## 4 Discussion
IRT1 has a wide range of substrates among divalent metals, including zinc. In _Arabidopsis_ grown in excess Zn, expression of IRT1 in the root is reduced, leading to decreased iron accumulation [44]. Comparison of the mineral composition of the plants obtained in the present study (Table 2) with the literature data revealed that the plantlets obtained from the in vitro culture in the control treatment had about 27 times as much zinc as mature leaves from blackberry shrubs grown in the ground (2.01 mg Zn 100 g\({}^{-1}\) DW, according to [45]), while plantlets from the treatment with the highest concentration of nanoparticles contained about 50 times as much zinc as mature blackberry fruits from that study. The control plantlets also contained 29 times as much zinc as Navaho blackberry buds grown in the ground (1.85 mg 100 g\({}^{-1}\) DW zinc, according to [3]). The control plantlets from in vitro culture also contained more iron and manganese, but less potassium, calcium, magnesium, and copper than blackberry leaves in the study cited [45]. The addition of ZnONPs to the medium reduced the content of iron (Table 2), but its lowest concentration, obtained in the treatment with 40 mg dm\({}^{-3}\) ZnONPs, was higher (40.5 mg 100 g\({}^{-1}\) DW) than in the buds (11.77 mg 100 g\({}^{-1}\) DW [3]) and leaves of blackberry grown in the ground (6.16 mg 100 g\({}^{-1}\) DW [45]). Given that zinc and iron are among the main deficient minerals in the human diet, plants grown in vitro could be used to produce dietary supplements, especially for vegetarians [46]. The blackberry plantlets obtained from a medium with 10 mg dm\({}^{-3}\) ZnONPs had very high content of zinc and iron, and at the same time had good growth parameters (Tables 1 and 2). Zinc oxide nanoparticles can affect plants not only as a nutrient, but also as elicitors modifying the metabolic reactions of plants. Owing to their nano-size, they can be absorbed into cell walls, and their physicochemical effects can influence the functions of the cell wall and cell membrane, the ultrastructure of the organelles, and the cell metabolism of plants [47]. Nanoparticles can induce the formation of pores in the cell wall structure, which allow them to enter plant cells [48], where they can generate reactive oxygen species and cause oxidative stress in cells [47]. In stress conditions, ROS play a key role in regulating the secondary metabolism of plants, which leads to increased production and storage of secondary metabolites [49]. The Navaho blackberry plantlets from the in vitro culture with the addition of zinc oxide nanoparticles had a reduced content of phenolic compounds, which are among the most important plant secondary metabolites (Figure 2). On the other hand, they were shown to have a positive effect on the content of antioxidants measured by the ABTS method (Figure 3). The literature data confirm that antioxidant capacity is closely correlated with the content of phenolic compounds [3,15,31]. A high positive correlation has been obtained between the TP content and TAC of blackberry bud extracts [3]. However, the antioxidant activity of plant extracts is believed not to be determined by the presence of polyphenols alone. Other compounds with antioxidant properties include antioxidant enzymes, ascorbic acid, glutathione, carotenoids, and salicylic acid. These antioxidant compounds work together to form a comprehensive defence system in plants, helping them survive various environmental stressors [41,50]. The role of ZnONPs as an elicitor of plant metabolites in in vitro culture is not yet well understood. Stevia plants from in vitro culture treated with 100 mg dm\({}^{-3}\) ZnONPs had the highest total phenolic content (TPC), total flavonoid content (TFC), and total antioxidant capacity (TAC) [15]. The callus of _Linum__ustitassium__cv_. [PERSON] growing on a medium with ZnONPs was shown to have increased concentrations of phenolic compounds and flavonoids, and a concentration of 10 mg dm\({}^{-3}\) increased the content of lignans and neoligrans in the callus [48].