Callus culture and in vitro biosynthesis of echitamine from Alstonia scholaris (L.) R. Br.

Alstonia scholaris (L.) R. Br., belonging to the family Apocynaceae, is a rich source of indole alkaloids among which echitamine is the most important. It is reported as an antiproliferative agent and targeted for cancer chemotherapy. However, echitamine and its derivatives are mainly concentrated in stem bark and root of the plant and bulk collection of these parts from nature is not recommended. The present study is the first attempt to standardize the induction and proliferation of callus from leaf explants of A. scholaris along with in vitro biosynthesis of echitamine. Quantitative estimation of echitamine was performed by using ultra performance liquid chromatography mass spectrometry. The medium used for callus induction and proliferation was Murashige and Skoog which was optimized with various combinations and concentrations of different auxins and cytokinins. Best induction and proliferation of callus was noted in 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-furfurylaminopurine (FAP) combination with their specific concentration at 0.5:0.5 mg L-1. Furthermore, the data indicated that both auxin/cytokinin ratio as well as their independent concentration was important for the same. Echitamine biosynthesis was observed in 0.5:0.5 and 0.5:0.3 mg L-1 of 2,4-D and FAP under 16:8 h light-dark cycle. However, production of echitamine was increased more than twofold in 0.5:0.3 mg L-1 of 2,4-D and FAP containing medium upon application of yeast extract at 150 mg L-1 with 5 days incubation period. Thus, in vitro biosynthesis may offer an alternative source of echitamine without harming natural plant population.