Cryopreservation in vitro of apple shoot tips following droplet-vitrification

Abstract

The aim of this study was to develop an efficient and rapid droplet-vitrification protocol to cryopreserve in vitro shoot tips of Malus x domestica Borkh. 'Gala Must'. Following excision, shoot tips were pre-cultured in liquid Murashige and Skoog (MS) medium enriched with sucrose at two concentrations (0.3 M for 15 h, then 0.7 M for 5 h). Loading took place at room temperature in a solution containing 1.9 M glycerol and 0.5 M sucrose for 30 min. Explants were dehydrated using either modified PVS2 solution (PVS A3 - 22.5% sucrose, 37.5% glycerol, 15% ethylene glycol and 15% DMSO, w/v) or PVS3 solution (50% sucrose and 50% glycerol, w/v). PVS A3-based dehydration was done for 30, 40 and 50 min either at room temperature or at 0 degrees C. As regards PVS3, explants were dehydrated at room temperature for 40, 50, 60, 90 and 120 min. Regrowth of the cryopreserved shoot tips dehydrated at room temperature with PVS A3 solution ranged between 0 and 14.5%, the highest rate being achieved with the shortest treatment duration. Dehydration with the same VS at 0 degrees C resulted in a considerably higher regrowth capacity of cryopreserved shoots (45-70%), the best results were achieved with 40-min treatment. Regrowth of PVS3-treated shoot tips (40-60 min) did not markedly vary (45-50%), while prolonged dehydration (90-120 min) resulted in a significant decrease of regeneration capacity of cryopreserved shoot tips (12.1-5%). After regrowth, shoots were successfully multiplied and rooted. These results prove the feasibility of the PVS A3-based dehydration at 0 degrees C for a long-term storage of this genotype using droplet-vitrification technique, although good results were also achieved with shorter PVS3 treatments at room temperature.