Cryopreservation of Chrysanthemum Shoot Tips by D Cryo-Plate Method

doi:10.20809/seisan.23.1_1

D cryo-plate method was successfully adapted using in vitro-grown chrysanthemum (Chrysanthemum morifolium) shoot tips. Shoots of chrysanthemum cultured on 1/2 MS medium were cold-hardened at 4oC for 30 days. Shoot tips (1.5-2.0 x 1.0 mm) were excised from shoot cultures and precultured at 4oC for 3 days on solidified 1/2 MS medium containing 0.3 M sucrose. Precultured shoot tips were placed in elliptical wells on an aluminium cryo-plate and embedded in calcium alginate gel. Osmoprotection was performed by immersing the cryo-plates for 30 min at 23oC in LS solution (2.0 M glycerol + 1.0 M sucrose). In the D cryo-plate method, the shoot tips were dehydrated by placing the cryo-plates in the air current of a laminar flow cabinet for 30 to 150 min. In this procedure, cooling was performed by transferring the cryo-plate in uncapped 2 ml cryotubes, which was held on a cryo-cane, and then directly plunging it into liquid nitrogen where it was kept for at least 1 h. For rewarming, shoot tips attached to the cryo-plates were immersed in petri dish containing 1.0 M sucrose solution at 23oC. Regrowth of cryopreserved shoot tips using D cryo-plate procedure was 96.7%. This method was successfully applied to six other rysanthemum lines. This protocol appears to be a promising technique for cryopreservation of chrysanthemum genetic resources.

Cryopreservation of Chrysanthemum Shoot Tips by D Cryo-Plate Method

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