## 1 Introduction  
Apples (_Malus domestic_ L.) have long been the most economically important fruit crop in the conditions of the central European temperate climate zone. In the territory of the Czech Republic, 5864 hectares of production plantations of apple trees were registered in 2022, which represents almost half of the total area of commercial orchards. The importance of this species is also evidenced by the fact that the fruit's nursery sector produced a total of 1,288,554 apple trees, which is more than the production of trees of all major commercial stone fruit species in the last statistically processed year of 2021 [1]. The nursery production of fruit trees is a stabilized, economically significant branch of crop production with slightly growing potential. However, apple trees can be infected with several viruses and phytoplasmas, often in mixed infections. The synergistic negative effect of these systemic pathogens then reduces fruit yields and quality, tree vigor, and fruit orchards' longevity. Trees infected with viruses and virus-like pathogens cannot be sanitized after planting in a permanent location. The infection is often systemic, and the economic damage persists to varying degrees throughout the lifetime of an individual tree in the production orchard [2]. Therefore, the nursery production of healthy, pathogen-free apple-starting propagation material has a demonstrable benefit in increasing the competitiveness of growers by producing fruits of better quality and yield. Apple chloride leaf spot virus (ACLSV), apple mosaic virus (ApMV), and _Candidatus_ Phytoplasma mali-causing apple proliferation (AP) are among the most important systemic infections of apple trees in Europe [2]. ACLSV is widespread and the most prevalent latent virus of apples in the Czech germplasm collection, with an infection rate exceeding 90% [3]. Cryopreservation of the shoot tips taken from in vitro-grown explants has been widely used as a potential tool for plant material sanitation. Rather inconsistent results have been reported, dependent on specific pathogens and plant material, and different opinions have been expressed on the applicability of cryotherapy. Numerous cryopreservation protocols have been published [4], of which vitrification is one of the most widely used. This procedure includes--in many variations--acclimatization, treatment with a loading solution (LS), the vitrification itself, freezing in liquid nitrogen, thawing, the unloading of the shoot tips with an unloading solution (ULS), and regeneration. To date, different compositions of plant vitrification solutions (PVS) have been used so far for different plant species [5]. The eradication of apple stem pitting virus (ASPV) and ACLSV from in vitro-grown apple cultures by cryopreservation was very successful [6]. An attempt to use cryotherapy for the apical buds of hop explants infected with ApMV did not yield results that would indicate the effectiveness of this approach [7], whereas ApMV was successfully eradicated for hazelnut [8]. For apple stem grooving virus (ASGV), present in apple rootstocks, exposure to liquid nitrogen was not effective unless preceded by thermotherapy. The combination of thermotherapy and cryotherapy has also been suggested for ACLSV [9]. The results of virus cryotherapy vary among apple cultivars [10], and different cryopreservation techniques may lead to different results [11]. On the other hand, cryopreservation can also be used for the opposite purpose--as a method to preserve some viruses. In support of this possibility, ASGV was shown to persist in the cryopreserved shoot tips of the apple cultivar Gala [12] be in agreement with the results obtained by [PERSON] et al. [9]. bases and from different production groups. These were two clones of the Golden Delicious variety as representatives of the main commercial segment of the apple market, two original Czech cultivars, Taborita (landrace) and Panenske zlepsene (Panenske ceske x Jonathan), and the Virginia Crab apple as a representative of the cider production segment.