Cryopreservation of Embryogenic Cell Suspensions by EncapsulationVitrification and EncapsulationDehydration

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.