Cryopreservation of embryogenic cell suspensions by encapsulation-vitrification and encapsulation-dehydration

Encapsulation-vitrification and encapsulation-dehydration are two newly developed techniques for cryopreservation of embryogenic cell suspensions. Here, we describe the two protocols using grapevine (Vitis) as a model plant. Cell suspensions at the exponential growth stage cultured in a cell suspension maintenance medium are encapsulated to form beads, each being about 4 mm in diameter and containing 25% cells. In the encapsulation-vitrification procedure, the beads are stepwise precultured in increasing concentrations of sucrose medium up to 0.75 M, with 1 day for each concentration. The precultured beads are treated with a loading solution for 60 min and then dehydrated with plant vitrification solution 2 at 0°C for 270 min before a direct immersion in liquid nitrogen. Following cryostorage, the beads are rapidly rewarmed at 40°C for 3 min and then unloaded with 1 M sucrose solution for 30 min. In the encapsulation-dehydration procedure, the beads are precultured in increasing concentrations of sucrose medium up to 1 M, with 1 day for each concentration, and then maintained on 1 M sucrose medium for 3 days. The precultured beads are dehydrated for 6 h under a sterile air flow, prior to rapid freezing in liquid nitrogen. The freezing and rewarming procedures are the same as used in the encapsulation-vitrification technique. The unloaded beads from encapsulation-vitrification and rewarmed beads from encapsulation-dehydration are postcultured on a recovery medium for 3 days at 25°C in the dark for survival. Surviving cells are transferred to a regrowth medium to induce cell proliferation. Embryogenic cell suspensions are reestablished by suspending the cells in a cell suspension maintenance medium maintained on a gyratory shaker at 25°C in the dark. For plant regeneration, surviving cells are transferred from the recovery medium to an embryo maturation medium and maintained at 25°C under light conditions. Embryos at the torpedo stage are cultured on a rooting medium until whole plantlet regenerates.