Development of the V cryo-plate method for cryopreservation of in vitro rakkyo (Allium chinense G. Don)

The V cryo-plate method was successfully adapted for the cryopreservation of in vitro grown rakkyo plants (Allium chinense G. Don, 'Shima-rakkyo'). Rakkyo is a cash crop of western Japan. The optimal V cryo-plate method for rakkyo is as follows. Apical shoot tips were collected from in vitro plants and precultured for 1-3 days at 25°C on solidified MS medium containing 0.3 M sucrose. Precultured shoot tips were placed in wells on an aluminium cryo-plate (No. 2) and embedded in calcium alginate gel. Osmoprotection was performed by immersing the cryo-plates for 30 min at 25°C in LS (2.0 M glycerol and 1.0 M sucrose). The optimal PVS2 exposure time was 20-40 min at 25°C. Cooling was performed by transferring the samples into uncapped 2-mL cryotubes held on a cryo-cane which was then directly plunged into liquid nitrogen. Regrowth was initiated when the cryopreserved shoot tips attached to the cryo-plates were rewarmed by immersion in a Petri dish containing 1.0 M sucrose solution at 25°C. Using this procedure, the regrowth level of cryopreserved shoot tips was 100%. This protocol appears to be a promising technique for cryopreservation of rakkyo genetic resources.