Optimization of Alkaloid Production in Catharanthus roseus Callus Culture using Statistically Designed Experiment Following Response Surface Methodology (RSM)

Abstract

The main principle used in medium engineering is creating the optimal operating condition of a parameter by changing one parameter at a time and keeping the others at a constant level which is tedious as well as can also lead to misinterpretation of results, especially when interactive effects between different factors have to be worked out. Response surface methodology (RSM), which comprises of experimental strategies, mathematical methods and statistical inference for constructing and exploring an approximate functional relationship between a response variable and a set of design variables is an ideal methodology to infer facts scientifically. The two step statistical methodologies such as Plackett Burman design (PBD) and Box - Behnken design (BBD) have proved an efficient and effective approach for systematic investigation on the desired experimental factors. The present optimization studies indicated 5- significant factors such as 2,4-Dichlorophenoxy acetic acid, Kinetin and naphthalene acetic acid, sucrose as principal carbon source and pH of the medium following PlackettBurman design (PBD) experiments. The remaining 3-factors temperature, glutamine as reduced nitrogen source and PEG 6000 as water stress were found insignificant among 8-factors subjected for experiments and result analysis. Further optimization process using these 5- significant factors by Box - Behnken design (BBD) experiments were used to develop a second order mathematical model which resulted comparable alkaloid production upon validation. It was concluded that RSM based statistically designed experiment using PBD and BBD models were found to be the most suitable tool for identification, screening, optimization and enhancing the alkaloid production response of significant variables in short period of time with least experimentation runs.