# Cryobiotechnology of Plants: A Hot Topic Not Only for Gene Banks
## 4 Cryobiotechnology of _Malus_ (Apple)  
Many protocols used for cryopreservation of fruit plants have been reported. However, long-term germplasm storage of economically important horticultural woody plants in cryopreserved collections remains well-established, mainly for apple and pear plants [73; 74]. Furthermore, among all plant species, _Malus_ (apple) is one of the most extensively studied with respect to cryopreservation of the plant germplasm over the last decades. Interestingly, many protocols applied to other species originated from cryopreservation procedures that were first demonstrated with apples [73]. Traditionally, field collections and in vitro culture are used for preservation of _Malus_ germplasm. Seed banking can be applicable only for the preservation of wild _Malus_ species germplasm, since apple is genetically highly heterozygous [75; 76]. Cryopreservation of apple seeds is possible after their drying to achieve 6%-19% moisture content [77]. It is valuable to cryopreserve pollen for immediate availability for breeding programs. Successful cryopreservation of _Malus_ pollen has been reported many times. According to the published data, this pollen stored in liquid nitrogen can retain viability for at least 15 years [78; 79; 80]. The cryopreservation of shoot tips of in vitro-grown plants and cryopreservation of dormant buds are the most commonly used techniques for ultra-low temperature long-term storage of vegetatively propagated species (cultivars) [81]. Up to date, all known techniques for cryopreservation of shoot tips were used for _Malus_. The very first experiments concerning _Malus_ in vitro shoot tip cryopreservation used classical two-step freezing [82; 83]. The first experiments failed to enable regrowth of frozen shoot tips, suggesting that excessive cryo-injury occurred in the treated cells during the cryoprocedure. [PERSON] et al. [84] overcame this problem in 1999 via optimization of the cryoprotocol by inserting the pretreatment of shoot tips with a cryoprotectant mixture and achieved a 66% shoot regrowth. Other early experiments were accomplished by using the PVS2 vitrification technique, with an average regrowth level of about 66% in different examined apple cultivars, rootstocks, and wild apple species [85]. Studies on the cryopreservation of in vitro shoot tips by encapsulation, linked with either dehydration or vitrification, have been reported. The first encapsulation-dehydration protocol was described in 1992 by [PERSON] and [PERSON] [86] and subsequently more cryoprotocols using this or encapsulation-vitrification method were developed or optimized for a wide spectrum of _Malus_ cultivars [84; 87; 88; 89]. Finally, in 2010 [PERSON] et al. tested [90] the efficiency of two droplet cryopreservation techniques, droplet-vitrification and droplet-freezing, that included in vitro-grown shoot tips as materials for long-term storage. This study showed that the droplet-vitrification method allowed a high regrowth level of cryopreserved shoot tips, and consequent study [91] pointed out the effects of preculture conditions on the level of regenerated shoot tips after cryopreservation using this method. [PERSON] and [PERSON] (1978) [92] were the first to mention the cryopreservation of in vivo apple dormant buds. In general, dormant bud cryopreservation procedures consist of desiccation and slow cooling to \(-\)35 \({}^{\circ}\)C prior to liquid nitrogen exposure [73]. Based on data from the National Laboratory for Genetic Resources Preservation (NLGRP), the lack of a need for aseptic cultures, high processing throughput, and approximately ten-times-lower cost of cryoprocessing compared to shoot tip cryopreservation are the major advantages of this method [74].