Withania somnifera (L.) Dunal is an important Ayurvedic and indigenous medicinal plant in India belonging to the Solanaceae family; the common name is ‘Ashwagandha’ or ‘Indian ginseng’. Due to the presence of a wide range of stimulant and curative properties, it has been used in modern scientific and different clinical studies, including anti-microbial, anti-cancerous, neuro-protective, anti-inflammatory, immunomodulatory, nephroprotective, cardiovascular, anti-diabetic activity etc. (Zahiruddin et al., 2020, Gaurav et al., 2023). Metabolic profiling of this plant obtains approximately 80 unique metabolites, most of which are withanolides, phenolic acids, and alkaloids (Chatterjee et al., 2010, Tousif et al., 2022). The pharmacological properties of this plant have been explored with the C28-steroidal lactone triterpenoids group of naturally occurring withanolides (Mirjalili et al., 2009, Gaurav et al., 2023). In India, the production of Withania dried plant material is estimated at 5905 tonnes from the cultivation field, but the actual demand is 9127 tonnes per year (Sharada et al., 2008, Rangaraju et al., 2018). In general, field growth plants are used to produce commercial withanolides, where environmental factors and genotypes affect secondary metabolite production (Sivanandhan et al., 2012a). Continuous, quality secondary metabolites production from field-grown plants is impossible to produce from all individual plants because all individuals are derived from single seeds and their genetic makeup is not the same. So, the clonal propagation of root organs is an alternative for continuous and same-quality secondary metabolites production. The root of this plant is the primary demand due to the accumulation of numerous active compounds. So transformed roots or hairy roots establishment is considerable as an excellent alternative source to the modern biotechnological system for continuous production of withanolides and phenolic acids under the aseptic conditions in absence of growth regulators in the culture medium (Shilpha et al., 2023).

As an alternative source for the production of secondary metabolites, hairy root cultures (HRC) can provide a continuous supply of secondary metabolites. Hairy roots are produced by the transfer of Ri (root inducing) plasmid containing different transgenes from Gram-negative soil-born bacterium Agrobacterium rhizogenes to plant cells and expression therein (White and Nester, 1980). Productions of withanolides from hairy root culture of W. somnifera have been reported by several authors (Ray et al., 1996, Bandyopadhyay et al., 2007, Murthy et al., 2008, Thilip et al., 2015), but there were no reports to study about phenolic acids from hairy root culture. It is the first time to study simultaneously of withanolides and Phenolic acids from hairy root culture along with elicitor treatment to produce more respective bioactive compounds from this plant. In addition to withanolides. W. somnifera plants contain some phenolic acids in field-growing plants, including benzoic acid, ferulic acid, caffeic acid, and catechin (Tomar et al., 2019). These phenolic acids play a critical role in preventing and treating various diseases. The phenolic acids in medicinal plants are generally produced in less quantities by differentiated tissues. Hence, it makes more sense to use biotechnological methods to produce them efficiently and more effectively in less time (Yousefian et al., 2020). Genetic and biochemical stability, large biomass production, and high biosynthetic capacity of hairy root cultures make them suitable sources of secondary metabolites for biosynthesis (Shilpha et al., 2023). A majority of phenolic acids are synthesized from phenylpropanoid and tyrosine-derived compounds; many of the genes in this pathway encode critical enzymes such as phenylalanine ammonia-lyase (PAL), tyrosine aminotransferase (TAT), and cinnamic acid 4-hydroxylase (C4H). It was reported that the synthesis of active compounds enhanced by the treatments of exogenous factors in in vitro culture is one of the most effective strategies for accumulating secondary metabolites with various medicinal plant species (Alcalde et al., 2022, Shilpha et al., 2023).

Elicitors are chemicals or biochemical compounds introduced in small amounts into a plant culture to stimulate the biosynthesis of target bioactive compounds. Plant cell, tissue and organ culture and the production of secondary metabolites using biotic and abiotic elicitor treatments are called elicitation (Khalili et al., 2010). Applying the biotic elicitors, i.e. Chitosan and Yeast extract, is considered adequate for enhancing bioactive compound production in different plant cells, tissue and organ cultures. Chitosan is a signal molecule source that activates the defensive enzymes in signal transduction pathway genes, including chitinases, peroxidase, phenylalanine ammonia-lyase, and polyphenol oxidase. (Coqueiro et al., 2015, Mukarram et al., 2023). Similarly, YE also increases the gene expression of key enzymes involved in tyrosine-derived pathways (TAT and HPPR) and phenylpropanoid-derived pathways (PAL and C4H). (Park et al., 2016). In addition, few reports are using biotic elicitors to improve withanolides production in in vitro shoot cultures (Vinod et al., 2022, Kaur et al., 2022), Adventitious root culture (Sivanandhan et al., 2012b, Kaur et al., 2022), and Hairy root culture (Sivanandhan et al., 2013, 2016).

Abiotic elicitors (Salicylic acid, Methyl jasmonate and Silver nitrate) also increase the plant active compounds by enhancing the expression of biosynthetic pathways involving genes. Salicylic acids play a vital role in antioxidant metabolism and tightly control cellular ROS production (Khan et al., 2015). A signalling process and activation of a protein kinase can occur when SA acts as a signal for developing systemic acquired resistance (SAR) (Durner et al., 1997). Meanwhile, MJ is the methyl ester of jasmonic acid. It is involved in signal transduction pathways to activate relevant promoters and different transcription factors correspond to produce a series of secondary metabolites, including steroidal lactones, terpenoids, alkaloids and polyphenolic compounds (Buraphaka et al., 2020; Sharifzadeh et al., 2021; Shoja et al., 2022). Silver nitrate can act as signal transduction molecules recognized by cell membrane-containing receptor molecules; they transfer the signal to internal cells to activate the forward signal transduction cascade to enhance the synthesis of various targeted active compounds (Tripathi et al., 2017). Recently, some studies have been reported to enhance secondary metabolites of respective abiotic elicitors in several solanaceous and other medicinal plants (Jeyasri et al., 2023, Biswas et al., 2023). The effect of SA and MJ have been used in W. somnifera shoot culture to produce withanolides (Singh et al. 2022). Similarly SA and MJ treatment on W. somnifera hairy root culture reported by a few researchers (Sharma et al. 2023) to enhance withanolides content, but they did not show any phenolic acids content. In this present study, for the first time use silver nitrate on hairy root culture of W. somnifera to enhance the accumulation of withanolides content. Several reports published regarding the induction of HR culture in W. somnifera; however, none have comprehensively documented the simultaneous presence of withanolides and phenolic acids. Furthermore, the study aims to investigate the effects of both biotic and abiotic elicitors on the enhanced accumulation of withanolides and phenolic acids production in transformed root cultures.