Apical meristems from adventitious buds induced by culturing of bulb-scale segments of Japanese Pink Lily (Liliumjaponicum 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.Abbreviations: DMSO -dimethylsulfoxide, E G -ethylene glycol, L N -liquid nitrogen, MS medium -Murashige & Skoog (1962) medium, PVS2 -vitrification solution
A cryopreservation protocol based on air dehydration of explants placed on aluminium cryo-plates, termed D cryo-plate, was successfully developed for in vitro mat rush (Juncus decipiens Nakai) lateral buds. The buds of line 'Hiroshima 4gou(1)' with basal stems were dissected from multiple shoots and precultured overnight at 25°C on solid MS medium containing 0.3 M sucrose. Precultured buds were placed on aluminium cryo-plates, each one with 10 elliptical wells (2.5 mm long, 1.5 mm wide and 0.75 mm deep) and embedded in calcium alginate gel with 0.4 M sucrose and 1 M glycerol. Osmoprotection was performed by immersing the cryo-plates with buds for 30 min at 25°C in loading solution (2 M glycerol+1.0 M sucrose). Buds were dehydrated to 26% moisture content (fresh weight) by placing the cryo-plates for 2.5 h either in the air current of a laminar flow cabinet or in Petri dishes containing silica gel. Cooling was performed by placing the cryo-plates in uncapped cryotubes, which were immersed in liquid nitrogen. For rewarming, cryo-plates were immersed in liquid medium with 1.0 M sucrose for 15 min at room temperature. Under these conditions, regrowth of cryopreserved buds of line, 'Hiroshima 4gou (1)' was 93% after four weeks culture. The average regrowth of 20 mat rush lines was 88%. The D cryo-plate procedure will facilitate cryostorage of mat rush germplasm.
Cryopreservation using an aluminium cryo-plate was successfully applied to in vitro-grown strawberry (Fragaria £ ananassa Duch.) shoot tips. The shoots were cold-hardened at 58C for 3 weeks with an 8-h photoperiod. The shoot tips (1.5 -2.0 mm £ 0.5-1.0 mm) were dissected from the shoot and pre-cultured at 58C for 2 d on Murashige and Skoog medium containing 2 M glycerol and 0.3 M sucrose. The pre-cultured shoot tips were placed on the aluminium cryo-plate containing ten wells embedded in alginate gel. Osmoprotection was performed by immersing the cryo-plates in a loading solution (2 M glycerol and 0.8 M sucrose) for 30 min at 258C. Dehydration was performed by immersing the cryo-plates in plant vitrification solution 2 for 50 min at 258C. Then, the cryo-plate with shoot tips was transferred into an uncapped cryotube that was held on a cryo-cane and directly immersed into liquid nitrogen (LN). After storage in LN, shoot tips attached to the cryo-plate were directly immersed into 2 ml of a 1 M sucrose solution for regeneration. Using this procedure, the average regrowth level of vitrified shoot tips of 15 strawberry cultivars reached 81%. This new method has many advantages and will facilitate the cryostorage of strawberry germplasm.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.