Development of a shoot-tip vitrification protocol and comparison with encapsulation-based procedures for plum (Prunus domestica L.) cryopreservation

Cryopreservation of plum (Prunus domestica L.), cv Regina Claudia, was obtained by a vitrification/one-step freezing procedure of shoot tips from cold-hardned in vitro-grown plants. Best survival (57%) was obtained when the shoot tips, consisting of the apical meristem ad 4-5 leaflets, were precultured at 4°C for 2 days on 0.09 M sucrose-containing QP medium, loaded for 30 min with a cryoprotectant (2 M glycerol and 0.4 M sucrose), incubated with the PVS2 vitrification solution at 0°C for 90 min, and directly plunged into liquid nitrogen. After thawing in waterbath at 40°C, the shoot tips were washed in a 1.2 M-sucrose MS solution for 20 min and finally plated on a semi-solid QP medium, lacking of NH4NO3 and containing 0.09 M sucrose, 1.5 µM N6-benzyladenine and 0.5 µM gibberellic acid. In comparison with the one-step freezing procedure, both the slow cooling (-0.5°C min-1 up to -45°C), and the two-step freezing (-160°C for 25 min, then –196°C) gave lower percentages of shoot-tip survival. Among the other tested cryogenic procedures, the vitrification-encapsulation method performed similarly to the vitrification protocol in terms of shoot-tip regrowth (47%), while encapsulation-dehydration was unsatisfactorily.