## 2 Materials and Methods
AP phytoplasma and virus detection was carried out in a Rotor-Gene Q (Qiagen) cycler utilizing the same temperature profile: denaturation, 94 \({}^{\circ}\)C/5 min; cycling, 45\(\times\) (94 \({}^{\circ}\)C/20 s, 58 \({}^{\circ}\)C/20 s with fluorescence reading, 72 \({}^{\circ}\)C/20 s). Primers and probes were designed using Geneious Prime software 2023.1 (Biomatters Ltd., Auckland, New Zealand);  
AP phytoplasma detection:  
primers 5\({}^{\prime}\)-GCAGCTGGCGTAATACATGG, 5\({}^{\prime}\)-GAATTCCACTTGCCCTATCCA, and a probe 6-FAM-5\({}^{\prime}\)-AGTTCAACGCTTTGTGGTAT. ApMV detection:  
primers 5\({}^{\prime}\)-CGAAACTTACCCACGAAAAGGATG, 5\({}^{\prime}\)-CATCTGCGCATGAAACCCTCTC, and a probe 6-FAM-5\({}^{\prime}\)-TCCTTAAAGCTCGAATYAAAAGGCAG. ACLSV detection:  
5\({}^{\prime}\)-GGTGAGAGGCCTCTATTCACATCTG, 5\({}^{\prime}\)-CAGAATAAATTCGAGCTTTTCACC, and a probe 6-FAM-5\({}^{\prime}\)-TCGCAGAAGGGGATTTCCAATTGCCT. The presence of chloroplast 16S rDNA and the mitochondrial Nad5 transcript was used as an internal control for AP phytoplasma and virus detection, respectively. The relative load of a pathogen was related to the level of an internal control using the formula:  
\[2^{\text{(Ct [internal control] - Ct [pathogen])}}.\]  
Cultivars Golden Delicious B and Taborita, which were positively tested for phytoplasma and viruses, were cryopreserved. Cryopreservation of both cultivars was carried out according to a modified protocol published by [PERSON] [16]. The cultures were transferred to an incubator (PHCbi MIR-154) to grow under short photoperiod conditions (8 h: 22 \({}^{\circ}\)C, light; 16 h: \(-1\)\({}^{\circ}\)C, darkness) after 2 month-growth in the cultivation room. After 12 days of acclimatization in the incubator, shoot tips (2 mm in length) were excised under a stereoscopic microscope. Excised shoot tips were put in Petri dishes on a growth medium containing 5 vol% of dimethyl sulfoxide (DMSO) and kept on this medium for 2 days in the incubator. In the middle of this period, they were transferred to a fresh medium to avoid adverse effects of the medium and explant browning. The tips were then immersed in 1 mL of LS containing 2 M of glycerol and 0.5 M of sucrose in a liquid growth medium in cryoyvials for 30 min. The LS was then exchanged for 0.75 mL of PVS2 containing 4 M of glycerol, 2 M of DMSO, 2.7 M of ethylene glycol, and 0.2 M of sucrose in a liquid growth medium. After 30 min, the vials were immersed in liquid nitrogen and stored in liquid nitrogen for 1 week. Thawing was performed by immersing the cryovials in a 40 \({}^{\circ}\)C water bath for 1 min. The PVS2 was replaced with 1 mL of ULS, containing 1.2 M of sucrose in a liquid growth medium. After 20 min, the shoot tips were dried on filter paper and planted in Petri dishes with the growth medium. They were kept in darkness for 1 week and then transferred to standard conditions in the cultivation room. The regenerating material was gradually replanted to Erlenmeyer flasks and kept on the standard growth medium with a subcultivation interval of 1 month. When a sufficient amount of biomass was reached, a mixed sample, consisting of all shoots derived from the successfully regenerated tips, was taken for both cultivars in order to perform PCR testing. The presence of all three initial pathogens was detected in both cultivars after cryotherapy.