Cryopreservation of apple

Abstract

In this work we employed two vitrification-based techniques (vitrification and droplet vitrification) to cryopreserve in vitro grown shoot tips of apple ‘Gala Must’ (Malus × domestica Borkh.). After preculture, shoot tips were osmoprotected at room temperature in a solution containing 2 M glycerol and 0.4 M sucrose for 20 min, and then dehydrated in the following plant vitrification solutions: PVS2 (13.7% sucrose, 30% glycerol, 15% ethylene glycol, 15% DMSO), PVS A3 (22.5% sucrose, 37.5% glycerol, 15% ethylene glycol and 15% DMSO) for 30, 40 and 50 min at 0 °C and PVS3 (50% glycerol and 50% sucrose) for 40, 50 and 60 min at room temperature. Explants dehydrated with PVS2 and PVS A3 were cryopreserved by vitrification while those dehydrated with PVS A3 and PVS3 were cryopreserved using droplet vitrification. In vitrification protocol, regrowth of the cryopreserved shoot tips dehydrated with PVS2 ranged between 20–40%. Dehydration with PVS A3 resulted in considerably higher regrowth rates (15–75%) using the same protocol. The highest values of regrowth were achieved with the longest treatment duration (50 min) for both vitrification solutions. As for droplet vitrification, regrowth of cryopreserved explants dehydrated with PVS A3 varied between 45–70%, and between 45–50% for those dehydrated with PVS3. The highest regrowth values, 70% and 50%, were achieved after 40-min PVS A3 treatment and 50-min PVS3 treatment, respectively. These results prove the feasibility of the PVS A3-based vitrification for the long-term storage of this genotype.