title: Cryopreservation of hairy roots of Rubia akane (Nakai) using a droplet-vitrification procedure.
abstract: An efficient protocol for the cryopreservation of madder (Rubia akane Nakai) hairy root cultures was developed using droplet-vitrification and alternative loading and vitrification solutions formulated previously in our laboratory. Among eight preculture treatments tested, the highest post-cryopreservation regeneration was obtained for explants incubated in liquid half-strength MS medium with progressively increased sucrose concentration (0.3 M for 54 h, then 0.5 M for 16 h). Loading of precultured explants improved their post-cryopreservation regeneration by 50-75% compared with non-loaded control. Combining loading solution C4 (35% PVS3) and vitrification solution B5 (80% PVS3) was the most effective, while applying six PVS2-based solutions at room temperature resulted in low post-cryo regeneration. Treatment duration was optimized to 30 min for loading and to 10-20 min for vitrification solution. Apices of primary and secondary hairy roots showed similar post-cryo regeneration (88 and 95%, respectively), which was significantly higher than regeneration of root sections without apices (65%). Droplet-vitrification produced higher post-cryo regeneration than 'classical' vitrification in cryovials. Our results suggest that droplet-vitrification using alternative loading and vitrification solutions is an efficient method for cryopreservation of R. akane hairy root cultures.
authors.affiliation: National Agrobiodiversity Center, National Academy of Agricultural Science, RDA, Suwon 441-707, Korea. cryohkim@korea.kr
authors.forename: Haeng-Hoon
authors.initials: HH
authors.lastname: Kim
authors.forename: Elena V
authors.initials: EV
authors.lastname: Popova
authors.forename: Jung-Yoon
authors.initials: JY
authors.lastname: Yi
authors.forename: Gyu-Taek
authors.initials: GT
authors.lastname: Cho
authors.forename: Sang-Un
authors.initials: SU
authors.lastname: Park
authors.forename: Sheong-Chun
authors.initials: SC
authors.lastname: Lee
authors.forename: Florent
authors.initials: F
authors.lastname: Engelmann
mesh_terms: D001709:Biotechnology; D015925:Cryopreservation / Q000379:methods; D003451:Cryoprotective Agents / Q000737:chemistry; D046508:Culture Techniques; D003890:Desiccation; D018517:Plant Roots; D045470:Rubia; D013395:Sucrose; D058989:Vitrification
chemical_list: D003451:Cryoprotective Agents; D013395:Sucrose