Cryopreservation of Winter-dormant Apple Buds Using Two-step Freezing

Winter apple buds germplasm was cryopreserved in the Korean Genebank to back up the genetic resources maintained by field collections. We examined the standard two-step freezing protocol for the cryopreservation of winter buds of apple germplasm developed by National Center for Genetic Resources Preservation (NCGRP) in the USA. This protocol requires desiccation of the stem explants containing a single dormant bud to 30% moisture content, cooling at a rate of 1°C/h to –30°C for 24 hours, followed by rapid immersion in liquid nitrogen. To evaluate the viability of cryopreserved buds after at least 24 hours, the thawed and rehydrated segments are transferred to a greenhouse and used for vegetative propagation by chip budding onto rootstock or by in vitro sprouting. To optimize the protocol for the cryopreservation of winter-dormant apple buds suitable for Korean climate, we tested several treatments, including different degrees of desiccation, pre-freezing temperatures, and pre-freezing rates. The highest regeneration rates were obtained from cryopreserved samples with 35% moisture content, –35°C pre-freezing temperature, and 1°C/h pre-freezing rate. Finally, using our two-step freezing method, 14 accessions of apple germplasm were cryopreserved successfully, with an average regeneration rate of 55.0%.