Cold acclimation improves regrowth of cryopreserved apple shoot tips

Cryopreservation is important for safeguarding the genetic resources of apple germplasm in Kazakhstan, the center of origin for apples. In this study, conducted with five apple genotypes [Malus domestica Borkh. and Malus sieversii (Ledeb.) M. Roem] we determined cold hardiness and the effect of cold acclimation on shoot tip recovery following cryopreservation using two techniques. Apple shoot tips were cold acclimated (CA) for 0 to 6 weeks and cryopreserved using PVS2 vitrification and encapsulation dehydration (ED). Cold hardiness was indicated by the temperature at which 50 percent of the shoot tips were lethally injured (LT50). For non-acclimated shoots, LT50 ranged from -6.7 degree C to -9.3 degree C. These LT50 values resembled the natural cold hardiness of field grown plants and resulted in 10-12 percent regrowth after cryopreservation. Acclimated plantlets had LT50 values of -12 degree C to -15 degree C after 1 to 3 weeks CA, and after 3 weeks CA, cryopreservation resulted in 65 percent regrowth. There were no significant differences between the two techniques for regrowth of shoot tips after each cold acclimation period. Overall, 2 to 5 weeks CA produced high regrowth for each of the five cultivars tested. Three weeks of alternating temperature CA can be recommended as a standard protocol for Malus germplasm cryopreservation. These conditions resulted in moderate (60 percent) to high (80 percent) recovery for all five genotypes tested with both cryopreservation methods used.