and estimation of camptothecin from Ophiorrhiza prostrata D. Don

Abstract

Camptothecin is an important molecule in the synthesis of some of major anticancer agents such as irinotecan and topotecan. Traditional sources of camptothecin are mainly woody plants, some of which are being overexploited to a level that threatens the existence of the respective species. Alternate sources are being identified. In the current study, a tissue culture protocol was developed for the mass multiplication of Ophiorrhiza prostrata, an herbaceous camptothecin-containing plant, using seed explants, in order to evaluate its potential as an alternate source of commercial camptothecin. The germination of seeds was standardized on half-strength solid basal MS medium (94.8 ± 2.65% success rate). Seedlings subcultured on half-strength MS agar medium supplemented with 1.5 mg l−1 benzylaminopurine (BAP) produced the most shoots (28.0 ± 2.58) from the nodal region of a single seedling. Mass multiplication of in vitro shoots was achieved by subculturing a single shoot as well as shoot clumps. Subculturing shoot clumps containing a minimum of 4–6 shoots yielded fewer shoots per node (32.2 ± 2.03) than when single shoots were subcultured (25.0 ± 2.0). Shoot elongation was achieved on half-strength MS basal medium in a period of 2–3 weeks. Various concentrations of auxins were tested for rooting, and 1 mg l−1 NAA gave the highest number of roots (44.0 ± 4.13). Rooted shoots were successfully established under field conditions. High-performance thin-layer chromatography (HPTLC) was employed to measure the camptothecin levels in wild-grown plants (1.46 ± 0.02 µg g−1 dry wt), in-vitro-regenerated plants (1.10 ± 0.02 µg g−1 dry wt), and in-vitro-derived hardened plants (1.83 ± 0.02 µg g−1 dry wt). The results of this study indicate that in vitro methods could be a potential path to the rapid expansion of plant biomass, which could serve as a potential source of commercial camptothecin.