Secondary Metabolites of Medicinal Plants. Bharat Singh

2.22.2 Culture Conditions

      Callus cultures of Cassia bicapsularis were established on solid MS basal medium supplemented with different growth regulators. Maximum growth of callus was obtained in medium with supplementation of 2,4-D and with dark periods. The formed callus was compact and yellowish brown in color and used for cell suspension culture studies. Maximum biomass of cells was achieved in medium supplemented with 2,4-D and kinetin. Initially the growth rate of cells was slow, but later growth of biomass increased gradually over a period of three weeks and reached maximum at fifth week (Abdel-Rahman et al. 2013). Effects of growth hormones on growth of cell biomass and production of phenolic compounds in C. fistula cells were examined. The production of polyphenols was largely dependent on the concentration of growth hormones in the culture medium. The accumulation of phenolic materials was essentially restricted to the most rapid phase of the growth cycle. The changes in peroxidase activity patterns were followed and their relationship with polyphenol synthesis is established (Shah et al. 1976). It has been reported that higher concentration of sucrose increased the production of polyphenols, but the combination of 2,4-D and kinetin inhibited the production (Bahorun et al. 2005).

Plant cells in suspension cultures often undergo spontaneous genetic variation in terms of accumulation of secondary metabolites, which leads to heterogeneous population of cells in a suspension culture. The genetic basis of somaclonal variation has not been extensively understood; however, it has been observed to be advantageous in crop improvement. Establishment of a high yielding genetically stable cell line would provide a suitable means for the large-scale production of plant metabolites. The regulatory mechanisms of secondary metabolism have not been fully understood. Yields of metabolites will improve with proper understanding of regulatory mechanisms, plant cell differentiation, intracellular organization, and cell physiological characteristics, as these are linked to secondary metabolism (Memelink et al. 2001; Zhao et al. 2016).

      The concentration of sennosides in cell cultures varies as per the type of cells and the composition of the culture medium. The optimized culture medium supports the induction of rapid biosynthesis of sennosides (Srivastava et al. 2006). The production of anthraquinones in the calli of Cassia senna was induced by addition of magnesium acetate, shikimic acid, 2,4-D, and kinetin in the culture medium (Spoke and Abdulahi 1978), but several authors have declined by expressing their views in the correlation of biosynthesis and growth hormones (Godoy-Hernandez and Loyola-Vargas 1991). Higher concentration of saccharose induces the growth of cells and synthesis of polyphenols in the C. fistula cell cultures. The induction of the biosynthesis of polyphenols is affected by the 2,4-D and kinetin, and the accumulation of polyphenols is dependent on the concentration of sugars in the growth medium (Mehta 2012).

      Hairy roots of Senna alata were transformed with Agrobacterium rhizogenes and grown in half-strength MS medium. Hairy roots were cultured on hormone-free half-strength MS medium supplemented with 5% sucrose and when incubated under dark condition mostly stimulated the growth of hairy roots and increased the yield of sennosides A and B (Putaluna et al. 2006; Dave and Ledwani 2012). The L-phenylalanine incorporation in the culture media of 36-month-old calli of C. fistula increased the production of polyphenolic compounds (Neergheen and Bahorun 2002). In this order, the quercetin and emodin were estimated in in vivo leaves and in vitro calli of C. tora (Nandani et al. 2013; Saito et al. 2012).

      Rotenoids are a group of ketonic compounds having chromanone ring structure. The recovery of rotenoids was found to be maximum in the roots and minimum in the stem of C. occidentalis. The isolated compounds were found to effective against Anopheles stephensi larvae with lethal concentration. Precursors were fed to increase the rotenoid production in vitro. Phenylalanine and methionine increased the production