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In vitro cultures have been effectively used as model system to study the production and accumulation of secondary metabolites in W. somnifera. The in vitro system possesses the capacity for higher production and accumulation of metabolites owing to the active growth and higher rate of metabolism in culture within a shorter period. It has been reported that withaferin A and withanone content in W. somnifera was significantly higher in tissues of in vitro raised plants as compared to ï¬eld-grown parent plants (Sivanandhan et al., 2011). A two-fold increase in withaferin A and withanolide A content while ten-fold enhancement in withanone content was observed in regenerated W. somnifera plantlets as compared to field-grown plants (Sharada et al., 2007). Similar to above reports a marked increase in withanolide A production in in vitro shoot cultures was observed as compared to the shoots of field-grown plants (Sabir et al., 2008). In the above background, it appears relevant to utilize in vitro propagation system as an alternative system for production of pharmacologically important withanolides throughout the year, which are otherwise severely limited in production.
Plant growth and production of secondary metabolites in in vitro system is dramatically regulated by media formulations, sucrose concentration and type and concentration of PGRs. Shoot tips of W. somnifera proliferating on B5 medium accumulated maximum withaferin A (0.09 %), while withanolide D accumulation was maximal (0.065%) in MS medium (Ray and Jha, 2001). Among different media tested (MS, B5, NN and Chu’s N6), half strength MS medium was found to be most suitable for both biomass accumulation and withanolide-A production in adventitious root cultures of W. somnifera (Praveen and Murthy, 2010). Further, it was observed that sucrose concentration in media was directly proportional to accumulation of withaferin A and withanolide D. Supplementing the media with 4% sucrose enhances accumulation of both withaferin A and withanolide D without adversely affecting growth (Ray and Jha, 2001).
The hormonal combinations also modulate the biogenesis of withanolides in the in vitro cultures of W. somnifera. Shoot cultures obtained from shoot tips of W. somnifera on MS medium supplemented with BAP (4.44 µM) accumulated both withaferin A (0.04 %) and withanolide D (0.06%) (Ray and Jha, 2001). However, further increase in BAP concentration lead to decline in withanolide content. Further, higher accumulation of withanolide A (0.24%) was reported in shoot cultures of W. somnifera grown on MS medium supplemented with BAP (4.44 µM) and KN (2.32 µM) (Sangwan et al., 2007). Studies also indicate that adventitious roots induced directly from leaf segments on half strength MS medium fortified with 2.46 µM IBA showed higher accumulation of withanolide-A content, almost 21-fold higher as compared in vivo leaves from the mother plant (Praveen and Murthy, 2010). Apart from shoot cultures, secondary metabolite production in callus was also significantly influenced by PGRs. The friable callus derived from shoot tips, nodes and leaf segments of in vitro germinated seedlings of W. somnifera in media containing 2,4-D and KN did not contain withanolide A and withaferin A, whereas solid callus induced in media supplemented with IBA and BAP had both of these withanolides (Chakraborty et al., 2013). The regenerating and non regenerating types of calli did not differ with respect to their chemical content suggesting, that the process of regeneration does not inhibit synthesis of secondary metabolites (Chakraborty et al., 2013).
In addition to above factors, supplementing media with casein hydrolysate (500 mg/l) in liquid system favoured withanolide D synthesis (0.10%) whereas addition of coconut milk 10% (v/v) favoured biomass accumulation (27 fold) and enhanced withaferin A synthesis (0.136%) (Ray and Jha, 2001). The shoot cultures of W. somnifera from 2 g/l FW of shoot inoculum cultured on liquid MS medium supplemented with 2.6 µM BAP and 20 mg/l spermidine showed increased amount of withanolides: 6-fold (withanolide A), 7.6-fold (withanolide B), 1.12-fold (withaferin A) (Sivanandhan, 2012a). Enhanced production of secondary metabolites could be attributed to the fact that polyamines along with PGRs mimic biosynthesis of secondary metabolites through activation of speciï¬c genes in shoot cultures (Sivanandhan et al., 2011).
Exogenous addition of elicitors of biotic and abiotic origin in culture is considered to be one of the most promising strategies for enhancing the production of secondary metabolites. Among the various abiotic elicitors, Methyl jasmonate (MeJ) and Salicylic acid (SA) have been commonly used as effective elicitors for the induction of secondary metabolites (Sivanandhan et al., 2012a). Multiple shoot cultures of W. somnifera grown in liquid medium and elicited with MeJ (100 µM) and SA (100 µM) for 4 hrs led to enhanced production of withanolide A (14 fold and 16 fold), withanolide B (11 fold and 13 fold), withaferin A (13 fold and 15 fold) and withanone (12 fold and 14 fold) respectively, when compared to control (Sivanandhan, 2012a). Comparison of MeJ and SA shows that elicitation by SA was signiï¬cantly higher than that by MeJ. Adventitious root cultures derived from leaf also resulted in the enhanced production of withanolide A (48 fold), withanolide B (29 fold), withaferin A (20 fold), withanone (37 fold), 12-deoxy withastramonolide (9 fold), withanoside V (7 fold), and withanoside IV (9 fold) when treated with 150 µM of SA for 4 hrs (Sivanandhan et al., 2012b).
5.2. Cell suspension culture
In medicinal plants, cell suspension cultures represent the best system for producing secondary metabolites (Mulabagal and Tsay, 2004). In W. somnifera, plant cell suspension culture holds promise, as it could be successfully used to enhance the commercial prospects for the production of withanolides as well as to study the cellular and molecular processes leading to the secondary metabolites accumulation. Normally, a relatively low yield of withanolides particularly, withanolide A and withaferin A is obtained in cell suspension cultures of W. somnifera (Ciddi, 2006; Baldi et al., 2008; Nagella and Murthy, 2011). To enhance withanolide production in cell suspension cultures, various factors such as media formulations, carbon source, PGRs, macro and micro nutrient compositions and inoculum mass are being explored (Nagella and Murthy, 2011; Sivanandhan et al., 2013b). Amongst different media, MS media was found to be most suitable for both biomass accumulation and withanolide A production (Nagella and Murthy, 2011). Another factor which has a profound effect on the withanolide production is the type and concentration of carbon source. Sucrose was found to be the best carbon source for the biomass accumulation as well as for the withanolide A production in comparison to glucose, fructose and maltose. The possible explanation for this might be that sucrose upon hydrolysis produces glucose, which is readily utilized for cell growth (Sivanandhan et al., 2013b). Several studies suggest that sucrose concentration between 2-4% was best suited for both biomass accumulation and withanolides production (Nagella and Murthy, 2011; Sivanandhan et al., 2013b).
Enhancement in withanolide content has also been achieved by manipulating medium salt concentrations. The ratio of ammonia/nitrate ions and overall levels of total nitrogen markedly affected the production of secondary metabolites. It was observed that higher NO3 and lower NH4 concentration favours both cell growth and withanolide production (Nagella and Murthy, 2011). In the cell suspension cultures of W. somnifera, the production of withanolide A was found to be maximum in MS medium containing 2.0X KNO3. Further, highest withanolide A content was recorded in the cell suspension cultures when ration of NH4/NO3 ions was set at 14.38/37.60 mM. Two fold increase in strength of CaCl2 concentration also resulted in enhanced withanolide A production (Nagella and Murthy, 2011).
The type and concentration of PGR in the culture medium regulates both growth and accumulation of secondary metabolites in the plant cell suspension cultures (Mantell and Smith, 1983). In W. somnifera, MS medium supplemented with 2, 4-D (9.05 µM) and KN (2.32 µM) showed highest biomass accumulation and withanolide A production in cell suspension cultures (Nagella and Murthy, 2011). However, a similar study revealed that picloram at a concentration of 4.14 µM also showed maximum biomass accumulation and withanolides production (Sivanandhan et al., 2013b). Besides the above mentioned parameters, agitation speed also has direct bearing on the biomass accumulation and production of withanolides in the cell suspension cultures. Of the different agitation speeds (80 to 160 rpm) tried, the highest biomass accumulation and withanolide production was achieved at 120 rpm (Sivanandhan et al., 2013b). Thus, optimization of the above mentioned factors is essential for large-scale culturing of cell suspension for production of important secondary metabolites from W. somnifera.
Elicitation has been another important strategy to enhance the production and accumulation of pharmaceutically important compounds in cell suspension cultures. Ions (calcium chloride, copper sulfate), inorganic compounds (arachidonic acid, MeJ, SA), biotic factors such as plant cell wall components and other components from a microbiological origin (culture filtrates of Alternia alternata, Fusarium solani, and Verticilium dahilae) act as potent elicitors (Sivanadhan et al., 2013b). In cell suspension culture of W. somnifera, maximum production of withaferin A was observed when treated with 100 µM of CuSO4 and 5% (v/v) V. dahilae cell extract (Baldi et al., 2008). This kind of media manipulation strategies could be applied for the development of cell culture-based bioprocesses for large-scale production of commercially important phytochemicals in W. somnifera.
The interconversion of compounds through biotransformation using cell suspension cultures is a promising way for obtaining important phytomolecules of interest. The possibility of biotransformation by interconversion of withanolides could provide important clues on the biogenetic route for the synthesis of important withanolides. The cell suspension cultures of W. somnifera obtained from leaf explant were used for the biotransformation of withanolides to different types of known withanolides and some new compounds (Sabir et al., 2011). Withaferin A, withanolide A, and withanone were fed to the synchronously growing cell suspension cultures of W. somnifera and it was observed that the interconversion of withanolide A to withanone was most significant whereas the interconversion in the opposite route occurs at low levels. This biotransformation mechanism probably involves substitution of hydroxyl group at C-20 position in withanolide A to C-17 in withanone (Sabir et al., 2011). Identification of genes involved in position-speciï¬c hydroxylation (cytochrome P450) would further enhance our understanding on biotransformation of withanolides.