Application of V and D cryo-plate methods for the cryopreservation of cherry rootstock Gisela 5

Abstract

The possibility of improving cryopreservation success using aluminium cryo-plates was investigated in cherry rootstock Gisela 5. The shoot tips were dissected from in vitro-grown shoots and precultured for one day at 25 C in the dark, on Murashige and Skoog (MS) medium containing 0.3 M sucrose. The explants were placed on aluminium cryo-plates with 12 wells and embedded in 2% alginate gel. Osmoprotection was performed by immersing the cryo-plates in loading solution (1.9 M glycerol + 0.5 M sucrose) for 30 min at room temperature. In the V cryo-plate protocol, dehydration was performed at room temperature using two types of plant vitrification solutions: modified PVS2 (37.5% glycerol, 15% dimethyl sulfoxide, 15% ethylene glycol and 22.5% sucrose) for 40 min and PVS3 (50% glycerol and 50% sucrose) for 60 min. In the D cryo-plate protocol, explants were desiccated for 2, 2.5 or 3 h in closed glass containers over silica gel. In both protocols, cryo-plates with explants were transferred in uncapped cryotubes and directly plunged into liquid nitrogen. Rewarming was done by direct immersion of cryo-plates in liquid MS medium containing 0.8 M sucrose (30 min at room temperature). In the V cryo-plate procedure regrowth of cryopreserved shoot tips dehydrated with PVS2 was 95.9%, while for those dehydrated with PVS3 was 85%. As for the D cryo-plate procedure, regrowth ranged between 45.8% and 66.7%. After regrowth, shoots were successfully multiplied and rooted. The results obtained clearly indicate that both cryopreservation procedures using aluminium cryo-plates can facilitate efficient cryostorage of Gisela 5 rootstock.