Further enrichment of targeted indole alkaloids through root-derived culture systems from Alstonia scholaris (L.) R.Br.

In our earlier experiment, we could successfully enrich the four bioactive indole alkaloids (IAs) to a certain limit, i.e. similar to 12-fold of echitamine and similar to 15-fold of acetylechitamine as well as similar to 4-fold of both picrinine and tubotaiwine in our lab through leaf-derived callus culture from wild Alstonia scholaris (L.) R.Br. The present study is an effort to achieve further enrichment of these targeted IAs through in vitro culture of normal roots from micropropagated plantlets and different systems like root-derived callus and suspension cultures. For this purpose, micropropagation protocols for this plant and the three culture systems were first standardized and established. Effect of various elicitors like yeast extract, chitosan, methyl jasmonate, salicylic acid, KCl, NaCl, mannitol, polyethylene glycol, CuSO4, MnSO4, NiSO4, CdSO4, and Pb(NO3)(2) with different concentrations and incubation periods were then evaluated to know their effect on the IA accumulation in these cultures. Tissues were extracted post-elicitation and analyzed through LC-MS for the identification and quantification of IAs. Multivariate statistical models like principal component analysis (PCA) and hierarchical clustering analysis (HCA) were used to analyze the data. Heatmap analysis revealed that a 2-d incubation period of 400 mM mannitol in the suspension culture had significant effect on the accumulation of acetylechitamine and tubotaiwine. The same culture with 100 mu M methyl jasmonate in 2-d period maximally favoured the accumulation of echitamine. The metabolite picrinine could be elicited to a substantial limit in root callus with a 5-d incubation period of 150 mu M salicylic acid. The fold enrichment of echitamine, acetylechitamine, picrinine, and tubotaiwine with respect to their own control in these culture systems averaged approximately 52.7-, 32-, 291-, and 16.8-fold, respectively.