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%.

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Newly developed shoot tips from adventitious buds induced by tissue cultured bulb-scale segments of five accessions of Lilium spp. were successfully cryopreserved by a droplet-vitrification method. Bulb-scale segments cultured on Murashige and Skoog (MS) medium with 0.1 mg·L⁻¹ IAA and 0.1 mg·L⁻¹ zeatin were then cold-hardened at 4°C for 7 days. The excised shoot tips were pre-cultured on solidified MS medium containing 0.3 M sucrose for 1 day at 23°C and then soaked in a mixture of 0.7 M sucrose for a day at 23°C. Pre-cultured shoot tips were cryoprotected with two loading solutions, LD1 and LD2, which included 35% and 40% plant vitrification solution (PVS3), respectively, for 40~60 min at 23°C. The cryoprotected shoot tips were then soaked in PVS2, modified PVS2 and PVS3 for 90~120 min at 23°C. The shoot tips, frozen in microdroplets of vitrification solution, were wrapped with aluminum foil strips, which were immersed rapidly in liquid nitrogen. The shoot tips were then rapidly warmed using unloading solution, transferred to a regeneration medium, stored in the dark for two weeks at 23°C, and then cultured under white fluorescent light at an intensity of 2000 lux with a 16-h photoperiod at 23°C. The average post-cryo regeneration rates of five accessions ranged from 52.7% to 87.5%.

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