Cryoprotective Effect of Carboxylated Poly-L-lysine on the Chrysanthemum Shoot Tips by Vitrification Based Method

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  • ガラス化法を用いたキク遺伝資源の超低温保存における両性電解質高分子ポリリジンの効果
  • ガラスカホウ オ モチイタ キク イデン シゲン ノ チョウテイオン ホゾン ニ オケル リョウセイ デンカイシツ コウブンシ ポリリジン ノ コウカ

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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.

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