Cryopreservation of apple shoot tips by vitrification and subsequent plant regeneration

This paper investigates the influence of composition of vitrification solution on the regrowth capacity of apple 'Gala Must' (Malus x domestica Borkh.) cryopreserved by a vitrification technique. In vitro-grown shoot tips were precultured in the dark at 23 degrees C, in a liquid MS medium with two sucrose treatments (0.3 M for 15 h, then 0.7 M for 5 h). After loading at room temperature with solution containing 2 M glycerol and 0.4 M sucrose for 20 min, explants were dehydrated at 0 degrees C for 30, 40 and 50 min. Dehydration was done using either original PVS2 (13.7% w/v sucrose, 30% w/v glycerol, 15% w/v ethylene glycol, 15% w/v DMSO) or modified PVS2 solution (PVS A3 - 22.5% w/v sucrose, 37.5% w/v glycerol, 15% w/v ethylene glycol and 15% w/v DMSO). The survival and regrowth of the cryopreserved shoot tips dehydrated with the original PVS2 solution ranged between 40 and 85 and 20-40%, respectively. The highest values of both parameters were achieved with longest treatment duration. Dehydration with the PVS A3 solution resulted in considerably higher survival rates (68.2-90%), as well as higher regrowth rates (15-75%) after cryopreservation. Fifty-min treatment with this vitrification solution led to a significant increase in the percentage of regrowth (up to 75%). After regrowth, shoots were successfully multiplied and rooted. These results prove the feasibility of the PVS A3 based vitrification technique for a long-term storage of this genotype