Development of an Encapsulation-Vitrification Protocol for Rubia Akane (Nakai) Hairy Roots: A Comparison with Nonencapsulation

BACKGROUND: A comparison of different cryopreservation techniques should be based on the characteristics of both the methodology and the material in question using an optimized procedure. OBJECTIVE: This study aimed at developing an encapsulation-vitrification procedure for hairy roots of Rubia akane using alternative loading and vitrification solutions, based on the existing optimized droplet-vitrification procedure. MATERIALS AND METHODS: Encapsulated roots were first precultured in liquid medium with 10% sucrose for 3 days, then with 17.5% sucrose for 1 day, after which they were osmoprotected with solution C6-40% (20% glycerol + 20% sucrose) for 50 min, cryoprotected with solution A3-90% (37.5% glycerol + 15% DMSO + 15% EG + 22.5% sucrose, w/v) on ice for 40 min, cooled and warmed in 2 ml cryovials, and unloaded in 35% sucrose solution for 60 min. RESULTS: Through the application of this procedure to aged-clustered roots, up to 97.5% post-cryopreservation regeneration was observed. In our previous study, droplet-vitrification of hairy roots of R. akane resulted in 83.8% post-rewarming regeneration following preculture with 10% sucrose for 2 days and 17.5% sucrose for 4-5 h, and osmoprotection with solution C4-35% (17.5% glycerol + 17.5% sucrose) for 30 min, and cryoprotection with solution A3-70% (29.2% glycerol + 11.7% DMSO + 11.7% EG + 17.4% sucrose, w/v) on ice for 20 min. In the present study, higher post-cryopreservation regeneration was observed by using a higher concentration of vitrification solution (A3-70% → A3-90%, B5-80% → B1-100%) and/or a longer cryoprotection duration (A3-70% at room temperature (RT) for 8 min → 15-30 min, on ice for 20 min → 40-80 min; B5-80% for 15 min → 30-60 min). CONCLUSION: Even though encapsulation provided some degree of protection from the cytotoxicity of vitrification solutions to cytotoxicity-sensitive R. akane hairy roots, an overall higher post-cryopreservation regrowth was obtained using the droplet-vitrification procedure under optimized conditions. This result implies that this sensitive material was not sufficiently cryoprotected, and thus, rapid cooling and warming using foil strips was more efficient than cryopreservation of encapsulated samples.