Kim H H, Lee Y G, Park S U, Lee S C, Baek H J, Cho E G, Engelmann F
National Academy of Agricultural Science, RDA, Suwon, Korea.
Cryo Letters. 2009 Jul-Aug;30(4):291-9.
In plant vitrification protocols, the loading treatment, which involves treating the explants with a moderately concentrated cryoprotectant solution, precedes dehydration of explants with highly concentrated vitrification solutions in order to reduce the toxicity which can be induced by their direct exposure to such highly concentrated solutions. This study aimed at developing alternative loading solutions composed of mixtures of glycerol and sucrose at various concentrations. Differential scanning calorimetry runs of loading solutions and of loaded and dehydrated explants were performed to assay thermal events occurring during cooling and warming. These loading solutions were applied to two model species, viz. garlic and chrysanthemum which were cryopreserved using a droplet-vitrification procedure. The loading treatment proved to be beneficial to both garlic and chrysanthemum and increased recovery of cryopreserved explants. However, response to the loading solutions tested varied between the two model species employed: with garlic, all the loading solutions had a similar effect, whereas survival of chrysanthemum shoot tips was significantly influenced by the composition of the loading solution employed. A loading solution comprising 1.9 M glycerol and 0.5 M sucrose was the most effective. The loading treatment may thus act as an osmotic stress neutralizer and/or induce the physiological adaptation of tissues and cells, including membranes, to both dehydration and freezing.
在植物玻璃化法方案中，加载处理（即先用适度浓缩的冷冻保护剂溶液处理外植体）先于用高浓度玻璃化溶液对外植体进行脱水处理，以降低外植体直接接触此类高浓度溶液可能诱导产生的毒性。本研究旨在开发由不同浓度甘油和蔗糖混合物组成的替代加载溶液。对加载溶液以及加载并脱水后的外植体进行差示扫描量热法分析，以测定冷却和升温过程中发生的热事件。将这些加载溶液应用于两种模式植物，即大蒜和菊花，采用液滴玻璃化法对其进行冷冻保存。结果表明，加载处理对大蒜和菊花均有益，且提高了冷冻保存外植体的回收率。然而，在所使用的两种模式植物中，对外加的加载溶液的反应有所不同：对于大蒜，所有加载溶液的效果相似，而菊花茎尖的存活率受所用加载溶液组成的显著影响。含1.9 M甘油和0.5 M蔗糖的加载溶液效果最佳。因此，加载处理可能起到渗透胁迫中和剂的作用和/或诱导组织和细胞（包括细胞膜）对脱水和冷冻的生理适应。
