Establishment and characterization of Stevia rebaudiana (Bertoni) cell suspension culture: an in vitro approach for production of stevioside

摘要

A protocol has been standardized for establishment and characterization of cell suspension cultures of Stevia rebaudiana in shake flasks, as a strategy to obtain an in vitro stevioside producing cell line. The effect of growth regulators, inoculum density and various concentrations of macro salts have been analyzed, to optimize the biomass growth. Dynamics of stevioside production has been investigated with culture growth in liquid suspensions. The callus used for this purpose was obtained from leaves of 15-day-old in vitro propagated plantlets, on MS medium fortified with benzyl aminopurine (8.9 mu M) and naphthalene acetic acid (10.7 μM). The optimal conditions for biomass growth in suspension cultures were found to be 10 g l~(-1) of inoculum density on fresh weight basis in full strength MS liquid basal medium of initial pH 5.8, augmented with 2,4-dichlorophenoxy acetic acid (0.27 μM), benzyl aminopurine (0.27 μM) and ascorbic acid (0.06 mu M), 1.0× NH4NO3 (24.7 mM), 3.0× KNO3 (56.4 mM), 3.0× MgSO4 (4.5 mM) and 3.0× KH2PO4 (3.75 mM), in 150 ml Erlenmeyer flask with 50 ml media and incubated in dark at 110 rpm. The growth kinetics of the cell suspension culture has shown a maximum specific cell growth rate of 3.26 day~(-1), doubling timeof 26.35 h and cell viability of 75 %, respectively. Stevioside content in cell suspension was high during exponential growth phase and decreased subsequently at the stationary phase. The results of present study are useful to scale-up process and augment the S. rebaudiana biological research.