acid."/> 2.24.2 Culture Conditions
Due to its versatile medicinal properties, C. asiatica has been increasingly required in pharmaceutical industries, thus leading to the over exploitation of this herb (Zheng and Qin 2007). The callus cultures of H. asiatica was established on MS medium with supplementation of 2,4-D and kinetin for enhancing the production of asiaticoside (Josekutty 1998). The cell growth and asiaticoside accumulation reached maximum peak after 24 days of suspension culture at an agitation speed of 150 r/min and aeration rate of 2.5 l/min in bioreactors (Loc and Nhat 2014). Alkaloid content was increased higher in the callus cultures of C. asiatica developed in MS medium with supplementation of 2,4-D + BAP (Rao et al. 2015). Maximum growth of callus was achieved on days 21 and 28 of inoculation on MS medium with addition of 2,4-D and BAP (Nath and Buragohain 2005). The maximum centelloside production was observed in the stationary growth phase at day 25 of the culture. The methyl jasmonate as elicitor did not change the centelloside pattern, with madecassoside being the main compound, followed by asiaticoside. The maximum production of centellosides was obtained at day 15, with a time lag between gene activation and centelloside biosynthesis (Bonfill et al. 2011). The centelloside profile in the elicited cell suspension cultures was the same as in calli (Mangas et al. 2009). Thus, the main compound was madecassoside followed by asiaticoside, with very low quantities of asiatic acid and madecassic acid (Mangas 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).
References
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8 Bunpo,
