Abstract
Vitrification based method combined with carboxylated poly-L-lysine (PLL) treatment was successfully
developed 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 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. Osmoprotection and PLL treatment were performed by immersing for 30 min at 25oC in loading
solution with PLL. Pretreated shoot tips were placed in elliptical wells on an aluminium cryo-plate and
embedded in calcium alginate gel. The shoot tips were dehydrated by plant vitrification solution 2 for 20 min.
In this procedure, cooling was performed by transferring the cryo-plate in uncapped 2.0 mL cryotubes, and
then directly plunging into liquid nitrogen. For rewarming, shoot tips attached to the cryo-plates were
immersed in petri dish containing 1.0 M sucrose solution at 25oC. Regrowth rate of cryopreserved shoot tips
using 5% and 10% PLL treatment were 83.3% and 85.0%, respectively. Although further investigation should
be required, PLL might protect membrane from the freezing or osmotic damage. This protocol appears to be a
promising technique for cryopreservation of chrysanthemum genetic resources.
