Evaluation and Production of Bacosides from Selected Clones of Bacopa Monnieri (L.) Wettst.

Abstract

Plant derived natural products represent some of the most important pharmaceuticals available today. However, uncertainty regarding the commercial supply due to the limited availability of many plants in nature has resulted in a dramatic reduction in the use of natural products. Plant cell suspension culture capable of large scale industrial production of such pharmaceutically important molecules is the alternative which promises sustained and assured supply of these important molecules. Hairy roots induced following infection with Agrobacterium rhizogenes are capable of unlimited growth in culture in the absence of plant growth regulator and exhibit higher potential for the production of secondary metabolite production. The objectives of the present study were to select elite clones of B. monnieri, investigate the production of ‘bacoside A’ using cell suspension cultures and hairy root cultures and finally enhancing the production of ‘bacoside A’ of cell suspension cultures and hairy root cultures. Fourteen accessions of B. monnieri (BM1- BM14) collected from different locations across India were maintained in nursery at Thapar University. Variation in the content of ‘bacoside A’ and biomass per plant in fourteen accessions of B. monnieri were studied during the different seasons of the year. Maximum biomass accumulation and ‘bacoside A’ contents were recorded in the samples processed in summer (June) in all the accessions and minimum biomass and ‘bacoside A’ content was recorded in winter (December). Amongst accessions, BM1 and BM7 recorded higher biomass accumulation, ‘bacoside A’ content, Relative growth rate (RGR) and Harvest Index (HI). These parameters showed minimum values in accession BM14. Molecular diversity was then investigated amongst these accessions using random amplified polymorphic DNA (RAPD) and Inter simple sequence repeats (ISSR). About, 35 % variations were detected in these populations based on combined data of RAPD and ISSR. Clustering based on molecular marker data grouped these accessions into two major groups and placed accession BM14 as an out group. Maximum shoot organogenic potential was observed in accession BM6 and maximum rooting potential was observed in accessions BM1, BM2, BM7, BM10 and BM14. Based on in vitro morphogenetic response, ‘bacoside A’ content and growth, accession BM6 was selected for the studies of cell suspension and hairy root cultures. Cell suspension cultures established on MS medium supplemented with α-Napthaleneacetic acid (NAA) at 5.0 µM and Kinetin (KIN) at 1.15 µM showed maximum cell growth and levels of ‘bacoside A’. Hairy roots induced by strain MTCC 2364 showed higher biomass accumulation and 'bacoside A' content by five-folds. Further, attempts were made to optimize growth and production of ‘bacoside A’ using cell suspension and hairy root cultures for commercial supply. Optimization of conditions for ‘bacoside A’ production was explored using conventional method (one-variable-at-a-time approach) and statistical method (Response surface methodology, RSM). Optimization of medium components using RSM leads to around two-fold increment in biomass accumulation and levels of ‘bacoside A’. The current study uncovered several aspects of the strategies for the conservation of this medicinally important herb and increased the ‘bacoside A’ production using cell suspension and hairy root cultures. These have the potential of up-scaling in the bioreactors for the production of ‘bacoside A’. Thus can be helpful to reduce the pressure on the wild populations.