C-2019: Cryopreservation of chrysanthemum using aluminium Cryo-plate protocol

Abstract

Two cryopreservation procedures using aluminium cryo-plates were successfully developed using in vitro-grown chrysanthemum shoot tips. Shoot cultures were cold-hardened at 5 °C on 1/2 MS medium for 30 days. Shoot tips with basal plate (1.5–2.0 × 1.0 mm) were excised from shoot cultures and precultured at 5 °C for 3 days on solidified 1/2 MS medium containing 0.3 M sucrose. Precultured shoot tips were placed on aluminium cryo-plate with 10 wells (diameter 1 mm) and embedded in calcium alginate gel. Osmoprotection was performed by immersing the cryo-plates for 30 min at 25 °C in a loading solution (2 M glycerol + 1.0 M sucrose). In the D-cryo-plate procedure, the shoot tips were dehydrated by placing the cryo-plates in the air current of a laminar flow cabinet for 30–150 min. In the V-cryo-plate procedure, buds were dehydrated by immersing the cryo-plates in PVS2 vitrification solution for 40 min at 25 °C. In both procedures, cooling was performed by placing the cryo-plates in uncapped cryotubes, which were immersed in liquid nitrogen. For rewarming, shoot tips attached to the cryo-plates were rewarmed by immersion in a petri dish containing 1.0 M sucrose solution at 25 °C. Regrowth of cryopreserved shoot tips using D-Cryo-plate and V-Cryo-plate procedures, was 90% and 80%, respectively. The two procedures were applied to 20 additional chrysanthemum lines. This protocol appears to be a promising technique for cryopreservation of chrysanthemum genetic resources.