*2.1. Plant Materials*

Research was conducted using blackberry cultivar 'Caˇ ˇ canska Bestrna' (*Rubus* subg. *Rubus* Watson) and blueberry cultivar 'Toro' (*Vaccinium corymbosum* L.). 'Caˇ ˇ canska Bestrna' is a cultivar developed at the Fruit Research Institute, Caˇ ˇ cak, which has been widely planted in Serbia; it displays excellent performance regarding cropping, fruit quality and resistance to diseases. In the group of semi-erect thornless blackberry cultivars, this cultivar reaches about 10% of the total world production. Although not widely grown in Serbia, 'Toro' belongs to the most common mid-season highbush blueberry cultivars in Central and Eastern European countries [33] and represents a self-fertile and heavy producer with large, juicy, sweet, and never tart berries. In addition to berries, the leaves of both cultivars appear to be good sources of antioxidants and have strong antibacterial activity [4,5].

Field-grown leaves as well as in vitro leaves and callus cultures of the two berry plant genotypes were obtained from the Fruit Research Institute, Caˇ ˇ cak, Serbia. In vitro shoots and calluses were cultivated on nutrient media in a growth room at 23 ± 1 ◦C, under 16 h-photoperiod and light intensity of 8.83 W/m2, using white fluorescent tubes (6500 K, 40 W) (Tissue Culture Laboratory of Fruit Research Institute), while field-grown leaves were obtained from plants grown in the Institute's research fields.

#### *2.2. In Vitro Shoot Cultivation and Callus Induction from Leaves*

Field grown plants of both cultivars were used as the source of initial explants for in vitro culture. Aseptic culture was established using single-node cuttings of newly formed shoots taken from branches during the spring. Cuttings with axillary buds were submerged in lukewarm water with a few drops of Tween 20 for 30 min and washed under running tap water for 2 h, followed by sterilization in 70% ethanol (1 min) and in a solution containing 0.1% HgCl2 and 0.01% Tween 20 (5 min), and finally washed with sterile distilled water (3 × 5 min) to remove all traces of disinfectants.

After establishment of aseptic cultures, shoots of examined genotypes were grown and multiplied on original or partially modified Murashige and Skoog (MS) medium [34], (Table S1). The axillary shoot proliferation of blackberry 'Caˇ ˇ canska Bestrna' and blueberry 'Toro' are presented in Figures S1 and S2. Composition of the media used for in vitro shoot multiplication of berry plant genotypes are listed in Table 1.

**Table 1.** Composition of media used for micropropagation of blackberry and blueberry.


MS—Murashige and Skoog basal medium.

Both blackberry and blueberry callus cultures were induced from in vitro leaves. Leaves were collected from the upper third of in vitro propagated shoots, cut three times transversely across the mid-vein and placed with the adaxial surface touching regeneration medium poured into Petri dishes (9 cm in diameter, around 50 mL of medium). The ingredients of media used for induction and maintaining of callus cultures are presented in Table 2.


**Table 2.** Composition of media used for callus induction.

MS—Murashige and Skoog basal medium.

In vitro shoot and callus cultures of blackberry 'Caˇ ˇ canska Bestrna' and blueberry 'Toro' are presented in Figure 1.

Field-grown and in vitro young and newly formed leaves of berry plants as well as their callus cultures were used for further phenolic characterization and the evaluation of antioxidant properties.

**Figure 1.** In vitro shoot and callus cultures of blackberry 'Caˇ ˇ canska Bestrna' (**<sup>a</sup>**,**b**) and blueberry 'Toro' (**<sup>c</sup>**,**d**).

### *2.3. Preparation of Leaves and Callus Culture Extracts*

Collected samples of leaves and calluses were finely ground and homogenized with liquid nitrogen using an Ika A11 basic mill. The extracts were then prepared using the extraction protocol previously described by Pavlovi´c et al. [35], with slight modifications. Briefly, previously ground samples (1 g) were extracted using 80% methanol with 0.1% HCl on a mechanical stirrer (Mechanical stirrer Thys 2) for 1 h. After that, the samples were centrifuged for 10 min at 4000× *g* (Janetzki T32c, Wallhausen, Germany) and filtered through Whatman No. 1 filter paper. The extraction procedure was repeated twice and supernatants were collected. Furthermore, combined supernatants were evaporated using a rotary evaporator to dryness (40 ◦C) (Laborota 4000, Heidolph Instruments, Schwabach, Germany) and reconstituted in 10 mL miliQ water for further analysis. The suspensions were filtrated through 0.45 μm syringe filters before further spectrophotometric and UHPLC-DAD MS/MS analysis. These extracts represented aqueous extracts of samples.

### *2.4. Total Phenolic and Flavonoid Content*

Total phenolic and flavonoid content in the blueberry/blackberry leaves and calluses were determined using a colorimetric assay with Folin-Ciocalteu's reagen<sup>t</sup> [36]; that is, assay with aluminium chloride [37]. Absorbance was measured at 765 nm for TPC and 510 nm for TFC, using a Shimadzu UV-1800 spectrophotometer (Shimadzu USA Manufacturing, Inc., Canby, OR, USA). Results for TPC were expressed in mg of gallic acid equivalent (mg GAE/g DW), while results for TFC were expressed as catechin equivalents (mg CE/g DW), both per g dry weight of samples.

### *2.5. UHPLC-DAD MS/MS Analysis of Leaves and Calluses*

The identification and quantification of phenolic compounds in leaves and calluses of 'Toro' and 'Caˇ ˇ canska Bestrna' was conducted using a Dionex Ultimate 3000 UHPLC system equipped with diode array detector (DAD) and TSQ Quantum Access Max triplequadrupole mass spectrometer (MS) (ThermoFisher Scientific, Basel, Switzerland), as previously detailed by Peši´c et al. [37]. A Syncronis C18 column (100 × 2.1 mm, 1.7 mm particle size) from Thermo Fisher Scientific was used as the analytical column for separation. The mass spectrometry data were acquired in the negative ion mode, in the m/z range from 100 to 1000. Full scanning and product ion scanning (PIS) were conducted for the qualitative analysis of the targeted phenolic compounds. The collision-induced dissociation experiments were performed using argon as the collision gas, and the collision energy varied depending on the compound. The time-selected reaction monitoring experiments for quantitative analysis were performed using two MS<sup>2</sup> fragments for each compound that was previously defined as dominant in the PIS experiments (Table S2). Other chromatographic and MS settings were the same as in Peši´c et al. [37]. However, it should be noted that in this study only MS data of commercially available standards were used for both identification and quantification of PCs. The Xcalibur software (version 2.2) was used for instrument control as well as for the acquisition and analysis of data. Calculation of the concentrations was based on the external standard method. Standards of phenolic compounds (gallic acid, vanillic acid, ferulic acid, syringic acid, chlorogenic acid, catechin, catechin gallate, gallocatechin, quercetin, quercetin-3- *O*-rutinoside, quercetin-3- *O*-glucoside, quercetin-3- *O*-rhamnoside, isohramnetin-3- *O*-rutinoside, isohramnetin-3- *O*-glucoside, kaempferol-3- *O*-glucoside, kaempferol, apigenin-7- *O*-glucoside, naringenin, aesculetin, and phlorizin) were obtained from Sigma Aldrich (Steinheim, Germany). The total amounts of each identified compound were evaluated via calculation of the peak areas and expressed as mg/kg dry weight (DW) of the sample.
