In vitro regulation of rotenoid biosynthesis from lablab purpureus

Introduction Lablab purpureus L. , belonging to family Fabaceae is a perennial herb alleged to exhibit various pharmacological activities such as antiviral, hypoglycemic, alpha amylase and others (Chau et al, 1967; Kabi et al, 1979; Baslas and Agha, 1986). Rotenoids are group of bioactive ketonic compounds with potent insecticidal activity, are mostly reported from Fabaceous sps. Derris, Lonchocarpus and Tephrosia sps producing rotenoids have been exploited commercially. Although Rotenoids have also been reported from other members of Fabaceous plants (Sharma and Khanna, 1975; Kamal and Jain, 1978; Kamal and Mehra, 1994; Kamal and Mathur, 2007; Kamal and Mathur, 2008) as well. Use of biotechnological applications to enhance the yield the metabolite of interest in culture is the need of the hour. The exogenous supply of a biosynthetic precursor to culture medium has been reported to increase the yield of the desired product. Attempts to induce or increase the production of plant secondary metabolites, by supplying precursor or intermediate compounds, have been effective in many cases (Moreno et al, 1993; Whitmer et al. 1998; Silvestrini et al, 2002). Cell immobilization is another approach, which has led to several commercial processes. Immobilization of microbial and animal cells has been performed for a number of years (Lindsey and Yeoman, 1983). Cells of Derris indica were also immobilized for production of rotenoids (Mathur and Kamal, 2001) and harvesting the bioactive metabolites periodically from nutrient medium and replenishing the fresh medium for sustained cell cultures in immobilized beads. The present study is in continuation of our previous work (Kamal and Mathur, 2010) was undertaken to isolate and identify and evaluate Rotenoids production and regulation in in vitro grown culture of L. purpureus using precursors and cell immobilization. Materials and Methods Tissue culture Unorganized static cultures of L. purpureus were established from its seeds. Seeds were pretreated with 50% sulphuric acid for 5 min, sterilized with 0·1% mercuric chloride, revised and inoculated in MS medium (Murashige and Skoog, 1962). Germination took place in 9–20 days and callus initiated from the radical portion. This callus was maintained for 12 months by periodic subculturings. The callus was harvested at different time intervals of 2, 4, 6, and 8 weeks of subculturings, dried at 60° C and growth index (GI) was calculated, separately. Three replicates of each of the samples were analyzed and mean values were taken. The dried samples were subjected to extraction of Rotenoids. Final wt. of the tissue – Initial wt. of the tissue GI = Initial wt. of the tissue Extraction of Rotenoids The callus harvested at different time intervals were separately dried and finely powdered. The extraction was carried out by percolating plant material in acetonitrile saturated with nhexane for 72 h at 22 o C, filtered and concentrated to dryness in vacuo (Delfel and Tallent, 1969). The concentrated semi-dried extract was dissolved in acetone, and the filtrate was mounted on a column of inert alumina to remove impurities. It was continuously eluted with acetone till the last elutant gave no spot on thin-layer chromatography (TLC). The various eluted fractions were pooled together dried in vacuo and weighed separately. Four sets of experiments were planned to evaluate rotenoid biosynthesis capacity of callus tissue with age and passage of subculturings. Callus tissues were maintained for 3, 6, 9 and 12 months (Set I, II, III, and IV, respectively) by periodic subculturings, separately. In each set, the calli were harvested at the age of 2, 4, 6 and 8 weeks after fresh subculturings. The various samples were dried at 100 C for 20 min to inactivate the enzymes and then at 60 0 C till constant dry weight was achieved. These were then subjected to qualitative and quantitative