Genes involved in biosynthesis of withanolides are Δ14-sterol reductase (EC 1.3.1.70), 1-deoxy-D-xylulose-5-phosphate reducto-isomerase/reductase (DXR; EC 1.1.1.267), 1-deoxy-D-xylulose-5-phosphate synthase (DXS; EC 2.2.1.7), 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MEcPP synthase, IspF, EC 4.6.1.12), 2-C-methyl-D-erythritol 4-phosphate cytidylyl transferase (EC 2.7.7.60), 3-hydroxy-3-methylglutaryl-coenzymeA reductase (HMGR; EC 1.1.1.34), 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (EC 2.7.1.148), 4-hydroxy-3-methylbut-2-enyldiphosphate reductase (EC 1.17.1.2), 4-hydroxy-3-methylbut-2-enyldiphosphate synthase (HMB-PPS, IspG, EC 1.17.7.1), acetyl-CoA acetyltransferase (ACT, EC 2.3.1.9), C-5-sterol desaturase (C5SD, EC 1.14.19.20), cycloartenol C-24 methyltransferase (EC 2.1.1.142), cycloartenol synthase (CAS; EC 5.4.99.8), cycloeucalenol cycloisomerase (EC 5.5.1.9), cytochrome-P450s reductase (CPR, EC 1.6.2.4), farnesyl diphosphate synthase (FPPS, EC 2.5.1.10), geranyl diphosphate synthase (GPPS, EC 2.5.1.1), geranyl-geranyl diphosphate synthase (GGPPS, EC 2.5.1.29), glycosyltransferases (GT, EC 2.4.-), hydroxymethyl glutaryl-CoA synthase (HMGS, EC 2.3.3.10), isopentenyl diphosphate isomerase (IPPI, EC 5.3.3.2), methyltransferase (MT, EC 2.1.1.), mevalonate diphosphosphate decarboxylase (EC 4.1.1.33), mevalonate kinase (MVAK, EC 2.7.1.36), obtusifoliol 14-demethylase (EC 1.14.13.70), phosphomevalonate kinase (EC 2.7.4.2), squalene synthase (SQS, EC 2.5.1.21), squalene monooxygenase/epoxidase (SQE, 1.14.14.17), sterol Δ7 reductase (DWF, EC 1.3.1.21), etc. (Senthil et al., 2010, 2015; Gupta et al., 2013b, 2015).

To understand the interactions of various molecular network in entirety, Dhar et al. (2015) and Singh et al. (2015b) summarized the available information of some in vitro studies with respect to regulation of pathway genes required for withanolide accumulation.

Among a number of enzymes, SQS and SQE are considered as an important enzymes in the biosynthesis of triterpenoids. Considering this, Gupta et al. (2012) characterize isoforms of SQS gene, while, Razdan et al. (2013) perform characterization and promoter analysis of SQE gene from W. somnifera. To confirm the functional activity, both studies also involved the cloning, expression and purification of gens/enzymes in E. coli. Genes encoding DXS, DXR and HMGR enzymes expressed their importance by catalyzing the key regulatory step of the isoprenoid biosynthesis. These genes revealed tissue specific, chemotype specific and modulated expression while exposed to SA, MeJA, as well as MI (Akhtar et al., 2013; Gupta et al., 2013c).

Few members of sterol-GT (SGT) gene family of W. somnifera, have been recognized and characterized (Sharma et al., 2007; Madina et al., 2007a,b; Chaturvedi et al., 2012). SGTs are responsible for diversified glycosylation of sterols (including withanolides). The identified SGTs expressed different level of expression in different tissues as well as under different stress conditions, to proove their physiological importance (Sharma et al., 2007; Chaturvedi et al., 2011, 2012). Purified SGTs showed broad substrate specificity for sugar acceptor but not for the sugar donor (Madina et al., 2007a,b). Similar pattern of glycosylation was observed by Singh et al. (2013) during functional characterization of flavonoid-GT gene from W. somnifera.

Relation among few pathway genes, withanolides accumulation with morphogenic transition has been studied by Sabir et al. (2013). In vitro tissues belongs to different stages of organogenesis (rhizogenesis and shoot organogenesis) were used for the experiment. Accumulation of major withanolides and expression of HMGR, FPP synthase (FPPS), SQS, SQE, cycloaretenol synthase (CAS), GTs were analyzed at different morphogenic transition states.

Detailed study on four-CYP450 has been performed by Srivastava et al. (2015) to illustrate involvement of these enzyme in some specialized secondary metabolite (withanolides). The expression profiles of these CYPs showed chemotype-specific and tissue-specific variation, as well as variation in response to physiological and developmental factors. To expand the understanding of expression of genes in relation to withanolide biosynthetic pathway, Pal et al. (2016) perform experiments with different concentrations of fertilizers on fresh twigs of W. somnifera. Treated twigs related to highest accumulation of withaferin-A has been selected to analyse expression pattern of CYPs, allene oxide cyclases (AOCs) and few other pathway related genes.

Numerous, campylotropous, whitish, disk shaped seeds are found inside red or orange colored fruit (berry) of W. somnifera. Earlier reports mentioned high dormancy with poor seed viability (Khanna et al., 2013; Viji et al., 2013), also seeds of W. somnifera showed low and erratic germination with heterogeneous seedlings (Vashistha et al., 2010) having higher mortality rate of seedlings under field conditions (Khanna et al., 2013). The problems with seed germination of W. somnifera (in vitro and in field) guided the researchers toward finding of simple techniques with optimized conditions, in order to get faster and more germination rate. These conditions include nutrient medium, light conditions and condition of seeds, etc. These findings will help nursery workers and poor farmers interested in developing mass planting stock.

Soaking of seeds in water, diluted sodium hypochlorite, nitrate solutions (of potassium, ammonium, cobalt, sodium, calcium and zinc), has been suggested to soften the hard seed coat of W. somnifera (Kattimani and Reddy, 2001; Vashistha et al., 2010). Improved germination has been observed at 25 ± 2°C and 16-h-light/8-h-dark photoperiod with the light intensity of 3,000 lux (Kambizi et al., 2006; Khanna et al., 2013; Viji et al., 2013). In addition to these conditions, incision on seed coat and few pre-incubation conditions (dark or 15°C) increases the germination percentage (Pandey et al., 2013; Viji et al., 2013; Kumar et al., 2016).

Seedlings, embryos, cotyledon, epicotyl, hypocotyl, petiole, leaves, nodes, internodes, stem, shoot tips and roots have been used in different experiments for callus induction, adventitious root induction, regeneration, differentiation, flower induction, and fruit setting (Sharada et al., 2008; Supe et al., 2011; Singh et al., 2017). Composition of gelling matrix was standardized for encapsulation of shoot tips of W. somnifera along with optimization of media composition (or soilrite) for conversion of encapsulated shoot tips into plantlets (Singh et al., 2006). Most studies with optimized in vitro tissue culture conditions of W. somnifera have been briefly summarized recently by Singh et al. (2017).

Hairy root cultures are a promising approach of bioprocess engineering for large scale production of valuable plant secondary metabolites. There are several reports available in order to modulate quantity of withanolides in hairy roots culture using A. rhizogenes mediated transformation (Pawar and Maheshwari, 2004; Bandyopadhyay et al., 2007; Murthy et al., 2008; Saravanakumar et al., 2012; Sivanandhan et al., 2013a, 2015b). It has been reported that different factors like carbohydrates (Doma et al., 2012), inorganic supplements (Praveen and Murthy, 2013), seaweed extracts (Gracilaria edulis and Sargassum wightii; Sivanandhan et al., 2015b), hormones, elicitation (like, chitosan, JA, SA; Chaudhuri et al., 2009; Doma et al., 2012; Sivanandhan et al., 2013a), etc. modulate biogeneration of withanolides in hairy root cultures.

Difference in hairy root emergence was observed illustrating resistance or susceptibility of W. somnifera toward different strains of A. rhizogenes (Pawar and Maheshwari, 2004; Bandyopadhyay et al., 2007; Saravanakumar et al., 2012) as well as transformation efficiency of different explants used for the experiment (Murthy et al., 2008; Saravanakumar et al., 2012). Leaves proved to be more appropriate for infection by different strains of A. rhizogene, since used as explant in various studies (Ray et al., 1996; Bandyopadhyay et al., 2007; Chaudhuri et al., 2009; Doma et al., 2012; Saravanakumar et al., 2012; Praveen and Murthy, 2013; Sil et al., 2015; Thilip et al., 2015). Recenlty, Pandey et al. (2015) induced hairy root from leaf explants of W. somnifera expressing sterol glucosyltransferase gene (clone-4) using A. rhizogenes. The transgenic hairy roots were observed to accumulate higher amount of withanolide-A when subjected to elicitation (salicylic acid and methyl jasmonate).

Numerous studies have helped in developing efficient methods for regeneration of W. somnifera, while only few reports are available for genetic transformation for this medicinal plant (Singh et al., 2017). Altered expressions of genes related to biosynthetic pathway, ultimately modulate quantity of plant secondary metabolites, which are of therapeutic importance. Ray and Jha (1999) infected leaves of in vitro grown plants (two genotypes) with wild type nopaline and octopine strains of A. tumefaciens. Different types of galls obtained due to different levels of virulence on the two genotypes. Two principle withanolides, withanolide D and withaferin A extracted from shooty teratoma cultures in higher amount, while, withanolide D alone was detected in rooty teratomas.

Pandey et al. (2010) performed successful A. tumefaciens mediated transformation with 1.67 efficiency using non-virulent strain. Leaves excised from 1-5-nodes of both in situ and in vitro grown 30 to 90-day-old seedlings of different accessions of W. somnifera were used for the study. LBA4404 containing the binary vector pIG121Hm showed more gus expression in second and third leaves of 75 day old seedlings. Leaf explants ultrasonicated at 47 KHz ± 6% for 10 s showed higher gus expression as compared to directly infected explants. The protocol was used to analyse in vivo enzymatic action of one SGT (WsSGTL1) of W. somnifera by Saema et al. (2015, 2016). RNAi silencing (Saema et al., 2015) as well as overexpression (Saema et al., 2016) of WsSGTL1 gene has been achieved in transgenic W. somnifera. As expected, reduction in the level of glycosylated products observed in transgenic with silenced WsSGTL1 transcript. However, transgenics with overexpressing WsSGTL1 showed early and enhanced growth, increased production of glycosterols, and glycowithanolides. These transgenics displayed biotic (Spodoptera litura) and abiotic (cold) stress tolerance as well as recovery after cold stress along with improved photosynthetic performance.

Patel et al. (2014) established A. tumefaciens mediated transformation, microprojectile bombardment and microprojectile bombardment assisted agroinfection. Apical and nodal explants obtained from multiplied culture after in vitro seed germination were used as explants. Modified vector pCAMBIA1301 used to confirm transgene expression. Pre-cultured explants were bombarded and immediately infected with A. tumefaciens for microprojectile bombardment assisted agroinfection. The transformation efficiencies achieved were 3.86, 3.62, and 8.71%, through A. tumefaciens mediated, microprojectile bombardment and with the combination of both, respectively.

The protocol (Patel et al., 2014) used to overexpress of WsSQS in W. somnifera (Patel et al., 2015). Grover et al. (2013) also transformed leaves and shoots of 4-6-weeks old seedlings with A. tumefaciens (GV3101 harboring pBI121H) containing SQS from W. somnifera. Transgenics were confirmed with enhance expression of WsSQS transcript and its enzymatic activity. Higher amount of different withanolides observed in transgenics to proove the involvement of SQS with enhanced withanolide biosynthesis.

Nodal explants of 3-month old filed grown plants were used to develop transformation protocol of W. somnifera by Sivanandhan et al. (2015a) with 10% efficiency. These explant were found as an ideal tissue for the production of higher number of multiple shoots, hence adopted for the production transgenics. Explants were precultured (6-days) to obtain maximum transformation efficiency using Agrobacterium suspension (strain LBA4404 harboring pCAMIBA2301) at 0.2 OD₆₀₀. The transformation frequency increased significantly with wounded nodal explants subjected to a sonication (10 s, longer treatment affected the viability of regenerating cells). Maximum transformation efficiency of 10.6% was observed by Mishra et al. (2015) using nodal explants infected with A. tumefaciens strain GV3101 harboring pIG121Hm. Explants were pre-cultured on MS supplemented with TDZ for 2 days and infected with Agrobacterium (0.2 OD₆₀₀) for 20 min and co-cultivated for 48 h at 22°C.

Virus induced gene silencing methods was adopted by several researchers to achive fast and efficient characterization of genes related to withanolide biosynthesis. Using this technology, successful silencing of SQS (Singh et al., 2015a), WsDWF-5 (Gupta et al., 2015) and three-WsSGTLs genes (Singh et al., 2016) were achived in W. somnifera. Ws-SQS silenced plants revealed positive and negative affects on expression of upstream asnd downstream pathway genes, which ultimately reduces the accumulation of phytosterols. Silencincing of WsDWF-5 was observed with reduced accumulation of withanolide while, 3-WsSGTLs gene silencing found associated with enhanced level of different withanolides and reduced level of glycowithanolides. Increased expression of other upstream genes of withanolide biosynthesis pathway also relates with the supressed activity of WsSGTLs, which leads to reduced tolerance toward biotic stress.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.