Cryopreservation of in vitro-grown shoot tips of carnation (Dianthus caryophyllus L.) by vitrification method using aluminium cryo-plates

Abstract

Cryopreservation using an aluminum cryo-plate was successfully applied to in vitro-grown carnation (Dianthus caryophyllus L.) shoot tips. The shoot tips (1-1.5 mm�1 mm) were dissected from the shoot and precultured at 25°C for 2 days on MS medium containing 0.3 M sucrose. The precultured shoot tips were placed on the aluminum cryo-plate containing ten wells embedded with alginate gel. Osmoprotection was performed by immersing the cryo-plates in loading solution (2 M glycerol and 1.4 M sucrose) for 90 min at 25°C. Then, dehydration was performed by immersing the cryo- plates in PVS2 for 25 min at 25°C. After storage in liquid nitrogen, shoot tips attached to cryoplate, were directly immersed into 2 ml 1 M sucrose solution for regeneration. Using this procedure, the average regrowth level of vitrified shoot tips of 4 carnation cultivars reached 95%. This new method has many advantages and will facilitate the cryostorage of reference cultivars of carnation.