Callus and biomass culture of Catharanthus roseus L. were established to check for the presence of total alkaloid and its subsequent yield. Various treatments like strength of nutrient salts, sucrose concentrations and combinations of plant growth regulators (PGR’s) were applied to both MS and B5 in agar as well as suspension medium to test their effects on enhanced alkaloid content and its yield. There was no significant difference in any of the observable parameters of fresh wt, dry wt, alkaloid content, production, productivity and yield if the culture were treated similarly in both types of media formulations (MS or B5 salts). Physical state (agar solidified or the liquid suspension) of the medium had significant effect on all the parameters particular on fresh wt, alkaloid content and yields. Although, the fresh wt. and dry wt. of biomass in suspension culture was 2-3 times less than that of callus obtained from agar medium. However, the alkaloid content and yield was 2-3 times higher in suspension culture compared to agar medium in similar treatments. The highest alkaloid content observed was 5.67mg/g dwt in B5 suspension medium containing 3% sucrose and modified with 0.5mg/l 2,4-Dichlorophenoxy acetic acid (2,4-D) + 1 mg/l Kinetin (KIN) + 2mg/l α- naphthalene acetic acid (NAA). The combined effects of these factors on the enhanced production of total alkaloids were expected to contain higher yield of anticancer vinblastine and vincristine in the cell suspension culture system.

Several elevated levels of nitrogen and phosphate at varying pH of the medium which impart a major influence on callus and biomass development and subsequent production of alkaloids was investigated using suspension culture system of Catharanthus roseus in the present study. The B5 medium was buffered at pH 4.51, 5.82 and 7.32 by addition of different levels of (A) diammonium hydrogen phosphate (NH4)2HPO4 and (B) ammonium dihydrogen orthophosphate (NH4H2PO4) representing the enhanced and varied supply of total nitrogen (NH4 + +NO3 ˉ) and phosphate compared to MS medium (as control) for cell biomass production and alkaloid yield. The pH of the medium have shown significant effects with maximum biomass fresh wt., dry wt. and total alkaloid yield at 5.82 medium pH with elevated phosphate levels and total nitrogen concentration of 3710.10 mg/L compared to control MS medium with 2850 mg/L total nitrogen. At 3667.33 and 3752.48 mg/L of total nitrogen with enhanced phosphate supply showed reduced biomass fresh wt., dry wt. and total alkaloid yield at lower (4.51) and higher (7.32) medium pH respectively. Inclusion of 200 mg/L of tryptophan or phenylalanine as reduced nitrogen source in B5 medium buffered at 5.82 ± 0.2 pH showed enhanced biomass and alkaloid production. Hence, addition of nitrogen, phosphate, tryptophan, phenylalanine as nutrient in suspension culture stimulate their uptake to enhance cell biomass and total alkaloids production but as a function of pH of the medium.

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