Development of a droplet-vitrification protocol for cryopreservation of Rubia akane (Nakai) hairy roots using a...

A systematic approach using a set of 13 treatments was applied to develop a droplet-vitrification protocol for Rubia akane hairy roots, based on their responses to preculture, loading, dehydration and cooling/rewarming steps. The roots were very sensitive to osmotic stress induced by both preculture in liquid sucrose-enriched medium (up to 0.5 M sucrose) and by dehydration with highly concentrated vitrification solutions (VSs). Loading was necessary before dehydration of explants with VS, and the composition of the loading solution (LS) significantly affected their post-cryopreservation regeneration. Due to high sensitivity of roots to both chemical cytotoxicity and osmotic stress produced by VSs, cryoprotection with alternative VSs, i.e. B5-80 percent (40 percent glycerol + 40 percent sucrose, w/v) at room temperature for 15 min or with A3-70 percent (29.2 percent glycerol + 11.7 percent DMSO + 11.7 percent EG + 17.4 percent sucrose, w/v) at 0 degree C for 20 min ensured the highest post-cryopreservation regeneration. However, when using these solutions, endothermic peaks (enthalpies) with -2.9 and -5.8 J per gram fresh weight, respectively, were recorded by differential scanning calorimetry (DSC) during the rewarming phase. Droplet-vitrification using foil strips showed higher post-cryopreservation regeneration (86 percent) compared with vitrification in cryovials (59 percent), possibly due to the higher cooling and rewarming rates achieved with droplet-vitrification. The developed protocol was applied to hairy roots of five other species with minor modifications in explant type, the duration of the last subculture before explant excision, and the dehydration duration with VS B5-80 percent.