Mass production and proliferation of medicinal and/or endangered plants by phytohormone supplemented micropropagation is commonly employed as a promising approach. In-vitro micropropagation in Withania somnifera has been previously performed by several researchers like Furmanowa et al., 2001; Rani et al., 2003. In the current study, firstly we have generated callus mass of W. somnifera and then exposed it with different concentrations of BAP supplemented MS medium to examine several responses and growth patterns. For maximum callus frequency (to induce microshoot production) and shoot morphological attributes, 2 mg L− 1 BAP was found to be best suited concentration (Table 1). Contrary to this, lower as well higher doses of BAP proved to be less effective. All these outcomes were in consistent with previous reports, performed on W. somnifera and Vaccinium corymbosum L. (Rani et al, 2003; Kharel et al., 2022). In these plants, they have also attested that an optimum dose of BAP is best suited for proliferation and maximum production of microshoots. According to Alagumanian et al. (2004) this may be due to the fact that average concentration of BAP; a cytokinin, suppresses the apical dominance effect of auxins and thus magnifies the emergence of lateral branches.
Salt stress in plants has been proven as a major constraint that limits plants’ vitality and yield. In spite of this, ROS toxicity induced after salt stress has proven as elicitor for several medicinal plants. For in-planta enhancement of phytoactive compounds, it is very important to know the effect of salt generated osmotic stress on its growth as well as secondary metabolism. In recent decades several research works has been performed on in-vitro regenerated medicinal plants to examine the impacts of salinity induced toxicity on plants’ machinery (Sabir et al., 2012; Dogan 2020). In addition, various reports revealed the detrimental impacts of high salinity on in-vitro cultured solanaceous plants such as Solanum tuberosum (Mohamed et al., 2010; Khenifi et al., 2011; Khalid and Aftab 2020), tomato (Amini et al., 2007), Withania coagulans (Ghahremani et al., 2019), and Nicotiana (Makkar et al., 2022) etc. In the present study, effect of different concentrations of NaCl on growth capabilities, ionic concentrations, photosynthetic machinery, anti-oxidative defence system, and secondary metabolism of in-vitro grown microshoots of W. somnifera was investigated along with several morphological and biochemical parameters. In addition, these outcomes were further validated and attested with the help of expression analysis of withanolide synthesising and photosynthetic genes. An adverse impact of saline condition on W. somnifera growth was observed throughout the presented study. However, mild doses of NaCl did not bring any significant changes. The pattern of decline in salt treated shoot length and biomass was in consistent to the report of Ahire et al. (2014) and Dogan (2020). Salinity also inhibited the shoot morphological attributes of in-vitro cultured W. somnifera as compared to control sets. Similar pattern of salt influenced inhibition was also reported in Bacopa monnieri (Sable et al., 2018). These outcomes suggested that salt-induced ROS toxicity inhibited cell growth and proliferation which finally retarded the growth and morphologies of the treated explants (Oukarroum et al. 2015).
Alteration in the relative water content of the plant is the signature response under any osmotic stress (Bertamini et al., 2006). In this study, we did not observe any significant changes in RWC till 100 mM NaCl. Nevertheless, 10.23%, 12.69%, and 12.99% declines occurred in the contents of relative water after 150, 200, and 250 mM NaCl treatments respectively. The osmotic imbalances caused by high salt concentrations in plant cells alter the ionic contents as well as water potentials by inhibiting the absorption and transport of water (Kumar et al., 2021). A high K+ to Na+ ratio is vital for plant as it provides optimum osmoticum to the cells for better performance of enzymes and proteins. In W. somnifera we observed marked enhancement in the Na+ ion concentration while K+ ion was declined according to the increasing doses of NaCl. According to Kumar et al (2021) this may be due to high cellular transport of Na+ as compared to K+ ions in NaCl exposed plant samples. Accordingly ionic ratio of treated plants also altered significantly as compared to control.
Plants are unique as they can perform the most vital reaction on our planet i.e. photosynthesis, on which we all are dependent. The rate of photosynthesis is greatly affected by total concentration of chlorophyll (Muranaka et al., 2002). According to Munns (2002) salinity imposes growth retardation in leaf which reduces leaf area that resulted in declined efficiency of photosynthetic apparatus. Besides this, salt induces over accumulation of Na+ ions that again reduces net photosynthesis (Rai et al., 2003; Tavakkoli et al., 2010). After notifying the significant decrease in growth and enhanced accumulation in Na+ ions, we have further analysed the photosynthetic attributes of saline-treated shoots of W. somnifera. A linear and significant decline in the chlorophyll and carotenoid content and Fv/Fm values in salt treated samples suggested the detrimental impacts of NaCl on various proteinaceous bodies that constitute the photosynthetic apparatus. These results, regarding decreasing trends of pigments were in accordance with the outcomes of Guiza et al. (2022) on Medicago sativa. In this context, we have also analyzed the expression levels of certain genes that encode the integral proteins of PSI and PSII (Fig. 8). Transcript levels of all the analyzed genes viz. PsaA, PsaB (both encode PSI core complex), PsbC (encodes CP43 protein i.e. the part of PSII), PsbA (encode PSIID1 protein), and PsbD (encode PSIID2 protein) were found to down-regulate after increasing concentrations of NaCl. These observations can be attested by Makeen et al. (2007) and Leelavathi et al. (2011) as they have concluded that mutations in PsaA and PsbA genes resulted in less chlorophyll contents with declined Fv/Fm values.
Several adaptive radiations are being developed by plants to cope the environmental stress and alteration in the biochemical behaviour is one of them. In this line, we have estimated the contents of several primary metabolites like proline and sugar along with stress markers such as membrane damage, lipid peroxidation, and H2O2 content. The results suggested sharp increment in the proline and phenol upto toxic doses of NaCl which was in consistent with the outcomes recoded by Guiza et al. (2022). According to Hayat et al. (2012) increased accumulation of cellular proline may act as of the quencher of singlet oxygen by participating in ROS scavenging activity. Like proline, phenol and flavonoids act as non-enzymatic defence system against stress mediated osmotic imbalance (Şirin and Aslım, 2019). We have observed significant up-regulation in the contents of both the metabolites after exposure of higher doses of salt. Similar to these observations, Kiani et al. (2021) depicted salt-induced increment in the phenol, proline, and flavonoid in wheat cultivars. Further, effect of salt stress on membrane damage intensity of in-vitro grown W. somnifera depicted increasing electrolyte leakage upto 200 mM NaCl which suggested higher accumulation of MDA content in salt stress (Akbari et al., 2020). To cope up these impacts, plants have developed more pronounced defence system such as free radical scavenging activity. In our finding, percent DPPH inhibition was significantly induced after increasing doses of NaCl which may depict the active participation of plants’ machinery to mitigate ROS burst.
Leaf surface trichomes are the repository and secretary sites of several metabolites of plants (Kumari et al., 2018). Their morphology and surface density depends upon the responses under several environmental constraints (Wagner et al. 2004). In this regard, we have estimated the leaf surface trichome densities from control and treated sets (that gave significant alterations i.e. 100–200 mM NaCl). The results revealed that salinity caused induction in the number as well as size of the GTs and NGTs upto 200 mM concentration. This may indicate the possible role of trichome in storage of secondary metabolites in W. somnifera (Munien et al., 2015). In a report of Zhou et al. (2018) they have also concluded that moderate and severe salt intensities significantly induced the leaf surface trichome density in Schizonepeta tenuifolia. Besides the trichomes, salt also affected the stem anatomical features of in-vitro propagated microshoots of W. somnifera. In our report, there was non-significant decline in the cortical cell diameter, which might suggest that salt upto 200 mM concentration does not severely affect the cortex. Contrary to this, a pronounced reduction in the pith area, thickness of vasculature, and thickness of stele was observed after 100–200 mM NaCl treated shoots when compared with control. These outcomes can be validated by the research performed by El-Taher et al. (2021) on Vigna ungiculata L.
Excess oxidative damage generated by salinity mediated ROS burst in plants can be mitigated by developing anti-oxidative defence systems (Abogadallah 2010). These activated defence responses ultimately maintains the growth and health status of the plant. In the presented study, we have recorded dose dependent increment in the activities of SOD, CAT, and Peroxidase. These findings were agreed with what had presented by Rahnama and Ebrahimzadeh (2005) and Wang et al., (2014) on effect of NaCl on potato seedling and Kandelia candel root respectively. However, maximum activity of APx was found after 150 mM NaCl, beyond that dose it was declined in non-significant manner.
In various research reports, researchers have tried to enhance the withanolides content by various abiotic stresses such as drought, low light regime, metal nanoparticles, UV-B etc. (Kannan et al., 2011; Jacob et al., 2014; Singh et al., 2019; Tripathi et al., 2021) In addition, NaCl-mediated up-regulation in secondary metabolism has been earlier reported in medicinal plants like Artemisia annua and Ocimum sanctum along with crop plants like cotton and Medicago (Yadav et al., 2017; Scagel and Lee, 2019; Ibrahim et al., 2019; Sarri et al., 2021). Likewise, we have also analyzed the impacts of salinity induced ROS stress on withanolide production in in-vitro grown W. somnifera by HPLC as well as gene expression analysis. Results suggested a positive correlation between optimum concentration of NaCl and production of withaferin A. Similarly, the Real-time PCR analysis of several withanolide synthesising genes also depicted the stimulatory effects of average NaCl concentration on gene expressivity which reveals the positive orchestration of genes towards synthesis of secondary metabolites in W. somnifera. In line with these outcomes, Yadav et al. (2017) also concluded that optimum dose of NaCl (100 mM) was more beneficial for artemisinin and other metabolites than toxic dose (200 mM) in A. annua. Additionally, they have also depicted the similar trend in the fold expressions of MVA and MEP pathway genes i.e. HMGR and DXR respectively. These findings were in consistent with what we have observed in the presented study. Besides these, other terpenoid-related genes such as DWFs, CYPs, CAS, ODM, SQE etc. also followed the similar trend of expression.