Park Sang-Un, Kong Hyunjung, Shin Dong-Jin, Bae Chang-Hwan, Lee Sheong-Chun, Bae Chang-Hyu, Rha Eui-Shik, Kim Haeng-Hoon
Division of Plant Science and Resources, Chungnam National University, Daejeon, Korea.
Dept. of Well-being Resources, Sunchon National University, Suncheon, Korea.
Cryo Letters. 2014 Mar-Apr;35(2):138-44.
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.
基于溶液的玻璃化冷冻方案是一个依次更换溶液的过程，在冷冻暴露前，冷冻保护剂的流入（加载）和水分的流出（脱水）均在此过程中完成。因此，我们需要妥善控制冷冻保护剂溶液的浓度/成分。
本研究采用系统方法，为茜草毛状根（一种对玻璃化溶液细胞毒性非常敏感的材料）制定一种方案。
由于体外保存10年的茜草毛状根对已有的冷冻保存方案反应不佳，因此设计了以下几组实验：1）基于PVS2（A3 - 70%）和PVS3（B5 - 80%）的玻璃化溶液进行预培养、渗透保护和冷冻保护的联合效应；2）不同的降温/升温速率和升温温度；3）改变卸载溶液（25%、35%和45%蔗糖）及其持续时间（7分钟和30分钟），且有或没有更换卸载溶液。
预培养和渗透保护处理对于在所测试的两种玻璃化溶液中获得细胞毒性耐受性是必要的，且渗透保护处理更为关键，尤其是在B5 - 80%溶液中。在常规渗透保护（C4 - 35%）之后进行顺序渗透保护处理（C10 - 50%），以提高冷冻保存后的再生能力。铝箔条优于冻存管，但所测试的升温温度（20℃和40℃）并不影响冷冻保存后的恢复。在卸载过程中，使用较高蔗糖溶液（S - 45%）且持续时间较长（30分钟）是有害的，这可能是由于渗透胁迫所致。
茜草毛状根对细胞毒性（包括渗透胁迫和化学毒性）非常敏感，因此，为了获得更高的冷冻保存后恢复率，需要一个适当的过程（预培养、渗透保护、冷冻保护和卸载）。
