## 3 Nano-Elicitation of Flavonoids
### ZnONPs as a Novel Elicitor of Flavonoids  
ZnONPs have also been investigated as abiotic elicitors of plant secondary metabolites (Table 3). Not only size, but also the morphology of NPs plays a role in effective elicitation. Licorice (_Glycyrrhiza glabra_) roots and rhizomes contain more than 100 various useful compounds, including phenolic compounds and glycyrrhizin [64]. In a study, the effects of bulk and nano sized ZnO and CuO on some secondary metabolites were compared in _G. glabra_ seedlings. Generally, both bulk and nano-sized particles enhanced the secondary metabolism in comparison to the control. It was observed that nano-sized ZnONPs were efficient in eliciting the anthocyanin content compared to bulk sized ZnO and the control [65]. Due to their nanometer-size, NPs can easily penetrate plant cell walls and plasma membranes as well as the cytoplasm and cell organelles, therefore affecting the plant metabolism effectively [66]. [PERSON] and Skoog Basal Medium; TFC: total flavonoid content; PVP: polyvinyl pyrrolidone; PEG: polyethylene glycol; QE: quercetin equivalent; DW: dry weight. S Seventy-five milligrams per liter of biogenic ZnONPs was reported to produce 3.7 mg/g DW flavonoids as compared to commercially available micro-sized ZnO, which produced 3.01 mg/g DW flavonoids in callus culture of _Echinacea purpurera_[67]. Biogenic ZnONPs effectively elicited in vitro flavonoid production in a cell suspension culture of Flax [68]. The shoot culture of _S. rebaudiana_ and _Lilium ledebourii_ showed increased secondary metabolism and TFC in response to engineered ZnONP elicitation [69; 70]. \begin{table}
\begin{tabular}{c c c c c c c c} \hline \hline
**Plant Species** & **Type of In Vitro Culture** & **Growth Media** & **NP Type** & **NP Size (nm)** & \begin{tabular}{c} **Effective** \\ **Concentration** \\ **of NPs** \\ \end{tabular} & **Effect on Flavonoid Production** & **References** \\ \hline _S. byczpersicum_ & Plantlets & Greenhouse & Cu & 50 & 50 mg/L & 99.67 mg QE 100 g\({}^{-1}\) of dry weight flavonoid production. & [51] \\ _O. busilicum_ & Seeds & MS media & CuSO4 & - & 10 \(\mu\)M/L & Enhanced total flavonoid production & [53] \\ _H. vulgar_ & Seedlings & MS media & CuO & \textless{}50 & 0.5 mM, 1 mM & Enhanced flavonoid production on day 20. & [54] \\ _G. sybestre_ & \begin{tabular}{c} Cell \\ suspension culture \\ \end{tabular} & MS media & CuO & 25-55 & 3 mg/L & TFC enhanced 2-fold. & [56] \\ _C. intylus_ & \begin{tabular}{c} Shoots and \\ roots \\ \end{tabular} & MS media & CuO & - & 1 ppm & 12.878 mg QE g\({}^{-1}\) extract TPC level in shoots and roots, respectively. \\ _W. seminifera_ & \begin{tabular}{c} Shoots and \\ roots \\ \end{tabular} & MS media & CuO & - & 1 ppm & TFC production increased. & [58] \\ _T._ & \
\begin{tabular}{c} Callus culture \\ \end{tabular} & MS media &
\begin{tabular}{c} NoC, PVP \\ and PEG \\ impedest \\ \end{tabular} & 47 & 5 mg/L & Flavonoid production enhanced. & [59] \\ _A. thaliana_ & Seedlings & MS media & CuO & - & 10–100 mg/L & Enhanced anthocyanin content. & [60] \\ _S. rebaudiana_ & Callus & MS media & CuOZnO & - & 100 mg/L & DW TFC & [62] \\ _S. rehudiana_ & Submerged & MS media with & Cu-Au & - & - & TFC increased. & [63] \\ _Bertoni_ & root culture & NAA & bimetallic & - & - & \multicolumn{1}{c}{} & \\ \hline MS: Murashige and Skoog Basal Medium; NAA: 1-Naphthaleneeacetic acid; TFC: total flavonoid content; PVP: polyvinyl pyrrolidone; PEG: polyethylene glycol; QE: quercetin equivalent; DW: dry weight. & & & & & \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Copper NPs (CuNPs) and copper oxide NPs (CuONPs) as elicitors of flavonoids.