## 4 Discussion
The shoot and root growth of the blackberry plantlets on media supplemented with nanoparticles was also poorer, resulting in a smaller increase in the fresh weight of the plants (Table 1). Statistically significant differences were shown most often at the highest concentrations of ZnONPs (30-40 mg dm\({}^{-3}\)). The reduction in the growth parameters of regenerated plants at higher concentrations of ZnONPs (Table 1, Figure 1) may be associated with the mechanism of action of metallic nanoparticles on plant cells. At the cellular level, they induce the formation of free radicals which interact with virtually all cellular mechanisms, leading to protein modifications, lipid peroxidation, and DNA damage, ultimately resulting in necrosis or cell death [32, 33]. ZnONPs can affect the functions of the cell wall and membrane of plant cells and restrict the electron transport chain in mitochondria and chloroplasts, leading to oxidative bursts, increased ROS concentrations, and cell death due to elevated levels of reactive oxygen species [33]. Zinc oxide nanoparticles had a smaller effect on shoot regeneration than on root growth and development in blackberry plantlets (Table 1). Zinc is essential for hormonal balance in plants, especially auxin (IAA) activity [39]. After three months of culture, plantlets growing on the medium with the highest concentration of ZnONPs had about 1.5 fewer shoots and were about 1.4 times shorter, while they produced about one fourth as many roots, which were 6.5 times shorter than in the control plantlets. The effect of ZnONPs on the regeneration of plants in in vitro culture depends on multiple factors, mainly the species of plant, the type of explant used, and the concentration and type of nanoparticles. In a culture of _Musa paradisical_ using shoot tips as explants, ZnONPs also stimulated callus formation, shoot and root regeneration, and the amount of biomass produced [40]. In an in vitro culture of _Solanum Lyopersicum_, in which cotyledons of plants were used as explants, regeneration of plants in media with ZnONPs (15 mg dm\({}^{3}\)) was rapid, and the plantlets also reacted better to salt stress [31]. In an in vitro culture of _Brassica nigra_, ZnONPs (1 mg dm\({}^{3}\)) reduced the fresh weight of the callus [41]. On the other hand, regeneration of _Brassica napus_ cv. [PERSON] from the hypocotyl in the presence of ZnONPs (10 mg/L) increased the amount of callus produced [42]. [PERSON] et al. [15] found that the regeneration of stevia plants was positively influenced only by concentrations of 0.1 and 1 mg dm\({}^{-3}\) ZnONPs, while higher concentrations caused various toxic effects. The addition of ZnONPs to the in vitro medium affected the mineral composition of regenerated blackberry plants (Table 1). Plantlets from the treatments with 10-30 mg dm\({}^{-3}\) ZnONPs contained significantly more potassium, calcium, zinc, manganese, and copper than the control, while the iron concentration was lower in all cultures with ZnONPs. The increase in the concentration of these elements (Table 1) can be explained by the fact that ZnO nanoparticles promote the production of organic acids secreted by the roots, mainly oxalic, citric, malic, and fumaric acids, which facilitate the absorption of nutrients by plants [43]. ZnONPs can also create new pores in cell walls, increasing access to water and nutrients and promoting uptake of minerals [43]. Plants from the treatments with ZnONPs had lower iron content in comparison to the control (Table 1). Zinc disrupts iron uptake by roots and its translocation to other parts of plants. An antagonistic relationship between Fe and Zn has been noted in many plants, while complex relationships in the uptake and distribution of these mineral nutrients have been demonstrated in others [44]. The Zn concentration affects Fe uptake, and excessive Zn has been shown to result in physiological Fe deficiency. Due to their chemical similarity, bivalent cations of Fe and Zn can interact with transport channel proteins, including iron-regulated transporter 1 (IRT1) [44].