Cryopreservation of in vitro-grown apical meristems of lily by vitrification

Abstract

Apical meristems from adventitious buds induced by culturing of bulb-scale segments of Japanese Pink Lily (Lilium japonicum Thunb.) were successfully cryopreserved by a vitrification. The excised apical meristems were precultured on a solidified Murashige & Skoog medium, containing 0.3 M sucrose, for 1 day at 25°C and then loaded in a mixture of 2 M glycerol plus 0.4 M sucrose for 20 min at 25°C. Cryoprotected meristems were then sufficiently dehydrated with a highly concentrated vitrification solution (designated PVS2) at 25°C for 20 min or at 0°C for 110 min prior to a plunge into liquid nitrogen. After rapid warming in a water bath at 40°C, the meristems were placed in 1.8 ml of 1.2 M sucrose for 20 min and then, placed on filter papers over gellan gum-solidified MS medium. The revived meristems resumed growth within 5 days and directly produced shoots. The rate of shoot formation was approximately 80% after 4 weeks. When bulb-scale segments with adventitious buds were cold-hardened at 0°C for more than 7 days before the procedure, the rates of shoot formation were significantly increased. This vitrification method was successfully applied to five other lily cultivars. Thus, this vitrification procedure for cryopreservation appears promising as a routine method for cryopreserving meristems of lily.