Establishment and standardization of in vitro regeneration protocol in Nothapodytes nimmoniana Graham and evaluation of camptothecine (CPT) in tissue culture plants

Nothapodytes nimmoniana Graham is an important medicinal tree species occurring in the Western Ghats, a mega diversity hotspot in Southern India. The inner stem bark of the tree contains an important anticancer alkaloid, camptothecine (CTP), for which the natural population of the trees is heavily extracted. In this paper, we report the development of a rapid, high frequency regeneration protocol from leaf and nodal explants of N. nimmoniana. Multiple shoot induction was carried out using leaf and nodal explants on Murashige and Skoog (MS) medium supplemented with different concentration/ combination of phyto-hormones. N6-benzyladenine (BA) was the most effective cytokinin for the induction of multiple shoots. The MS medium with 8.87 lM BA yielded the highest number of shoots from leaf and nodal explants, respectively. Further, proliferation and elongation of adventitious buds were observed in secondary medium containing MS supplemented with 4.44 lM BA and 0.87 lM gibberellic acid (GA3). Shoots were rooted on half strength MS medium containing 4.9 lM indole-3-butyric acid. The plantlets were acclimatized in a growth chamber at 25 "C, 60 % relative humidity, with 16/8 h light/dark photoperiod. Regenerated plants were free of any noticeable phenotypic variability and showed a survival rate of 90 %. The in vitro regenerated plants accumulated substantial amount of camptothecine (ranging from 0.08 to 0.2 %). These results suggest the possibility of using in vitro regenerated plants as a possible alternative source of CPT. This is the first report of direct regeneration in N. nimmoniana with significantly high plant regeneration frequency and with high CPT yiel