Cryopreservation of genetic resources of plum and apple

Genetic resources of clonally propagated fruit species cannot be stored as seed and alternative storage methods that are long-term and effective for a wide range of genotypes are important for breeding teams and genebank curators. Cryopreservation in liquid nitrogen is one of these techniques. In our study, in vitro shoot tips of 2 temperate fruit crops (plum and apple) encapsulated in sodium alginate were successfully cryopreserved using 3 different methods based on vitrification and two-step freezing. Initial in vitro shoots were previously established and multiplied on MS medium in Erlenmeyer flasks with 25 mL of MS medium. Basic shoot tip cultures were cold-hardened for 4 weeks in 4°C. Explants (3 mm) aseptically dissected from cold hardened in vitro cultures were cold treated in MS medium supplemented with 2.0 M sucrose and for 48 h at 4°C. Shoot tips encapsulated in sodium alginate were dehydrated by exposure to a sterile air flow for a period of 8 h. Two freezing methods were tested and compared: fast cooling by direct immersion in liquid nitrogen (vitrification) and two variants of two-step freezing. The best results were obtained with a two-step freezing protocol in variant b) with progressive cooling at a rate of -4°C min^‑1 to -40°C followed by immersion in liquid nitrogen. In this variant, total average survival rate used for apple and plum cultivars was 71.0%. High values of survival were also observed in the case of the two-step freezing protocol in variant a) with an average survival of 65.1%. Encapsulated shoot tips immersed directly to liquid nitrogen were cryopreserved with 59.4% survival. Described cryopreservation procedures were successfully applied for 6 cultivars of plum and apple.