## 3 Results and Discussion
### Biomass, Cu, and GA II Content in CuO NP Treated and Non-Treated CSC  
Cu accumulation was noted when the cells were exposed to CuO NPs. The Cu level was higher (1.1 mg/L) in 5 mg/L CuO NP treated CSC compared with the value in 1 or 3 mg/L of CuO NP treated CSC (Figure 2a). Similarly, Cu accumulation in _B. rapa_ was reportedly enhanced depending on treatment with CuO NPs [29]. A high concentration of Cu ions may be toxic; for example, binding of Cu to sulfhydryl groups of proteins can inhibit enzyme activities and may induce a deficiency of other elements through competition with ions like iron or by imposing oxidative stress in plant cells [39]. The redox reactions that make Cu a significant essential element is also responsible for its toxicity [13]. Thus, the transition from Cu\({}^{2+}\) to Cu\({}^{+}\) and the reverse (i.e., the redox cycle) catalyzes the interaction between superoxide ion (O\({}_{2}\)\({}^{-}\)) and hydrogen peroxide (H\({}_{2}\)O\({}_{2}\)) to produce hydroxyl radicals (OH), which are highly toxic and can cause oxidative stress that involves cell proteins, lipids and DNA [40]. Presently, the maximum level of biomass (14.51 g DCM)  
Figure 1: Callus induction and biomass accumulation in _Gymnema sylvestre_. (**a**) Effect of different concentrations of 2,4-dichlorophenoxyoxyacetic acid (2,4-D) in combination with 0.1 mg/L kinetin (KIN) for callus induction in _G. sylvestre_. (**b**) Biomass accumulation. (**c**) Gymnemic Acid II (GA II) production on MS liquid medium with 2,4-D (2 mg/L), KIN (0.1 mg/L), and sucrose (30 g/L) with time. Different letters indicate a significant difference at \(p\leq 0.05\).  
accumulation was obtained on medium with 1 mg/L CuO NPs compared to the levels in the control and in samples containing \(>\)1 mg/L CuO NPs. Increasing the level of CuO NPs above 1 mg/L decreased the DCM (Figure 2b). A similar observation was also noted in the CSC of bitter gourd after elicitation with silver NPs (AgNPs). The authors corroborated that a higher dose of CuO NPs decreased the biomass accumulation in hairy root culture of Chinese cabbage [25]. Cu has been studied as a stimulatory element for in vitro culture in many plants [41; 42]. For example, CuO NPs stimulate phytochemical production in _Stevia reboundana_ in vitro [42]. The present study focused on the impact of CuO NPs on GA II content in CSC as determined using HPLC. The content of GA II in elicited CSC was significantly higher (four- to nine-fold) than non-elicited CSC (Figure 3a). The nine-fold increase of GA II (89.25 mg/g DCM) was evident with 3 mg/L CuO NPs (Figure 3a). However, 5 mg/L of CuO NPs inhibited the synthesis of GA II (Figure 3a). The abiotic elicitation of cadmium chloride produced the maximum amount of GA (59.97 mg/g DCM), i.e., a 6.8-fold increase in comparison to the non-treated CSC of _G. sylvestre_[12]. GA content was improved in the CSC of _G. sylvestre_ using signaling molecules, such as methyl jasmonate and salicylic acid [43]. An increase in the GA content (7.78-fold) was evident in the presence of linoleic acid treatment compared to that of the non-elicited roots of _G. sylvestre_[4]. The results indicate that CuO NPs are effective in improving the production of GA II when compared to metal salts and signaling molecules in the CSC of _G. sylvestre_. CuO NPs have a useful role, which is mainly due to the release of Cu ions from the NPs that are taken up by the cells and which play a pivotal role in plant biochemistry [44]. Significant variations in cell biomass, GA II, PC, and pharmacological activities were observed, indicating that CuO NPs are abiotic stress elicitors that enhance the quantity of GA II content in _G. sylvestre_.  
Figure 2: Effect of copper oxide nanoparticles (CuO NPs) on copper content and biomass accumulation in cell suspension cultures (CSC) of _G. sylvestre_. (**a**) Copper content, (**b**) biomass accumulation. Different letters indicate a significant difference at \(p\leq 0.05\).