Development of vitrification protocol in Rubia akane (nakai) hairy roots using a systematic approach

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.

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.

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.

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.

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.