Lee Yoon-Geol, Popov Elena, Cui Hai-Yan, Kim Haeng-Hoon, Park Sang-Un, Bae Chang-Hyu, Lee Sheony-Chun, Engelmann Florent
National Agrobiodiversity Center, National Academy of Agricultural Science, Suwon, Korea.
Cryo Letters. 2011 Nov-Dec;32(6):487-97.
A droplet-vitrification protocol has been established for cryopreserving Chrysanthemum morifolium cv. Peak using axillary shoot tips and apical shoots of in vitro plants. In the optimized procedure, explants were submitted to a step-wise preculture in liquid sucrose-enriched medium (0.3, 0.5 and 0.7 M for 31,17 and 7 h, respectively). Precultured explants were treated for 40 min with C4 loading solution comprising (w/v) 17.5 percent glycerol + 17.5 percent sucrose, then dehydrated with PVS3 vitrification solution (w/v, 50 percent glycerol + 50 percent sucrose) for 60 min (axillary shoot tips) or 90 min (apical shoots). Explants were cryopreserved by direct immersion in liquid nitrogen in minute drops of PVS3 attached to aluminum foil strips. The optimal age of donor plants was 4-5.5 weeks for apical shoots and 7 weeks for axillary shoot tips, producing post-cryopreservation regeneration percentages of 81.9 percent and 84.9 percernt, respectively. Plants regenerated from cryopreserved samples showed no phenotypical abnormalities and similar profiles of relative DNA content were recorded for control and cryopreserved plants. Our results suggest that the modified droplet-vitrification protocol described in this paper is highly effective and may prove user-friendlier than the cryopreservation protocols already published for chrysanthemum.
已建立一种玻璃化滴冻法，用于冷冻保存菊花品种“Peak”的离体植株腋芽茎尖和顶芽。在优化程序中，外植体先在富含蔗糖的液体培养基中进行逐步预培养（分别在0.3 M、0.5 M和0.7 M蔗糖溶液中培养31小时、17小时和7小时）。预培养后的外植体用含有（w/v）17.5%甘油 + 17.5%蔗糖的C4装载溶液处理40分钟，然后用PVS3玻璃化溶液（w/v，50%甘油 + 50%蔗糖）脱水60分钟（腋芽茎尖）或90分钟（顶芽）。外植体通过直接浸入附着在铝箔条上的微量PVS3液滴中，投入液氮进行冷冻保存。供体植株的最佳苗龄对于顶芽为4 - 5.5周，对于腋芽茎尖为7周，冷冻保存后的再生率分别为81.9%和84.9%。从冷冻保存样本再生的植株未表现出表型异常，对照植株和冷冻保存植株的相对DNA含量谱相似。我们的结果表明，本文所述的改良玻璃化滴冻法非常有效，可能比已发表的菊花冷冻保存方法更便于使用。
