# Cryobiotechnology of Plants: A Hot Topic Not Only for Gene Banks
## 3 Pathogen-Free Plant Material
### Cryotherapy  
Cryotherapy of shoot tips as a method for pathogen elimination from infected plants was first reported in 1997 by [PERSON] 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 butatas_ L.) [52], banana (_Mus_ spp.) [53], raspberry (_Rubus ideaus_ 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 chloride 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]. [PERSON] 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.