Cryopreservation of apple in vitro axillary buds using droplet-vitrification

In vitro axillary buds of two apple cultivars, Pinova and Jonagold, were successfully cryopreserved by droplet-vitrification. In vitro axillary buds of cv. Pinova were subjected to PVS2 for 15, 30, 45, 60, 80 or 100 min, while Jonagold buds were treated with PVS2 for 15, 30, 45 or 60 min. In addition, the effect of age of in vitro mother-plants on recovery after cryopreservation was evaluated. Recovery was performed on medium with various combinations of BA, IBA and GA3. Regrowth percentages for cv. Pinova increased in line with increasing PVS2 exposure durations, from 15 to 60 min. Cv. Jonagold showed a similar trend with an increase in regrowth from 30 to 60 min PVS2 exposure. Improved regrowth was observed when axillary buds were excised from aged mother-plants in comparison to those excised from plantlets that were regularly subcultured. The highest shoot regrowth was obtained when applying a 60 min PVS2 treatment to axillary buds excised from non-preconditioned 4-month old in vitro shoots and performing regrowth on recovery medium containing 4.50 microM BA and 0.50 microM IBA. This optimal protocol was also successfully applied to apple rootstocks M26 and Jork 9.