Background: A solution-based vitrification protocol is a process of sequentially changing-solutions from which both influx of cryoprotectants (loading) and efflux of water (dehydration) were accomplished before cryo-exposure. Hence, we need to properly control the concentration /composition of the cryoprotectant solutions.
Objective: The study was, using a systematic approach, to develop a protocol for Rubia akane hairy roots, a very sensitive material to cytotoxicity of vitrification solutions.
Methods: Due to the poor response of 10-year in vitro maintained R. akane hairy roots to already established cryopreservation protocols, the following sets of experiments were designed: 1) combinational effect of preculture, osmoprotection and cryoprotection with PVS2-based (A3-70%) and PVS3-based (B5-80%) vitrification solutions; 2) different cooling/warming rates and warming temperature; 3) varying unloading solutions (25%, 35%and 45% sucrose) and durations (7 min and 30 min) with or without changing the unloading solutions.
Results: Preculture and osmoprotection treatments were necessary to acquire cytotoxicity tolerance in both vitrification solutions tested and osmoprotection treatment was more critical, especially in B5-80%. A sequential osmoprotection treatment (C10-50%) following conventional osmoprotection (C4-35%) was needed to increase the post-cryopreservation regrowth. Aluminum foil strips were superior to cryovials, but the warming temperature tested (20 degree C and 40 degree C) did not affect post-cryopreservation recovery. In the unloading procedure, a longer duration (30 min) with a higher sucrose solution (S-45%) was harmful, possibly due to osmotic stress.
Conclusion: R. akane hairy roots are very sensitive to cytotoxicity (both osmotic stress and chemical toxicity) and thus a proper process (preculture, osmoprotection, cryoprotection and unloading) is necessary for higher post-cryopreservation recovery.