## 4 Discussion  
The topic of the micropropagation of blackberry has been taken up by a number of researchers [5; 6; 9; 10; 11]. In the present study, the direct organogenesis of the Navaho cultivar of blackberry (_Rubus fruticosus_ L.) from shoot tips and the use of MS medium supplemented with 0.5 mg dm\({}^{-3}\) IBA and 0.01 mg dm\({}^{-3}\) GA\({}_{3}\) enabled the efficient micropropagation of this plant, and the addition of activated carbon in the amount of 0.5 g dm\({}^{-3}\) stimulated root formation. Other authors have also generally conducted in vitro cultures of blackberry from shoots on MS media supplemented with cytokins and auxins. BA concentrations proposed by other authors as optimal for the micropropagation of blackberry are 1.0 mg dm\({}^{-3}\) BA [8; 11], 0.6 mg dm\({}^{-3}\) BA [7], 0.5 mg dm\({}^{-3}\) BA [6], and 0.3 mg dm\({}^{-3}\) BA [13]. Navaho blackberry plantlets obtained in the control had on average 4.67 shoots per explant with a length of about 2 cm after two months of in vitro culture, and 7.25 shoots per explant with a length of over 3 cm after three months (Table 1). The growth parameters of _Rubus fruticosus_ plantlets growing on the same medium, such as shoot number and length or root number and length, may be cultivar traits, as shown for the 'Castanska bestra', 'Chester Thornless', 'Loch Ness', and 'Navaho' blackberry varieties [13]. Various factors are used to stimulate the micropropagation of blackberry. Apart from changes in the type and concentration of plant hormones, silicon and Fe-EDTA are added to the medium. Media other than classic MS are also used, such as starch medium [12; 13; 14]. To the best of our knowledge, the effect of ZnONPs on the micropropagation of blackberry in in vitro culture has not yet been investigated. Other authors have used ZnONPs in in vitro culture to improve the effectiveness of propagation of other plant species, including _Phoenix dactylifera_ L., _Coffea arabica_ L., _Solanum Lyopersicum_ MILL, and _Panicum virgatum_ L. [31; 32; 33; 34]. In the present study, four concentrations of zinc oxide nanoparticles were used to assess their effect on the growth and selected biological quality characteristics of blackberry plantlets in in vitro culture. Many studies have confirmed that ZnONPs can stimulate plant growth at various stages of development, supplying plants with zinc, an important nutrient. Zinc regulates the growth and development of plants by taking part in carbohydrate metabolism, biosynthesis of proteins, respiration, DNA/RNA metabolism, and photosynthesis [19; 35]. Zinc contributes to the production of growth substances--auxins, and a lack of zinc disturbs the balance between plant hormones--auxins and cytokins, and cytokins and abscisic acid [20]. \begin{table}
\begin{tabular}{c c c c c c c} \hline \multirow{2}{*}{**Biometrical Feature**} & \multirow{2}{*}{ \begin{tabular}{c} **Subsoil without or** \\ **with Added Bacteria** \\ **(–/+)** \\ \end{tabular}  } & \multicolumn{5}{c}{**Planlets from In Vitro Culture in Nutrient Medium Supplemented with ZnONPs**} \\  & & & & & **[mg dm\({}^{-3}\)]** & & \\  & & & & & **[mg dm\({}^{-3}\)]** & & \\  & & & & & & **[mg = 0.05]** \\ \hline Number of & – & 6.67 \({}^{\text{a}}\) & 7.23 \({}^{\text{a}}\) & 5.46 \({}^{\text{b}}\) & 5.18 \({}  
Zinc oxide nanoparticles stimulate the germination of seeds of various plants and improve seedling vigour and growth in plants such as maize, onion, and mung bean [36, 37, 38]. They also increase the content of microelements, protein, and phenols in mung bean shoots [38]. The addition of zinc oxide nanoparticles to the medium in in vitro culture can positively influence callus development, rooting, and somatic embryogenesis in various plant species [31, 33, 34]. In the present study, the addition of zinc oxide nanoparticles reduced the number of shoots formed, except for the concentration of 10 mg dm\({}^{-3}\) ZnONPs.