# Cryobiotechnology of Plants: A Hot Topic Not Only for Gene Banks
## 1 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 \({}^{\circ}\)C)or in its vapor phase (\(-150\,^{\circ}\)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, [PERSON] 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.