## 2 Materials and Methods  
Experimental cultures of the Taborita cultivar infected with AP phytoplasma were established using actively growing explants from the in vitro microorganism gene bank maintained at the Research and Breeding Institute of Pomology (RBIP), Holovousy Ltd. (Czech Republic). The starting material of the apple cultivars Golden Delicious (clones A and B), Virginia Crab, and Panenske zlepsene was obtained from phytoplasma-infected plants growing in technical isolation of the RBIP within the in vivo microorganism bank. The plant material of these cultivars had been tested before the start of this project (Table 1). The scions were cut at the end of the dormancy phase and then kept in water under laboratory conditions. At the time of first sprouting, the apical parts of buds (5-10 mm) containing a meristematic area were excised, and the covering scales were peeled off. The resulting explants were then immersed in 0.15% of HgCl\({}_{2}\) for 1 min. Following sterilization, they were rinsed twice with demineralized water. The buds were dried on sterile filtration papers in a laminar flow cabinet, and 10 explants of each cultivar were planted on a growth medium in Erlenmeyer flasks. The composition of the cultivation medium was based on Murashige and Skoog [15]. Two plant growth regulators were added: 1.5 mg/L of 6-benzylaminopurine and 0.1 mg/L of indole-3-butyric acid. The medium was solidified with 8.5 g/L of a Difco Bacto agar and autoclaved at 121 \({}^{\circ}\)C and 200 kPa for 20 min. The material was maintained in a cultivation room in a 12 h photoperiod and 22 \(\pm\) 2 \({}^{\circ}\)C. All manipulations with explants were performed under sterile conditions in a laminar flow cabinet. The material was transferred to a fresh medium every day due to leakage and oxidation of polyphenic substances manifested by the browning of explants and the medium at the beginning of cultivation. Consequently, the subcultivation interval was extended to 1 month until a sufficient number of shoots was obtained. Prior to cryopreservation, random mixed samples consisting of stem and leaf material from 4-5 mericlones were collected from the Erlenmeyer flasks containing the in vitro cultures of all apple cultivars tested in order to perform PCR detection of AP phytoplasma. If positive, the material was also tested for the presence of ApMV and ACLSV. For Golden Delicious A, 2 samples were collected for phytoplasma testing. For Golden Delicious B, 4 samples were taken for phytoplasma testing and 1 for virus testing. For Virginia Crab, 1 sample was collected for phytoplasma testing, and for Taborita, 2 samples were collected for phytoplasma testing and 1 for virus testing. Briefly, the total DNA was isolated from 100 mg of leaves ground in liquid nitrogen using an Exgene Plant SV isolation kit (GeneAll Biotechnology, Seoul, Republic of Korea), \begin{table}
\begin{tabular}{c c c} \hline \hline
**Sample** & \
\begin{tabular}{c} **DNA Internal** \\ **Control Ø Ct** \\ \end{tabular} &
\begin{tabular}{c} **AP Phytoplasma** \\ **Ø Ct** \\ \end{tabular} \\ \hline Golden Delicious A & 12.29 & 14.99 \\ Golden Delicious B & 11.09 & 15.09 \\ Virginia Crab & 11.94 & 15.69 \\ Panenske zlepsene & 12.40 & 16.05 \\ \hline \hline \end{tabular} \(\mathcal{O}\) Ct—average PCR cycle threshold value (samples run in triplicate); AP—apple proliferation.  
\end{table} 
Table 1: Phytoplasma detection in initial in vivo plants by PCR testing. according to the manufacturer's instructions. For virus diagnostics, RNA was isolated from 50 mg of pulverized leaves ground in liquid nitrogen using a Ribospin Plant kit (GeneAll Biotechnology), following the manual. The RNA was then reverse-transcribed using M-MLV Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA, USA). For real-time PCR detection, a qPCR 2x Blue Master Mix (Top-Bio) was used under the following general conditions: samples run in triplicate; final volume, 20 \(\mu\)L; final concentration of primers/probes, 0.5 \(\mu\)M; template, 2 \(\mu\)L of isolated DNA/cDNA.