Secondary Metabolites of Medicinal Plants. Bharat Singh

et al. 2006, 2008). In vitro cultured shoots on semisolid produced maximum fresh weight of calli and asiatic acid, whereas leaf callus produced a maximum amount of asiatic acid (Gandi and Giri 2013). Among tested elicitors like yeast extract, CdCl₂, CuCl₂, and methyl jasmonate for enhancing the production of asiaticoside in cell cultures of C. asiatica, the methyl jasmonate and yeast extract stimulated the maximum accumulation of asiaticoside (1.5-fold). It was also reported that methyl jasmonate not only induced the production of asiaticoside but also played an important role in senescence of C. asiatica (Kim et al. 2004). The presence of asiaticoside in callus and regenerated roots of C. asiatica was detected by thin layer chromatography as well as by high-performance liquid chromatography (Inamdar et al. 1996), and the accumulation of a significant quantity of asiaticoside was demonstrated by spectrophotometric analysis (Mercy et al. 2012; Nath and Buragohain 2005). The maximum callus induction frequency was obtained in MS medium containing 2,4-D, while the combination of 2,4-D and kinetin gave the highest biomass yield. The quercetin, kaempferol, luteolin, and rutin in callus was estimated and found luteolin as a major compound. This is the first report on the use of growth hormones on the establishment of cell suspension cultures in flavonoid production of C. asiatica (Tan 2010). In the first sub-cultured callus, the accumulation of asiaticoside was highest among other subcultures, but this accumulation of asiaticoside was lower than that obtained from whole plant material (Sholapur and Dasankoppa 2011).

      The production of asiaticoside in hairy roots of C. asiatica was enhanced by feeding of chitosan and its derivatives (chito-oligosaccharide, carboxymethyl chitosan) with different concentrations. It has been reported that chito-oligosaccharide induced the maximum fresh weight of hairy roots as well as asiaticoside concentration (Zahanis et al. 2016), but in the presence of sucrose (60 g/dm³), the transformed hairy roots were unable to increase the accumulation of asiaticoside and madecassoside (Aziz et al. 2007). It had been reported that the production of asiaticoside enhanced by feeding of methyl jasmonate to hairy root cultures of C. asiatica (Kim et al. 2007). Cell cultures were initiated with the general MS medium supplemented by growth hormone IAA and BAP. Pectin as elicitor had an effect on asiaticoside production. As per the mechanism of genetic transformation, the bacterial DNA is integrated into plant DNA that may not only induce the hairy root formation but also affect the production of secondary metabolites (Ruslan et al. 2012).

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