Abstract
In vitro morphogenic response of mature seed embryo-derived callus cultures of Chonemorpha fragrans was studied using solid and liquid Murashige and Skoog medium amended with cytokinins or their combinations with naphthalene acetic acid at 0.5 mg L-1. The plant growth regulators (PGRs) combination and concentrations tested could not stimulate organogenesis after three subcultivations of the callus cultures on the same PGRs-amended solid medium and when cultivated in the liquid but, formation of morphogenic callus was observed. Evaluation of biomass and camptothecin production showed that the PGRs influenced biomass and CPT yield of the callus cultures. The alkaloid yield of various explants of 3-4 weeks old axenic seedlings was higher in roots (0.019% CPT) followed by mature seed embryos (0.0053%), cotyledons (0.0039%), hypocotyls (0.0024%) and leaves (0.0017%), and no significant difference was observed in yield of CPT from callus induced from the various explants. Camptothecin yield of morphogenic callus cultures cultivated in liquid medium was lower than that of solid due to extracellular leaching effect of the alkaloid. Amount of synthesized CPT in the callus cultures also varied with PGR type and concentration amended in the cultivation medium, and was association with biomass production. Results of the present study suggest that callus cultures offer alternative tissue source for in vitro CPT yield enhancement through biotechnological approaches, with application in the large-scale production of the alkaloid to conserve the ever-decimated natural population of the medicinal woody climber for CPT.
Highlights
Chonemorpha fragrans is an evergreen laticiferous and endangered woody perennial vine of the family Apocynaceae endemic in its distributions (Khan et al, 2005)
Callus cultures derived from the zygotic embryo explants were cultivated on MS medium supplemented with cytokinins or their combinations with naphthalene acetic acid (NAA) at lower levels; for the morphogenic effects of BAP, TDZ or Kin on the callus cultures, callus was cultivated on MS medium supplemented with the various concentrations or when in combination with NAA (0.5 mg L-1)
In the present study, when green non-organogenic callus cultures were cultivated in liquid medium (Figure 1 y-z, 1) with a weekly replenishment of medium nutrients, in vitro morphogenesis was not stimulated after four weeks and subsequent sub-cultivations of the callus cultures, suggesting that C. fragrans is recalcitrant to indirect organogenesis at least from seed embryo-derived callus cultures under the experimental conditions of the present study
Summary
Chonemorpha fragrans is an evergreen laticiferous and endangered woody perennial vine of the family Apocynaceae endemic in its distributions (Khan et al, 2005). Due to slow growth of this woody climber that needs several years to reach maturity coupled with rare distribution, application of plant cell, tissue and organ culture (PCTOC) techniques offer alternative strategies for the production of CPT and other phytochemicals isolated from the species (Banerji and Chatterji, 1973; Rastogi and Mehrotra, 1993; Kedari and Malpathak, 2014). Extensive use of C. fragrans in traditional systems of medicine coupled with many phytochemicals it produces calls for alternative strategies for its rapid clonal multiplication to meet demand of CPT raw material supply for anticancer drugs production by the pharmaceutical industry. Application of PCTOC techniques, influence of plant growth regulators (PGRs) in production of the alkaloid is yet unexplored in C. fragrans
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