**1. Introduction**

Blueberry and blackberry have high economic importance, particularly in hilly and mountainous regions of Serbia. The special economic importance of these fruit species is determined by the high usage value of their fruits, the profitability of production, high margins, contribution to additional employment, and more [1]. Furthermore, the small edible and colored berries of genera *Vaccinium* and *Rubus* are recognized as a good source of bioactive compounds (BCs), primarily phenolics, which contribute to the berries' organoleptic properties and after consumption exert a positive impact on human health [2,3]. In addition to their highly valuable fruits, the leaves of berry plants are also a rich source of phenolics and are often used in traditional medicine to treat numerous diseases such as colds, various inflammations, diabetes [4]. Leaves are present as a byproduct of growing berries and can be used as an alternative source of bioactive compounds which can be applied further for the development of functional food products and nutraceuticals [4–6]. Moreover, the European Medicines Agency (EMA) has approved the marketing and use of some *Rubus* leaf extracts and infusions for medical purposes [4]. Due to the mentioned characteristics of both fruits and leaves there has been increasing interest in growing berries in recent years, with a special emphasis on blueberries and blackberries. Furthermore, there is grea<sup>t</sup> interest in evaluating the BC composition of berry leaves, in vitro screening of their antioxidant and antimicrobial properties, and evaluation of in vivo biological activities and conduction of clinical trials [5–11]. For example, the highest values of antioxidant properties evaluated with DPPH• and ABTS•*+* scavenging or FRAP assays were 586.6 μmol TEAC/g DW, 862.4 μmol TEAC/g DW and 2674 μmol FEAC/g DW for 'Nanjin' variety among the 73 investigated blueberry cultivars [11]; that is, 45.0, 257, and 139 mg/g DW for leaves of 'Bluerain' and 'Vernon' varieties among 104 selected blueberry cultivars [12], respectively. On the other hand, some wild blackberry cultivars showed high ABTS•*+* scavenging activity (212.69 mmol TE/g dm) and FRAP (192.91 mmol TE/g dm), as well as high correlations with total phenolic content and content of ellagitannins [9]. Leaf extracts of six commercial blueberry varieties [6] and *Vaccinium corymbosum* variety [13] showed good antimicrobial properties (MIC and MBC) against some gram negative and gram-positive bacteria strains. Thus far, the phenolic profiles, antioxidant and antimicrobial properties of field-grown leaves of different blueberry and blackberry cultivars, in particular, 'Toro' [6,11] and 'Caˇ ˇ canska Bestrna' [5], have been successfully evaluated. More precisely, the DPPH• scavenging activity of blackberry ('Caˇ ˇ canska Bestrna') and blueberry ('Toro') leaves were 83.77% [5] and 305.0 μmol TEAC/g DW [11]. In addition, extracts of the leaves of Toro blueberry cultivar have shown good antibacterial potential against some bacterial strains such as *Staphylococcus aureus*, *Rhodococcus equi*, *K. pneumonia* and *E. fecalis* [6]. Furthermore, the extract of 'Toro' blueberry showed a good antimutagenic effect against different *Salmonella typhimurium* strains, with percent inhibitions of 32.98% (TA98) and 38.68% (TA100), which gives them the possibility of potential application as a safe and useful alternative for the prevention of mutations [6].

However, the use of highly valuable leaves from field-grown berry plants is often limited due to the frequent use of pesticides [14], as well as their seasonal availability. This is why there is growing interest in and questions about the potential propagation and growing of berry plants in vitro as a source of secondary metabolites, or the induction of callus cultures using a specific nutrient medium for targeted production of some BCs which have significant potential as antioxidants [15–18].

Several methods are available in plant tissue culture, among which organogenesis and callogenesis are the most commonly used [15]. Organogenesis involves the production of plant organs (shoots or roots), directly from meristems or indirectly from dedifferentiated cells which are known as callus [15]. The obtained in vitro plant cultures can be used as a sustainable and alternative source of valuable BCs, primarily phenolic compounds, which have potential application as food additives [17]. Some blueberry and blackberry cultivars have been successfully micropropagated via axillary shoot culture and through indirect shoot organogenesis [19–23]. However, according to our knowledge, the pheno-

lic profile and antioxidant properties of blueberry and blackberry leaves obtained from in vitro shoots grown on specific nutrient medium have not ye<sup>t</sup> been examined. On the other hand, callogenesis creates an amorphous cell mass in response to the exposure of explants to various biotic and abiotic elicitors which further initiate or enhance the biosynthesis of specific BCs [15,24]. The produced calluses can be used for plant regeneration or for targeted production of important metabolites in the cell suspension [15,16,18]. The biosynthesis of phenolic compounds in plant calluses depends on numerous factors such as nutrient media, plant growth regulators (PGRs), precursor feeding and elicitors [17]. Plant callus/cell cultures have the ability to accumulate secondary metabolites, which is a very promising system for biotechnological production of specific phenolic compounds [16]. For example, callus culture of *Vitis vinifera* has shown promising potential for the production of phenolic compounds [25] or specific phenolic classes such as anthocyanins [26], resveratrol [27] or stilbenes [28]. Moreover, phenolic compounds from the callus culture of different grape varieties have been successfully used to enrich food products such as yogur<sup>t</sup> [29]. Furthermore, callus cultures of different blueberry [30,31], and blackberry [20] cultivars have already been successfully created. The antioxidant properties and total phenolic content of blackberry calluses [20], that is, the phenolic profile of some varieties of blackberry calluses [31], were determined, as a prerequisite for further biotechnological production of highly valuable active compounds. However, according to our knowledge, calluses of blueberry 'Toro' and blackberry 'Caˇ ˇ canska Bestrna' have not ye<sup>t</sup> been produced and analyzed. Furthermore, the antioxidant properties of in vitro leaves and calluses of these two berry cultivars were not determined by FRP, ABTS•<sup>+</sup> and DPPH• scavenging activity assays until now.

Therefore, the aim of this study was to evaluate and to compare the content and profile of phenolic compounds of field-grown and in vitro leaves and callus cultures of blackberry 'Caˇ ˇ canska Bestrna' and blueberry 'Toro', as well as their antioxidant properties, using three common antioxidant assays: FRP, ABTS•<sup>+</sup> and DPPH• scavenging activities. Bearing in mind a number of advantages of the plant tissue culture-based production of secondary metabolites in comparison with conventional agricultural production, including controlled production via standardized protocols independent from seasonal variation, low water and carbon input, no use of pesticide and herbicides, etc. [32], and given the economic importance of these fruit species and their potential for targeted BC production, the results could help to estimate their possible use in the food and pharmaceutical industry.

### **2. Materials and Methods**
