Popova Elena, Bukhov Nikolai, Popov Alexandr, Kim Haeng-Hoon
National Agrobiodiversity Center, National Academy of Agricultural Science, RDA, Korea.
Cryo Letters. 2010 Sep-Oct;31(5):426-37.
In this study, cryopreservation of Bratonia (Miltonia flavescens (Lindl.) Lindl. × Brassia longissima (Reichb.) Nash), a hybrid tropical orchid, was achieved using protocorm-like bodies (PLBs) multiplied in vitro. Cryopreservation was performed using a vitrification protocol including pretreatment of PLBs with a loading solution (LS, 2.0 M glycerol + 0.4 M sucrose) for 15 min followed by treatment with modified PVS2 vitrification solution (containing PEG instead of ethylene glycol) for 1 h. Increasing benzyladenine (BA) concentration in the recovery medium to 5.0 or 10.0 mg l⁻¹ during the initial 3 weeks after rewarming provided 20.4 % post-cryopreservation regrowth. By contrast, preliminary culture of PLBs with abscisic acid (ABA) and high sucrose concentrations (up to 0.3 M) as well as addition of reduced glutathione during the preculture, loading and post-culture steps were not beneficial. Forty to 45 plants were regenerated from each PLB which withstood cryopreservation. No morphological differences were observed between plants regenerated from cryopreserved and untreated PLBs. Investigations into the functional activity of photosystems I and II in PLBs suggest that electron transport was retained in the reaction centers of both photosystems shortly after cryopreservation.
在本研究中，利用体外增殖的原球茎状体（PLB）实现了热带杂交兰花布拉托尼亚（Miltonia flavescens (Lindl.) Lindl. × Brassia longissima (Reichb.) Nash）的冷冻保存。冷冻保存采用玻璃化方案，包括用装载液（LS，2.0 M甘油 + 0.4 M蔗糖）对PLB进行15分钟预处理，然后用改良的PVS2玻璃化溶液（含聚乙二醇而非乙二醇）处理1小时。解冻后最初3周内，将恢复培养基中的苄基腺嘌呤（BA）浓度提高到5.0或10.0 mg l⁻¹，可使冷冻保存后的再生率达到20.4%。相比之下，用脱落酸（ABA）和高蔗糖浓度（高达0.3 M）对PLB进行预培养，以及在预培养、装载和培养后步骤中添加还原型谷胱甘肽并无益处。每个经受冷冻保存的PLB可再生40至45株植株。冷冻保存的PLB和未处理的PLB再生的植株之间未观察到形态差异。对PLB中光系统I和II功能活性的研究表明，冷冻保存后不久，两个光系统的反应中心均保留了电子传递。
