Cryobiotechnology of Plants: A Hot Topic Not Only for Gene Banks

: Agriculture has always been an important part of human evolution. Traditionally, farming is changing and developing with regard to challenges it faces. The major challenges of modern agriculture are food and nutrition safety for the growing world population. Promoting species and genetic diversity in agriculture appears to be an important approach to dealing with those challenges. Gene banks all around the world play a crucial role in preserving plant genetic resources for future crop improvements. The plant germplasm can be preserved in di ﬀ erent ways, depending on the species or form of stored plant tissue. This review focuses on a special preservation method—cryopreservation. Cryopreservation is an e ﬀ ective technique for storing living systems at ultra-low temperatures, usually in liquid nitrogen or its vapor phase. This conservation method is crucial for plants that do not produce seeds or that produce non germinating seeds, as well as for plants that propagate vegetatively. Moreover, based on the cryopreservation method, a novel plant biotechnology tool for pathogen eradication called cryotherapy has been developed. The use of liquid nitrogen eliminates plant pathogens such as viruses, phytoplasmas, and bacteria. Our article reviews recent advances in cryo-biotechnologies such as cryopreservation and cryotherapy, with special focus on studies concerning fruit plants.


Introduction
Plant genetic resources are highly important for the maintenance of agro-biodiversity and for food safety. These genetic resources, as donors of valuable traits, can be used to breed new, more productive crops with better resistance to biotic or abiotic stresses [1]. Seed storage is one of the most convenient methods for long-term conservation of plant genetic resources. However, a large number of plant species are not suitable for seed banking because they are highly heterozygous or have recalcitrant seeds that cannot be desiccated. These species are usually conserved in field collections. This type of collection is beneficial because it provides immediate access to plant material during all phenophases. However, plants in field collections are exposed to many environmental threats such as pests, diseases, and adverse weather conditions. Maintenance of this type of collection is also labor-intensive and expensive [2].
In vitro gene banks, where plants are vegetatively propagated and grown on a medium under sterile conditions, are an alternative to seed or field banking. In vitro-grown plants are stored in growing chambers that significantly save storage space and protect plants against harmful environmental factors. However, because the plantlets require periodical subcultivation on fresh medium, this method is not ideal for long-term storage [3].
Cryopreservation is the most valuable method for long-term conservation of plant germplasm. It is based on the storage of biological material at ultra-low temperatures in liquid nitrogen (−196 • C) or in its vapor phase (−150 • C). This temperature suspends cell division and metabolic and biochemical activities, thus preventing genetic alteration during long-term storage [4].
More than 60 years ago, the first work on plant cryopreservation was published. In 1956, Akira Sakai reported the successful survival of cold hardened and prefrozen mulberry twigs after exposure to liquid nitrogen [5]. The next challenging step in the field of cryopreservation was to freeze fully hydrated tissues such as callus and suspension cells, where there is a high risk of the formation of lethal intracellular ice-crystals. For this purpose, slow freezing protocols were developed. However, this method was not sufficient for cryopreservation of organized tissues (e.g., meristems), which led to the development of fast freezing protocols (encapsulation-dehydration, droplet vitrification, etc.) [6]. To date, several methods and techniques of cryopreservation have been reported. Nevertheless, the nub is always the same. Plant tissue is first physically or osmotically dehydrated to remove all freezable water, and to avoid water crystallization and lethal injuries during the following freezing in liquid nitrogen [7].
Ultra-low temperatures can also be successfully used for pathogen annihilation. This novel progressive technique is called cryotherapy and is based on protocols that have been originally established for cryopreservation. In the process of cryotherapy, infected plant cells are eliminated using the fatal efficacy of the liquid nitrogen (the ultra-low temperature) in combination with subsequent warming [8]. One of the major advantages of using ultra-low temperature treatment for pathogen eradication is that there is no need for special equipment other than that which is used in a basic plant tissue culture laboratory. This means that cryotherapy can be efficiently incorporated into the cryopreservation methods already used in gene banks [9].
In this review article, we present updated and comprehensive information concerning the development and progress of plant cryopreservation and cryotherapy, with a focus on horticultural crops and a special focus on apples, which belong to one of the most extensively studied species in this field.

Cryopreservation of Plants
Originally, plant cryopreservation was based on studies of the basic biology of freezing [10]. Gradual improvements in the technology and intensive research work over the past decades have resulted in great progress in the field of ultra-low temperature preservation of plant germplasm [11]. The development and improvement of cryopreservation techniques and their application to new plant species remains the center of attention of many cryogenic labs and gene banks around the world.

Plant Cryopreservation Methods
Nowadays, several methods of cryopreservation exist, including both classical and new techniques ( Figure 1). Advantages and disadvantages of each method, as well as other factors such as available facilities, plant species, and the type of stored germplasm, have to be considered during the process of method selection (Table 1). Often more than a single cryoprotocol is suitable for successful plant cryopreservation, considering some modifications of established methods [12]. The first step in cryopreservation is removing freezable water from tissues by dehydration. A water content of less than 0.25 g H 2 O g/dm (dm; dry mass) is often termed 'unfreezable' water, and plant cells containing 0.25-0.4 g H 2 O g/dm usually survive the liquid nitrogen exposure [13]. Proper dehydration can be achieved osmotically by treatment with highly concentrated solutions, in which case the driving force for dehydration is the concentration gradient between the solution and the intracellular liquid. The second widely used method of dehydration is air-drying, where the water is removed by the air flow [14]. In any case, the process of dehydration is essential for successful cryopreservation to avoid intracellular freezing and irreversible injury of cells caused by the formation of ice crystals [1]. Appl. Sci. 2020, 10, x FOR PEER REVIEW 3 of 14

Classical Cryopreservation Methods
Classical cryopreservation methods were developed more than 40 years ago, and two main techniques are traditionally used: (i) slow freezing, and (ii) simple one-step freezing [7]. Slow freezing (also known as the two-step freezing method) includes pretreatment of samples with cryoprotectants such as dimethylsulfoxide (DMSO), glycerol, ethylene glycol, and sucrose. The samples are then slowly cooled at a controlled rate of 0.3-0.5 °C/min to −40 °C, and then rapidly immersed in liquid nitrogen. This method requires a programmable freezer [15]. On the other hand, no special equipment is required for simple one-step freezing. Samples pretreated with cryoprotectants are simply frozen at −30 °C for dehydration, and then immersed directly in liquid nitrogen [16]. Although these classical methods have been successfully applied to a range of plant materials, new and gentler cryogenic methods such as vitrification, encapsulation, and cryo-plates have been developed recently.

Vitrification
This approach is one of the most widely applied plant cryopreservation methods because it is relatively easy to carry out, no special equipment is needed, and it usually displays a high percentage of recovery. Vitrification is based on the formation of an amorphous glassy structure from intracellular solutes [1]. The plant material is treated with a highly concentrated vitrification solution that removes most or all freezable water from cells. The dehydrated material is then ultra-rapidly frozen by immersion in liquid nitrogen. The combination of dehydration and rapid freezing of cells causes residual water to solidify without crystallization, which could injure the living cells [17].
Among them, PVS2 is the most widespread, because of its less toxic effect on plant cells. Nevertheless, it cannot be used without any osmoprotection, because direct dehydration by PVS2

Classical Cryopreservation Methods
Classical cryopreservation methods were developed more than 40 years ago, and two main techniques are traditionally used: (i) slow freezing, and (ii) simple one-step freezing [7]. Slow freezing (also known as the two-step freezing method) includes pretreatment of samples with cryoprotectants such as dimethylsulfoxide (DMSO), glycerol, ethylene glycol, and sucrose. The samples are then slowly cooled at a controlled rate of 0.3-0.5 • C/min to −40 • C, and then rapidly immersed in liquid nitrogen. This method requires a programmable freezer [15]. On the other hand, no special equipment is required for simple one-step freezing. Samples pretreated with cryoprotectants are simply frozen at −30 • C for dehydration, and then immersed directly in liquid nitrogen [16]. Although these classical methods have been successfully applied to a range of plant materials, new and gentler cryogenic methods such as vitrification, encapsulation, and cryo-plates have been developed recently.

Vitrification
This approach is one of the most widely applied plant cryopreservation methods because it is relatively easy to carry out, no special equipment is needed, and it usually displays a high percentage of recovery. Vitrification is based on the formation of an amorphous glassy structure from intracellular solutes [1]. The plant material is treated with a highly concentrated vitrification solution that removes most or all freezable water from cells. The dehydrated material is then ultra-rapidly frozen by immersion in liquid nitrogen. The combination of dehydration and rapid freezing of cells causes residual water to solidify without crystallization, which could injure the living cells [17].
Among them, PVS2 is the most widespread, because of its less toxic effect on plant cells. Nevertheless, it cannot be used without any osmoprotection, because direct dehydration by PVS2 causes damages to the cells and tissues of many plants. For successful cryopreservation, it is necessary to implement osmoprotection pretreatment with a loading solution (LS) containing 2 M glycerol and 0.4 M sucrose that induces osmo-tolerance and enables plants to achieve higher rates of recovery after cryopreservation [21,22].
Two main vitrification-based methods, namely encapsulation-vitrification and droplet vitrification, have been developed via the modification and optimization of vitrification techniques. The encapsulation-vitrification method was first reported by Matsumoto et al. (1995) [23] and combines encapsulation of plant germplasm within alginate beads and dehydration using a highly concentrated vitrification solution. The major merits of this mixed technique are better protection of encapsulated plant samples during vitrification and reduction of time needed for dehydration, compared to that of the classical encapsulation-dehydration method [22]. Using the method called droplet vitrification, shoot tips pretreated with LS and subsequently treated with vitrification solution are inserted individually into droplets of PVS2 that are placed on a strip of aluminum foil. The whole aluminum foil is then directly immersed in liquid nitrogen. The main benefit of this technique is the achievement of very high cooling/warming rates [24,25].

Encapsulation-Dehydration
This method was first reported by Fabre and Dereuddre (1990) [26] and combines the encapsulation of plant samples with alginate beads and physical dehydration carried out with silica gel or in the air flow in a laminar flow cabinet [1]. Applying this method, plant material is precultured with 0.3-0.6 M sucrose medium for one to three days and then incubated in a liquid medium supplemented with sucrose and sodium alginate. Finally, this mixture is released drop by drop into liquid medium containing calcium chloride. Alginate beads with explants encapsulated inside them are formed during this process. The bead formation is followed by culturing in highly concentrated sucrose solution to achieve the osmoprotection, subsequent physical dehydration to a water content of 20%-30%, and direct immersion in liquid nitrogen ( Figure 2) [26]. The encapsulation-dehydration method is relatively simple; however, it requires more time-consuming handling of encapsulated samples. The main advantage of this method is elimination of the need for other cryoprotectants such as DMSO and ethylene glycol that could be toxic for plants and could cause genetic changes after regrowth [22]. sucrose solution to achieve the osmoprotection, subsequent physical dehydration to a water content of 20%-30%, and direct immersion in liquid nitrogen (Figure 2) [26]. The encapsulation-dehydration method is relatively simple; however, it requires more time-consuming handling of encapsulated samples. The main advantage of this method is elimination of the need for other cryoprotectants such as DMSO and ethylene glycol that could be toxic for plants and could cause genetic changes after regrowth [22].

Cryo-Plate Methods
The newest cryogenic procedures use cryo-plates. Two main cryo-plate methods, known as the V and D cryo-plate methods, can be distinguished [22] based on the dehydration process. Using the former method, explants are dehydrated on cryo-plates using PVS2 vitrification [27], whereas the latter method uses air dehydration of explants [28]. In both methods, shoot tips are placed in small wells of an aluminum cryo-plate with alginate beads and dehydrated there with PVS2 solution or with air flow in a laminar flow cabinet, depending on the particular method. Afterwards, the cryo-plate is directly immersed into liquid nitrogen. The main advantage of cryo-plate techniques is their user-friendliness, mainly due to the easy handling of samples on the aluminum plates [27,28]. Successful cryopreservation using cryo-plates methods has been reported for many plant species including strawberry [29], sugarcane [30], date palm [31], mat rush [32], and potato [33].

Pathogen-Free Plant Material
Phytoplasmas, bacteria, viruses, and other plant pathogens cause harmful plant diseases that negatively affect crop yield, crop industry, and food safety every year [8,34]. Vegetatively propagated plants tend to accumulate pathogens that are transmitted to new plants in infected cuttings, tubers, roots, and other vegetative propagules [35]. Pathogen-free plant material is essential not only for higher productivity of agricultural and horticultural corps, but is also pivotal for long-term preservation of plant germplasm [36].

Convential Methods for Pathogen Eradication
Conventional methods for pathogen eradication are based on in vitro meristem culture, as well as heat treatment (thermotherapy) and chemical treatment (chemotherapy), both of which are followed by meristem culture [37][38][39][40][41]. Although these traditional methods have been widely used for acquiring virus-free plants, all of them have some drawbacks.
The meristem culture method is based on the assumption that the youngest meristematic cells are free of viruses and other plant pathogens. Therefore, the extension and regeneration of small shoot tips containing the meristem (0.2-0.4 mm) and two to four leaf primordia should lead to pathogen-free plants [36,39]. The major limitations of meristem culture techniques are difficulties in excising tiny meristems and a low regeneration rate of shoot tips.
The use of heat treatment is another well-known conventional method for pathogen eradication in plants. Generally, thermotherapy is based on keeping the target in vitro cultivated plants at a temperature of 35 • C-42 • C for four to six weeks. Both temperature range and duration of thermotherapy are virus-type-and plant-species-dependent [42,43]. The need for specific and expensive laboratory equipment such as a growth chamber with precise temperature control is the major limitation of thermotherapy [44]. Moreover, this method cannot be used for infected plants that are not resistant to higher temperatures and it does not work for all viruses [45].
Another method used for plant pathogen elimination is chemotherapy. In this technique, in vitro-grown plants are treated with antiviral chemicals like ribavirin, whose positive effect on virus eradication has been reported [46,47]. In this method, optimization of the dose and duration of the treatment is crucial because a high concentration of antiviral compounds in the culture medium can negatively affect the growth of in vitro plants through their phytotoxic activity. The sensitivity of in vitro plants to antivirotics is species-and genotype-specific [45,47]. Because of the abovementioned limitations linked to traditional methods for pathogen eradication, it would be beneficial to develop some more efficient and simpler methods for obtaining pathogen-free plants. In this respect, cryotherapy, i.e., treatment with ultra-low temperatures, could be a useful biotechnological tool with great potential.

Cryotherapy
Cryotherapy of shoot tips as a method for pathogen elimination from infected plants was first reported in 1997 by Brison et al. [48], who successfully eradicated Plum pox virus (PPV) from in vitro-grown infected shoot tips of interspecific Prunus rootstock [48]. The crux of this technique consists in the treatment of infected materials in liquid nitrogen for a short period of time [49,50]. Cryotherapy relies on plant cryopreservation protocols that are available for a wide range of vegetatively propagated and economically important plant species. Thus, cryotherapy has been successfully applied to many plant species such as potato (Solanum tuberosum L.) [51], sweet potato (Ipomea batatas L.) [52], banana (Musa spp.) [53], raspberry (Rubus idaeus L.) [54], grapevine (Vitis vinifera L.) [55,56], and apple (Malus spp.) [57][58][59].
To date, large numbers of pathogens (mostly viruses) have been eradicated via cryotherapy. These include, for example, two common viruses infecting sweet potatoes-sweet potato feathery mottle virus (SPFMV) and sweet potato chlorotic stunt virus (SPCVS)-that interact synergistically and cause the sweet potato virus disease (SPVD). Both these viruses can be eliminated with 100% efficiency using cryotherapy [60,61]. Cryotherapy was also successfully applied to the eradication of two viruses infecting potato plants: potato leafroll virus (PLRV) and potato virus Y (PVY), which were among the most important viruses limiting sustainable and profitable potato production [51]. Cucumber mosaic virus (CMV) and banana streak virus (BSV) viruses, which infect bananas and cause diseases that are linked to a reduction in fruit yield or obstruction of breeding and germplasm dissemination, were effectively eradicated by cryotherapy [53]. Successful applications of cryotherapy was also reported for viruses infecting grapes, such as grape virus A (GVA) and grapevine leafroll-associated viruses (GLRaV), that cause economic losses in viticulture [55,56,62]. In addition, viruses infecting less common crops such as garlic and artichoke were recently eradicated using ultra-low temperature treatment [63,64]. Other pathogens such as phytoplasmas and bacteria can also be eradicated from plants via cryotherapy. For example, the sweet potato little leaf phytoplasma (SPLL), which causes heavy yield losses in infected plants, was efficiently eliminated from all sweet potato shoot tips via treatment with liquid nitrogen [60]. Another, more recent example of eradication of phytoplasma by using cryotherapy was reported in 2015. Jujube witches' broom phytoplasma (Candidatus Phytoplasma ziziphi) was fully eliminated from infected Chinese jujube (Ziziphus jujuba) plants [65]. So far, only one successful elimination of bacteria-a Gram-negative bacteria attacking Citrus, Fortunella, and Poncirus species and causing citrus Huanglongbing disease (HLB) disease, also called "citrus greening"-from infected plants by cryotherapy was reported [66]. Ding et al. demonstrated that cryotherapy is a powerful tool for elimination of this bacteria from infected sweet orange (Citrus sinensis L.) and other citrus species such as mandarin, pomelo, and Beijing lemon.

Mechanism
Cryotherapy is mostly applied to shoot tips, because they contain a unique meristematic zone consisting of small cells with small vacuoles and a higher nucleo-cytoplasmic ratio [67]. Those cells are more resistant to dehydration, which prevents the formation of ice crystals in cells during freezing [68]. Simply, cryo-treatment with liquid nitrogen destroys the differentiated cells, while meristematic cells survive and are able to self-renew, divide, differentiate, and regenerate to new virus-free plants (Figure 3). Because of this phenomena, cryotherapy is most effective for the elimination of pathogens infecting differentiated cells such as banana streak virus (BSV), cucumber mosaic virus (CMV), grape virus A (GVA), potato leafroll virus (PLRV), and potato virus Y (PVY) [51,53,62]. On the other hand, the eradication of pathogens that are able to infect meristematic cells, such as raspberry bushy dwarf virus (RBDV), pelargonium flower break virus (PFBV), and pelargonium line pattern virus (PLPV), is significantly more complicated [67,69]. It has been reported that a combination of cryotherapy and thermotherapy led to virus-free plants. Thermotherapy first inhibits movement of the virus toward the meristematic cells of the shoot tips and at the same time causes subcellular alterations, for example, enlargement of vacuoles, which results in much fewer surviving cells after subsequent cryotherapy. The combination of these two techniques enables an enhancement in the eradication of viruses localized in the meristem [67].

Merits and Demerits
Compared to other methods for pathogen eradication in plants, cryotherapy of shoot tips has the following advantages: (i) easier handling with meristems due to the lack of correlation between the size of the shoot tip and pathogen eradication rate [49,70]; (ii) high efficiency of pathogen eradication [49,71]; (iii) no need for special and expensive equipment, and (iv) it is easy to handle a large number of samples [36,49]. The major limitation of the wider application of the cryotherapy to produce pathogen-free plant material is the genotype-or often also cultivar-dependent response of plants to cryo-treatment. This means that each cryoprotocol needs to be developed and optimized for every single plant species or cultivar [68,72]. Although the risk of somaclonal variation during shortterm cryotherapy is minimal, it is important to verify the genetic stability of regenerated plants [72].

Cryobiotechnology of Malus (Apple)
Many protocols used for cryopreservation of fruit plants have been reported. However, longterm 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

Merits and Demerits
Compared to other methods for pathogen eradication in plants, cryotherapy of shoot tips has the following advantages: (i) easier handling with meristems due to the lack of correlation between the size of the shoot tip and pathogen eradication rate [49,70]; (ii) high efficiency of pathogen eradication [49,71]; (iii) no need for special and expensive equipment, and (iv) it is easy to handle a large number of samples [36,49]. The major limitation of the wider application of the cryotherapy to produce pathogen-free plant material is the genotype-or often also cultivar-dependent response of plants to cryo-treatment. This means that each cryoprotocol needs to be developed and optimized for every single plant species or cultivar [68,72]. Although the risk of somaclonal variation during short-term cryotherapy is minimal, it is important to verify the genetic stability of regenerated plants [72].

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. Wu 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 Niino and Sakai [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 Halmagyi 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. Sakai and Nishiyama (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 • 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]. Most protocols used for ultra-low temperature storage of dormant apple buds are based on the original outlines by Forsline [93,94], involving: cutting of winter hardened twigs in segments containing the dormant bud, desiccation at a temperature from −4 • C to −5 • C of the segments until moisture content is reduced to about 30%, cooling in a programmable slow cooler to a final temperature of −30 • C, and placing the cooled segments in liquid nitrogen vapor or liquid nitrogen for long-term storage. For plant regeneration, recovered scions with dormant buds are grafted directly on pot-grown rootstocks, or it is also possible to excise shoot tips from cryopreserved buds and place them on culture medium [95]. Successful regeneration of cryopreserved buds is tightly linked with the cold hardening of these buds before cryopreservation, rather than with the phase of endodormancy [96].
Several papers in recent years focused on pathogen eradication in apples by using cryotherapy. For example, the vitrification cryotherapy technique was reported by Romadanova et al. (2016) [97]. They infected apple cultivars and rootstocks and treated them with ultra-low temperatures to eliminate four different viruses (apple chlorotic leaf spot closterovirus (ACLSV), apple stem pitting virus (ASPV), apple stem grooving virus (ASGV), apple mosaic virus (ApMV)). Their therapy resulted in the production of virus-free plants for seven out of nine tested cultivars. In another study, the encapsulation-dehydration method was applied to in vitro-grown apple rootstocks M9 and M26 to eliminate ASPV and ASGV viruses. Cryotherapy was successful for ASPV but failed for ASGV. A probable explanation of this result was the different distribution of viruses in shoot tips. Although ASPV was not detected in the upper part of the apical dome and leaf primordia, ASGV was [89]. Results obtained two years later also showed that elimination of ASGV by cryotherapy had some limitations due to its localization in the shoot tip [58]. Finally, a recent study from the same research group examined the effect of another cryotherapy technique, i.e., droplet-vitrification, on the eradication of three latent apple viruses (ACLSV, ASGV, ASPV) from an infected 'Monalisa' apple cultivar. Cryotherapy was successful for the elimination of ASPV and ACLSV with 100% and 95% efficiencies, respectively. However, only 35% of regenerated plants were free of ASGV [59].

Conclusions
Cryopreservation is a progressive method for long-term storage of plant germplasm that uses the ultra-low temperature of liquid nitrogen to minimalize the metabolism of living cells. This technique has great application potential in biotechnology, agriculture, horticulture, and breeding programs. Several methods and protocols for cryopreservation of plant germplasm were reviewed in this manuscript, and each of them has its merits and demerits, which should be considered before cryopreservation (Table 1). Cryopreservation can serve as an alternative storage solution to the traditional field collections. It also has possible applications in the conservation of endangered plant species germplasm. Ultra-low temperatures can be used not only for the storage of germplasm, but also for the eradication of plant pathogens, for which the term "cryotherapy" was coined. Cryotherapy of shoot tips is a promising method for plant pathogen eradication that can be easily used for different plant species and cultivars with available cryopreservation protocols. It can be carried out in a basically-equipped tissue culture laboratory, shows promising results in production of pathogen-free regenerants, and minimizes the risk of genetic changes during treatment compared to the classical methods. To date, more than 10,000 accessions of in vitro propagated crop plants are safely cryopreserved for the long term, and over 80% of these belong to five main crops (potato, cassava, bananas, mulberry, and garlic). In this review, we show that much of the work has already been done in the field of plant cryobiotechnology. However, some important challenges still remain and limit a more scaled applications of cryopreservation. For example, post-thaw regeneration of some important crop species (cassava, sweet potato) is still extremely low. Some plant species survive cryopreservation, but they are not able to root and stop the growth and development [6]. Altogether, for the future, the optimization and modification of the existing protocols will be required for more plant species and cultivars.