## 3 Results  
Of Golden Delicious A and B, Virginia Crab, and Panenske zlepsene, only Golden Delicious B tested positive for AP phytoplasma after the establishment of the in vitro cultures. This result shows that the dissection of the apical part of the explant combined with the sterilization process was able to eliminate the AP phytoplasma infection in three of the total four apple genotypes. PCR testing of Taborita, after multiplication from the initial in vitro culture kept in the microorganism bank, confirmed the presence of phytoplasma. ApMV and ACSLV were also detected in both cultivars. In the Taborita cultivar, all three pathogens were present at high levels. In the case of Golden Delicious, AP phytoplasma and ACLSV were also present at high levels, while the test result for ApMV was only slightly positive (a Ct value of 29, Table 2). The appearance of the infected cultures varied: Golden Delicious showed no significant symptoms in growth and shoot morphology, except for smaller and narrower leaves compared to the uninfected in vitro cultures of this cultivar (Figure 1a). Taborita showed stunted growth, dwarfed undeveloped leaves, and extreme lateral shoot proliferation (Figure 1b). After the cryopreservation and subsequent thawing, five out of thirteen Golden Delicious shoot tips and five out of ten cryopreserved Taborita shoot tips were regenerated (Figure 2). Three Golden Delicious tips and four Taborita tips that showed a high ability to form viable shoots were selected for culture establishment. A shoot size sufficient for PCR testing was achieved after five months and five consecutive subcultures. The regenerated Golden Delicious shoot tips produced visually healthy shoots with apparently larger leaves than before the cryopreservation (Figure 3a). The Taborita cultures did not thrive after thawing; although it was possible to obtain a sufficient amount of material for PCR testing, the viability of all the shoots declined over the following months, followed by the gradual death of all the material (Figure 3b). \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Sample** & **DNA Internal** & **AP Phytoplasma** & **RNA Internal** & **ApMV** & **ACLSV** \\  & **Control O Ct** & \(\mathcal{O}\)**Ct** & **Control \(\mathcal{O}\) Ct** & \(\mathcal{O}\)**Ct** & \(\mathcal{O}\)**Ct** \\ \hline \multicolumn{6}{c}{_Before cryotherapy_} \\ Golden Delicious A (1) & 15.82 & Negative & - & - & - \\ Golden Delicious A (2) & 13.47 & Negative & - & - & - \\ Golden Delicious B (1) & 10.74 & 15.74 & - & - & - \\ Golden Delicious B (2) & 9.97 & 14.81 & - & - & - \\ Golden Delicious B (3) & 10.13 & 15.03 & - & - & - \\ Golden Delicious B (4) & 11.89 & 16.73 & - & - & - \\ Golden Delicious B (5) & - & - & 17.58 & 29.00 & 13.95 \\ Virginia Crab & 9.12 & Negative & - & - & - \\ Panenske zlepsene (1) & 11.03 & Negative & - & - & - \\ Panenske zlepsene (2) & 11.93 & Negative & - & - & - \\ Taborita (1) & 11.12 & 14.75 & - & - & - \\ Taborita (2) & 9.97 & 13.66 & - & - & - \\ Taborita (3) & - & - & 17.00 & 13.42 & 14.04 \\ _After cryotherapy_ & - & - & - & - & - \\ Golden Delicious B & 10.00 & Negative & 16.74 & Negative & Negative \\ Taborita & 10.01 & 30.88 & 16.35 & 14.73 & 13.90 \\ \hline \hline \end{tabular} \(\mathcal{O}\) Ct—average PCR cycle threshold value (samples run in triplicate); AP—apple proliferation; ApMV—apple mosaic virus; ACLSV—apple chlorotic leaf spot virus.  
\end{table} 
Table 2: Detection of phytoplasma and viruses in the explant cultures before and after cryotherapy by PCR testing. None of the infections found in the original material, i.e., AP phytoplasma, ApMV, and ACSLV, were detected in the regenerated Golden Delicious cultures. On the other hand, in the case of the Taborita cultivar, the tests showed that all the pathogens were still present, although AP phytoplasma was present at very low levels (a Ct value of 30.9), and ApMV and ACSLV also showed a moderate relative decrease (Table 2). Figure 1: Initial in vitro cultures of: (**a**) Golden Delicious; (**b**) Taborita.