Cryotherapy of shoot tips is a new method for pathogen eradication based on cryopreservation techniques. Cryopreservation refers to the storage of biological samples at ultra‐low temperature, usually that of liquid nitrogen (−196°C), and is considered as an ideal means for long‐term storage of plant germplasm. In cryotherapy, plant pathogens such as viruses, phytoplasmas and bacteria are eradicated from shoot tips by exposing them briefly to liquid nitrogen. Uneven distribution of viruses and obligate vasculature‐limited microbes in shoot tips allows elimination of the infected cells by injuring them with the cryo‐treatment and regeneration of healthy shoots from the surviving pathogen‐free meristematic cells. Thermotherapy followed by cryotherapy of shoot tips can be used to enhance virus eradication. Cryotherapy of shoot tips is easy to implement. It allows treatment of large numbers of samples and results in a high frequency of pathogen‐free regenerants. Difficulties related to excision and regeneration of small meristems are largely circumvented. To date, severe pathogens in banana (Musa spp.), Citrus spp., grapevine (Vitis vinifera), Prunus spp., raspberry (Rubus idaeus), potato (Solanum tuberosum) and sweet potato (Ipomoea batatas) have been eradicated using cryotherapy. These pathogens include nine viruses (banana streak virus, cucumber mosaic virus, grapevine virus A, plum pox virus, potato leaf roll virus, potato virus Y, raspberry bushy dwarf virus, sweet potato feathery mottle virus and sweet potato chlorotic stunt virus), sweet potato little leaf phytoplasma and Huanglongbing bacterium causing ‘citrus greening’. Cryopreservation protocols have been developed for a wide variety of plant species, including agricultural and horticultural crops and ornamental plants, and can be used as such or adjusted for the purpose of cryotherapy.

Shoot tips with 3-4 leaf primordia were excised from in vitro -grown sweetpotato plants ( Ipomoea batatas ) infected with little leaf phytoplasma ( Candidatus Phytoplasma aurantifolia) and subjected to cryotherapy. All plants regenerated from the cryo-treated shoot tips were free of phytoplasma, whereas shoot tip culture or dehydration of shoot tips without subsequent cryotherapy resulted in phytoplasma-free plants at a frequency of only 7-10%. Histological and ultrastructural studies with light and transmission electron microscopy, respectively, indicated that cryotherapy was lethal to all cells except those in the apical dome of the meristem and the two youngest leaf primordia. These surviving parts of the shoot tip contained vascular tissue and sieve elements, but electron microscopy showed no phytoplasma in them. In contrast, an abundance of phytoplasma was found in sieve elements located at the lower, non-surviving parts of the shoot tip 1·0 or 1·5 mm from the apical dome. In the greenhouse, the plants in which phytoplasmas were not detected were healthy-looking, grew vigorously and were readily distinguished from the infected plants that exhibited little leaf and chlorosis symptoms, proliferation of axillary shoots and roots, stunting, and heavily reduced number and size of storage roots. In this study efficient elimination of phytoplasma and production of pathogen-tested plant stocks were achieved with the novel cryotherapy-based approach. The proposed advantage of the technique is that it can be simultaneously used for long-term storage of plant germplasm and for production of pathogen-free plants.

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