Cryopreservation of apple shoot tips by vitrification and subsequent plant regeneration

Abstract

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.