Cryopreservation of Prunus spp. using aluminium cryo-plates

Cryopreservation using aluminium cryo-plates was successfully applied to in vitro-grown shoot tips of two Prunus genotypes, cherry plum (Prunus cerasifera Ehrh.) and the plum cultivar 'Požegača' (Prunus domestica L.). Shoot tips were dissected from the shoots and precultured for 1 day at 23 °C in the dark on Murashige and Skoog medium containing 0.3 M sucrose. The precultured shoot tips were placed on aluminium cryo-plates containing 10 or 12 wells and embedded in alginate gel. Osmoprotection was performed by immersing the cryo-plates in two types of loading solution (LS1 − 2 M glycerol + 0.4 M sucrose or C4 − 1.9 M glycerol + 0.5 M sucrose) for 30 min at room temperature. In the V cryo-plate protocol, dehydration was performed for 30 min at room temperature in a plant vitrification solution containing 37.5% (w/v) glycerol, 15% (w/v) dimethylsulfoxide, 15% (w/v) ethylene glycol and 22.5% (w/v) sucrose. In the D cryo-plate protocol, dehydration was performed by placing the cryo-plates for 2, 2.5 or 3 h under the air current of the laminar flow cabinet or in closed glass containers over silica gel. In both protocols, cooling was performed by placing the cryo-plates in uncapped 2 mL cryotubes, which were immersed in liquid nitrogen. Rewarming was done by direct plunging of cryo-plates in liquid MS medium containing 0.8 M sucrose at room temperature for 30 min (plum cultivar) or 60 min (cherry plum). All experiments were conducted twice in two different laboratories (IRD Montpellier, France and FRI Čačak, Serbia). In the V cryo-plate procedure regrowth (calculated as average values for two laboratories) of cryopreserved shoot tips loaded with C4 solution was 41.7% (cherry plum) and 34.2% (plum cultivar 'Požegača'), while in those loaded with LS1 solution regrowth was 56.1% and 44.6%, respectively. As for the D cryo-plate procedure, the average regrowth of cryopreserved explants ranged between 57.7% and 77.5% in cherry plum and between 28.5% and 47.5% in plum cultivar 'Požegača'. Although during the first subculture after regrowth, the multiplication capacity of cryopreserved explants was lower compared with those originating from dissection controls, by the third subculture they regained and even exceeded the multiplication capacity of shoots regenerated from explants placed on regrowth directly after dissection. The results obtained clearly indicate that both cryopreservation procedures using aluminium cryo-plates can facilitate efficient cryostorage of Prunus germplasm.