The first step of HR culture depends on the induction frequency of hairy root from explants; otherwise, further experiments related to the production of useful bioactive compounds are impossible. Acetosyringone is one of the important phenolic inducers of the different virulence genes of Agrobacterium. The addition of exogenous acetosyringone in the culture of A. rhizogenes in right concentration (200 µM) boosted the optimum infectivity followed by the induction frequency of hairy root in P. minima. Similar role of acetosyringone is also reported in different solanaceous plants like Withania somnifera, Solanum melongena, S. trilobatum and S. mauritianum (Drewes and van Staden 1995; Saravanakumar et al. 2012; Jain and Singh, 2015; Shilpha et al. 2015), and other non-solanaceous medicinal plants such as Plantago lanceolata (Rahamooz-Haghighi et al. 2020), Scutellaria bornmuelleri (Gharari et al. 2020) and Crocus sativus (Sharma et al. 2021). Concentrations of acetosyringone are very useful to optimise the culture for the enhancement of transformation rate. Some earlier workers reported that the number of brunching patterns of transformed roots depends on many meristems that are influenced for increased growth rate and biomass accumulations; these are the common features of some medicinal plants such as Artemisia annua (Mukherjee et al. 1995; Giri et al. 2001), Gentiana macrophylla (Tiwari et al. 2007), Catharanthus roseus (Benyammi et al. 2016) and Ficus caric (Amani et al. 2020).
For the production of more bioactive compounds, manipulation of plant genome by the Agrobacterium-mediated transformation is a useful technique in the modern biotechnology field (Mukherjee et al. 2000; Chaudhuri et al. 2005, 2006). Successful establishment of hairy root culture depends on several factors including explants types (Saravanakumar et al. 2012), bacterial concentrations (Tao and Li, 2006), quality of A. rhizogenes strains (Veena and Taylor, 2007; Ooi et al. 2013) and culture medium (Mano et al. 1989); all show good influences on hairy root induction from any types of medicinal plant.
Previously, many researches have been studied Agrobacterium-mediated transfection. Normally, many researchers have used different types of explants such as seedling roots, stems, hypocotyls, cotyledonary nodal segments, cotyledons and young leaves (Murthy et al. 2008; Saravanakumar et al. 2012; Mahendran et al. 2022). In our experiments, we have used leaves from in vitro growing micropropagated plants from the elite clone of P. minima. Similarly, leaf explants used for the production of more root biomass and root number from the infected leaves have been reported in Plumbago zeylanica (Sivanesan and Jeong, 2009), Azadirachta indica (Srivastava and Srivastava, 2012), Anisodus luridus (Qin et al. 2014), Salvia bulleyana (Wojciechowska et al. 2020) and Swertia chirayita (Mahendran et al. 2022).
For the establishment of axenic roots, the use of antibiotics in the medium is an effective strategy for the elimination of bacterial growth from the transformed root culture. So far, various antibiotics were tested, out of them cefotaxime has been more appropriate for the elimination of bacteria from HR culture of different plant species such as Withania somnifera (Murthy et al. 2008), Bacopa monnieri (Paul et al. 2015), Catharanthus roseus (Benyammi et al. 2016) and Papaver armeniacum (Naeini et al. 2020). In our experiments, MS medium containing 500 mg l− 1 cefotaxime showed best responses, i.e. without hampering the growth rate.
In this work, maximum hairy root induction was achieved after 15 d of infection. Achievements of hairy root induction varied from plant to plant. Similarly, A. rhizogenes treatments in Catharanthus roseus were initiated the roots after 12 d from the infection (Benyammi et al. 2016), whereas, 10–17 d required in plant species in Ficus caric (Amani et al. 2020). In the control culture, during the culture incubation, no root originated from the wound sides.
Morphological studies were observed between transformed HR and NTR, where we find distinct characters in HR such as high branching point, ageotropic in nature and first growth rate. Similar types of results were found from different plant transformed roots in Solanum melongena (Jain and Singh, 2015), Catharanthus roseus (Benyammi et al. 2016), Echinacea purpurea (Demirci et al. 2020) and Ficus caric (Amani et al. 2020). It is also reported that phenotypic behaviour among the hairy roots is arising due to the levels of integration of T-DNA genes (Bathoju et al. 2017).
The PCR amplification of transformed hairy root DNA by different transformed gene-specific primers used is the most important method to confirm the integration of Ri T-DNA genes. Bacterial Ri-plasmid gene transformation to the plant genome can show some typical characteristics features. In this experiment, we have used seven different gene (rolA, rolB, rolC, rolD, aux1, ags and virD1)-specific primers for the confirmation of transgenic status of hairy roots. The amplicon size of each genes is approximately 344 bp (rolA), 780 bp (rolB), 545 bp (rolC), 402 bp (rolD), 1814 bp (aux1), 975 bp (ags) and 450 bp (virD1). Putative results of amplified product of DNA were observed in Bacopa monnieri hairy roots with different gene-specific primers (Paul et al. 2015).
Transformed roots show potential sources for the production of secondary metabolites by bypassing the natural habitat and without loss of genetic uniformity along with continuous production of bioactive compounds. This strategy is possible to use in the genetic engineering field to enhance their biosynthetic capacity. Based on our observations, so many factors are influenced and optimised to enhance the ability of individual active compound production. In this study, different root lines were used. To study their reach in high biomass levels under the MS basal medium, the same amount of inoculums was used in every set of root lines. Secondary metabolites production in the plant depends on the physiological status of organs, culture time, carbon source, nitrogen source, optimum temperature, supplementation with plant growth regulators (PGRs) in culture medium, etc. (Dörnenburg and Knorr 1995). Different root lines in MS medium culture to establish elite clones were reported on the different medicinal plants for secondary metabolites (Chaudhuri et al. 2005; Benyammi et al. 2016; Amani et al. 2020).
Withaferin A is an important active compound of W. somnifera; it has lots of medicinal activities on different cancer cells and nerve cell research (Behl et al. 2020). This active compound was first isolated from W. somnifera in the mid-1970s; from that time to now, this compound has lots of demands throughout the world. The productivity of this active compound depends on the natural source, because it is not possible to isolate by chemical synthesis. In this experiment, we established high-yielding HR culture. The content of withaferin A from the elite rhizoclones showed 891.79 ± 36.21 µg g− 1 DW as compared with NTR (121.32 ± 17.25 µg g− 1 DW), and it has produced 7.35-fold higher amounts of withaferin A content.
The use of various elicitors such as biotic and abiotic elicitors has affected the production of more secondary metabolites under in vitro culture. Chitosan is the source of some signal molecules that activate the defensive enzymes in signal transduction pathway genes, including chitinases, peroxidase, phenylalanine ammonia lyase and polyphenol oxidase. (Coqueiro et al. 2015). These genes are associated with the transformed root growth and enhancement of bioactive compounds. In this work, chitosan-elicited roots have produced a maximum 13418.91 ± 32.65 µg g− 1 DW (15.5-fold) withaferin A content as compared with non-treated TR. Similar results of chitosan-elicited treatment on hairy root culture to enhance secondary metabolites were reported by several workers on different medicinal plant species (Jiao et al. 2018; Thilip et al. 2019). The chitosan elicitation for the accumulation of withaferin A was reported on W. somnifera root culture, i.e. nine times higher than the untreated root culture. Though W. somnifera is the main source of withaferin A, P. minima can be used for the production of withaferin A as underutilised plants for the same time. Increasing the concentrations of chitosan gradually decreases the synthesis of active compounds (Sivanandhan et al. 2012). Recently, Thilip et al. (2019) reported that 100 mg l− 1 chitosan produced 4.03-fold higher withaferin A content from the HR culture of W. somnifera. Earlier reports on Isatis tinctoria L. by elicitation with 150 mg l− 1 chitosan on hairy root cultures were conducted to enhance 7.08-fold production of pharmacologically active flavonoids (Jiao et al. 2018). Another report of chitosan elicitor treatment on Gentiana dinarica hairy root culture produced 24-fold norswertianin content (Krstić-Milošević et al. 2017). It is also reported that B5 medium with different concentrations (200, 400 and 800 mg l− 1) of chitosan on HR culture of Silybum marianum produced more silymarin content than control culture (Gabr et al. 2016).
The present study was carried out to investigate the effect of another biotic elicitor YE on the elite HR line of P. minima. For its extensive modulation of secondary metabolism, YE is believed to be the most effective biotic elicitor frequently utilised in plant cell and tissue cultures (Shi et al. 2007). We showed that enhanced yield of withaferin A was produced in presence of 150 mg l‒1 YE (12239.45 ± 81.65 µg g‒1 DW) which was 13.72-fold higher than the control culture. Some similar reports found in the presence of the YE-induced active compounds on hairy root culture of Salvia miltiorrhiza for tanshinone (Shi et al. 2007), Catharanthus roseus for vinblastine and vincristine (Maqsood and Abdul 2017), Astragalus membranaceus for astragalosides (Park et al. 2021). Elicitors can trigger a variety of plant defence responses, such as the production of reactive oxygen species (ROS), the hypersensitive response, phytoalexin synthesis, antimicrobial secondary chemicals and other defensive manifestations (Montesano et al. 2003). However, the process of elicitation differed amongst plant species, and in most cases, a “elicitor-receptor” complex was produced, resulting in a wide range of biochemical responses (Radman et al. 2003; Cai et al. 2012).
The effect of SA has induced different genes that are associated with the signal transduction pathways in several plant secondary metabolites biosynthesis. The SAs are important signalling molecules shown to express local defence reaction at the infection site and initiation of systemic acquired resistance (SAR) (Durner et al. 1997). In our present work, treatment with 30 µM of SA indicated the most effective elicitor concentration for HR culture of this plant for the synthesis of more bioactive compounds among the other concentrations of SA (Table 3). It was studied that the increased concentration of SA under the in vitro culture can decrease the accumulation of active compounds (Coqueiro et al. 2015; Rajan et al. 2020). Mukundan and Hjorosto (1990) reported that high doses of elicitor to induce hypersensitive responses on culture can accumulate toxic molecules to increase the cell death percentages, and thus, the optimum concentration of any types of elicitors is effective for the induction of plant secondary metabolites. The SA-induced plant secondary metabolites have been reported in several medicinal plants such as Centella asiatica, Solanum erianthum, Withania somnifera, Swertia chirayita and Cynara cardunculus (Thilip et al. 2019; Baek et al. 2020; Folgado et al. 2021; Mahendran et al. 2022).
Withaferin A accumulation was increased from 891.79 ± 36.74 µg g‒1 DW in non-treated hairy roots to 18186.55 ± 45.46 µg g‒1 DW in transformed roots in a 4-d treatment with 30 µM MJ, i.e. 20.39-fold above than control (Table 3). The MJ is the methyl ester of jasmonic acid, and it is involved in signal transduction pathways to activate relevant promoters and different transcription factors correspondingly to produce a series of secondary metabolites including terpenoids, alkaloids and polyphenolic compounds (Buraphaka et al. 2020; Sharifzadeh et al. 2021; Shoja et al. 2022). In hairy root cultures of ginseng, MJ increased triterpene saponin concentration and stimulated associated gene expression. (Kim et al. 2009). The MJ also enhanced tanshinone and salvianolic acid A content by promoting the expression of related genes in hairy root cultures of Salvia miltiorrhiza, Salvia castanea and Salvia virgata (Hou et al. 2021; Attaran Dowom et al. 2022).
Among the various AlCl3 concentrations studied, 200 mg l‒1 AlCl3 with a 4-d treatment resulted to increased withaferin A (11123.45 ± 16.33 µg g− 1 DW) accumulation. As compared with control culture, it has increased 12.47-fold higher amounts of withaferin A content (Table 3). Some previous reports of 25 mM and 250 mM aluminium chloride enhanced the accumulation of scopolamine and hyoscyamine in Brugmanisa candida (Spollansky et al. 2000). Adventitious root culture along with 125-µM AlCl3 increased colchicine production in Gloriosa superba that was reported by Ghosh et al. (2002). Recently, Sivanandhan et al. (2012) reported that 10 mg l− 1 of AlCl3 with 4-h exposure time on 6th week culture resulted to enhance 7-fold withaferin A production in adventitious root culture of Withania somnifera.