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首页> 外文期刊>Korean Journal of Horticultural Science & Technology >Improved Cryopreservation Using Droplet-vitrification and Histological Changes Associated with Cryopreservation of Madder(Rubia akane Nakai)
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Improved Cryopreservation Using Droplet-vitrification and Histological Changes Associated with Cryopreservation of Madder(Rubia akane Nakai)

机译：利用微滴玻璃化法改进冷冻保存和与马德冷冻保存相关的组织学变化（Rubia akane Nakai）

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摘要

An efficient protocol for cryopreservation of madder hairy root cultures has been developed using droplet-vitrification. In previous study, combining loading solution C4 (35% PVS3) and vitrification solution B5 (80% PVS3) was the most effective method. In this study, we tried three types of vitrification solution, B5, A3 (90% PVS2, on ice), and A5 (70% PVS2, on ice). Combining loading solution C4 and vitrification solution A5 (on ice) showed the best regeneration rate in this study. Histological changes of the cells within the hairy root of madder were also observed in different steps. The cells from the hairy roots of the control treatment were full and intact with different size of vacuoles and obvious cell nucleus having a dark nucleolus. After the stage of preparing for cryopreservation (after preculturing, loading, followed by dehydration solution A5 or B5), intercellular spaces had become distinct, and within cells, the cytoplasms had become denser and week plasmolyses had appeared. The cellplasmolyses were much more apparent and we measured the degree of plasmolysis by calculating, the area of cell/the area of cytoplasm. The value of plasmolysis degree was the highest in the combination of preculture, loading solution C4, and dehydration solution A5, 1.97. Because the highest regeneration rates appeared in the treatment of A5 for 20 min, we could assume that the optimal degree of plasmolysis for cryopreservation might be around 1.97. The changes in cell structure during cryopreservation might be a useful basis for the development of a proper long-term preservation method for madder germplasms.
机译：使用液滴玻璃化已开发出一种用于冷冻保存茜草毛状根培养物的有效方案。在先前的研究中，将加料溶液C4（35％PVS3）和玻璃化溶液B5（80％PVS3）结合使用是最有效的方法。在这项研究中，我们尝试了三种类型的玻璃化溶液：B5，A3（在冰上90％的PVS2）和A5（在冰上70％的PVS2）。在本研究中，结合加料溶液C4和玻璃化溶液A5（在冰上）显示出最佳的再生速率。在不同步骤中也观察到了茜草毛状根内细胞的组织学变化。来自对照处理的毛状根的细胞完整且完整，具有不同大小的液泡和具有深色核仁的明显细胞核。在准备冷冻保存的阶段（预培养，上样，然后脱水溶液A5或B5）之后，细胞间空间变得明显，并且细胞内的细胞质变得更稠密，出现了一周的溶酶。细胞质的溶解更加明显，我们通过计算细胞面积/细胞质的面积来测量细胞质溶解的程度。在预培养，上样溶液C4和脱水溶液A5的组合中，溶酶度的值最高，为1.97。因为在A5处理20分钟中出现了最高的再生速率，所以我们可以假设冷冻保存的最佳溶胞度可能在1.97左右。冷冻保存过程中细胞结构的变化可能是开发适当的madder种质长期保存方法的有用基础。

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