Hairy root cultures are appealing systems for the stable and continuous production of secondary metabolites, offering higher yields (Halder et al. 2018). This study evaluated the effects of R. rhizogenes bacterial density, inoculation time, and acetosyringone concentrations for the first time to optimize the maximum percentage of root induction frequency from W. somnifera. The induction potential of R. rhizogenes hairy roots is controlled by the density of the suspension and is strain-specific (Sarkar et al. 2020; Mahendran et al. 2022; Ji et al. 2023). In the case of W. somnifera, the results showed that the highest induction frequency occurred at a bacterial cell density of 0.8 (OD). Similarly, an OD of 0.8 plays a key role in effective transformation in Ocimum tenuiflorum (Sharan et al. 2019), Amaranthus hybridus (Castellanos-Arevalo et al. 2020), and Swertia chirayita (Mahendran et al. 2022).
Infection time is also crucial in hairy root formation and may vary with the bacterial strain and plant species. In this study, a 20 min infection time was found to be suitable for achieving the maximum transformation frequency (25.00% ± 3.64%) with the R. rhizogenes ATCC 43057 strain in W. somnifera. No prior studies have examined hairy root production from leaf explants using R. rhizogenes ATCC 43057. Many researchers have stated that the optimal infection time for achieving the highest root induction depends on the plant type and bacterial strain (Boobalan and Kamalanathan 2020; Mahendran et al. 2022). In this study, a 30 min infection time resulted in necrosis of the infected leaf explants and a decrease in transformation frequency. Earlier reports from Boobalan and Kamalanathan (2020) evidenced that 20 min of infection time for Aerva lanata, 20 min for Swertia chirayita (Mahendran et al. 2022), 10 min for Raphanus sativus (Balasubramanian et al. 2018), 15 min for Curcuma longa (Sandhya and Archana Giri 2022), and 30 min for Prunus persica (Xu et al. 2020) were appropriate for achieving the highest transformation efficiency.
Acetosyringone, an activator of virulence genes, is applied exogenously during the infection time to stimulate R. rhizogenes T-DNA transfer to the wounded area of the host, thereby triggering a high rate of hairy root induction (Gai et al. 2015; Mahendran et al. 2022; Ji et al. 2023). In this study, acetosyringone was supplemented at concentrations of 50, 100, 200, and 400 µM during the infection time to increase the transformation efficiency. The application of acetosyringone significantly enhanced the transformation efficiency of the W. somnifera leaf. The highest transformation frequency, 31.66% ± 2.98%, was detected at 200 µM acetosyringone. This corroborates the findings of Sharma et al. (2021), Mahendran et al. 2022, and Ji et al. 2023, who observed that the exogenous application of 200 µM acetosyringone during the infection time increased the hairy root induction frequency.
The accumulation of biomass in W. somnifera hairy roots was influenced by different culture conditions. Many researchers have revealed that the optimization of the culture medium plays a vital role in boosting the accumulation of hairy root biomass (Sarkar et al. 2020; Makowczynska et al. 2021; Mahendran and Vimolmangkang 2024). In this study, the highest hairy root biomass was observed in the MS medium compared to the B5 medium. Our results concur with the conclusions of studies on Aster scaber (Ghimire et al. 2019), Turbinicarpus lophophoroides (Solis-Castañeda et al. 2020), Rubia yunnanensis (Miao et al. 2020), and Cannabis sativa (Mahendran and Vimolmangkang, 2024).
The growth/biomass curve passage results indicated that different harvest times within a 30-day culture period in the MS medium significantly affected the biomass accumulation of W. somnifera hairy root culture. The W. somnifera hairy root was found to have three distinct phases, with biomass continuing to accumulate up to the stationary phase. In this study, the growth curve of W. somnifera hairy roots exhibited slow biomass accumulation from 0 to 10 days, followed by rapid growth of hairy root biomass until 25 days (the exponential growth phase). Beyond 25 days of growth, it reached the stationary phase, and no further biomass improvement was observed, indicating that the hairy root culture had entered the stationary phase by 30 days. The death or decline phase was not observed in this study. The investigation of growth time optimization in the growth curve led to the identification of the ideal time for elicitation application, which corresponds to the end of the exponential phase. According to the growth curve, the highest biomass was achieved during the stationary phase after 25 days, which was about 10.46 times greater in FW than the initial inoculum. These results corroborate earlier studies in Hybanthus enneaspermus (Sathish et al. 2023), Plumbago zeylanica (Kumar et al. 2023), Physalis minima (Halder and Ghosh 2023), and Cannabis sativa (Mahendran and Vimolmangkang 2024).
In this study, the hairy root biomass was harvested at various time periods to determine the optimum culture stage for producing the highest contents of withanolide A and withaferin A. The study showed that the highest contents of withanolide A and withaferin A were obtained after the 25th day of culture (exponential growth phase). Increasing the harvest time in the hairy root curve of W. somnifera led to a decrease in the production of withanolide A and withaferin A. This is similar to the findings of Zhang et al (2020), who noted that higher production of wilforgine and wilforine was achieved at 35 days (exponential growth phase) of Tripterygium wilfordii hairy roots. The higher yield of L-Dopa was found at the 40th day in the growth curve of Hybanthus enneaspermus hairy root (Sathish et al. 2023). In another study, Kumar et al. (2023) reported that the highest production of plumbagin in Plumbago zeylanica hairy root cultures was achieved at day 25.
Recent research has indicated that the exogenous application of elicitors is a potential tool for interacting with membrane receptors and activating specific genes, leading to the stimulation of target bioactive secondary metabolite production (Gharari et al. 2020; Jeyasri et al. 2023; Biswas et al. 2023). This study demonstrated that the elicitation of MJ or SA in W. somnifera hairy root cultures significantly enhanced the production of withanolide A and withaferin A. The application of MJ and SA is a common and potent signaling elicitor used to improve the production of targeted specialized bioactive metabolites in solanaceous hairy roots (Biswas et al. 2023). Recent results have shown that the exogenous elicitation of MJ or SA stimulates the accumulation of endogenous defense responses to wounds, leading to the production of enhanced metabolites (Jeyasri et al. 2023).
As stated in prior research, the time of elicitation, exposure period, and type and concentration of the elicitor play a pivotal role in the pattern of targeted/specific metabolite accumulation in hairy root cultures (Maciel et al. 2022; Mahendran and Vimolmangkang 2024). According to our growth curve and metabolite accumulation results, the 25th day of hairy root growth at the end of the exponential stage was identified as the most effective day for elicitation. Similarly, studies have reported that the 21st day of elicitation at the end of the exponential phase of Eclipta prostrata hairy roots was found to be the ideal time for achieving the greatest biomass and caffeoylquinic acid derivative production (Maciel et al. 2022). The study by Sandhya and Giri (2022) revealed that the 24th day-old hairy root of Curcuma longa, treated with an elicitor at the end of the exponential phase, produced the maximum amount of curcuminoids. In another study, Mahendran and Vimolmangkang (2024) described that the greatest hairy root biomass and friedelin and epifriedelanol contents were found after 28 days, which was the ideal time for elicitation in C. sativa.
In this study, we observed that the highest content of withanolide A (1.96 ± 0.03 mg/g DW) and withaferin A (1.68 ± 0.07 mg/g DW) was enhanced at 50 µM SA after 48 h of treatment. These results are in agreement with those obtained by Mahendran et al. (2022), who stated that the elicitation of Swertia chirayita hairy roots with 50 µM SA increased the yield of 1,2,5,6-tetrahydroxyxanthone (1.56-fold) and swerchirin (4.8-fold) compared to that in control hairy roots on the 6th day after elicitation. In our earlier study, we found that 50 µM SA was the most effective for the production of friedelin in the hairy root culture of C. sativa (Mahendran and Vimolmangkang 2024).
As proven in a prior study, 100 µM MJ stimulates the accumulation of rosmarinic acid in hairy root cultures of Prunella vulgaris (Ru et al. 2022) and the greatest tanshinone and rosmarinic acid in hairy root cultures of Salvia miltiorrhiza (Rastegarnejad et al. 2024). The combination of chitosan (50 mg/L) and MJ (100 µM) as elicitors on Scutellaria bornmuelleri hairy root was more effective in increasing the accumulation of chrysin, wogonin, and baicalein (Gharari et al. 2020). Similar to these results, our investigation revealed that compared to the control, treatment with MJ at 25, 50, and 100 µM and in combination with 50 µM SA significantly, increased the accumulation of withanolide A and withaferin A in hairy root cultures. In line with a study conducted by Rastegarnejad et al. (2024), our results specified that, the application of 100 µM MJ could enhance the production of withanolide A and withaferin A compared to 25 and 50 µM MJ. It was observed that withanolide A and withaferin A contents of 2.90 ± 0.08 mg/g DW and 3.40 ± 0.01 mg/g DW at 48 h after elicitation were 3.86 and 2.61-fold higher compared to the control, respectively. In this study, 100 µM MJ was combined with 50 µM SA to study on the combined effects of SA and MJ on withanolide A and withaferin A contents of W. somnifera.