Cryopreservation of mat rush lateral buds by air dehydration using aluminum cryo-plate

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 degrees 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 degrees 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