Abstract
A vitrification procedure using aluminum cryo-plates (V-Cryo-plate procedure) was successfully adopted and adjusted to in vitro-grown mulberry shoot tips (Morus spp.) from the tropics and subtropics. The excised shoot tips with basal part (1-1.5×1.0mm) were precultured at 25°C for 1 day on the MS medium containing 0.3M sucrose and 0.2mg/l benzyladenine. Precultured shoot tips were placed on an aluminum cryo-plate with ten wells and embedded in alginate gel. Osmoprotection was performed by immersing the cryo-plates with shoot tips for 30min at 25°C in loading solution (2M glycerol+0.6M sucrose). Then, cryo-plates were transferred and immersed into PVS2 solution for 50min at 25°C for dehydration. After this, the cryo-plates were transferred in an uncapped 2ml cryotube and directly plunged into liquid nitrogen one by one. For regeneration, the cryo-plate with shoot tips was directly rewarmed by immersion into cryotubes containing 2ml of 1M sucrose solution at room temperature. Using this procedure, the regrowth of cryopreserved shoot tips of line ‘Amami 07’ reached 97% or more. This protocol was successfully applied to other 12 different Morus lines, which had high regrowth levels ranging from 73% to 97%. This V-Cryo-plate method will facilitate the cryostorage of tropical and subtropical mulberry germplasm.
