Cryopreservation of shoot tips of endangered Hayachine-usuyukiso ( Leontopodium hayachinense (Takeda) Hara et Kitam.) using a vitrification protocol

Hayachine-usuyukiso (Leontopodium hayachinense) is an alpine plant native to Mt Hayachine. This unique chrysanthemum is listed as an endangered plant by the Department of Conservation, Iwate Prefecture, and as a threatened plant by the Ministry of the Environment, Japan. We successfully cryopreserved the shoot tips from in vitro-grown L. hayachinense shoots using a vitrification protocol. Cold-hardened shoot tips were excised and pre-cultured on a solidified Murashige–Skoog medium containing 0.3M sucrose for 1 d at 58C. The shoot tips were then treated with loading solution for 20min at 258C, dehydrated in plant vitrification solution 2 for 120min at 258C and immersed in liquid nitrogen. The survival rate of the vitrified shoot tips was 63.3% after 30 d of regrowth. This protocol appears to be a promising technique for the cryopreservation of in vitro-grown shoots of this endangered plant.