共生藻因與珊瑚有著不可或缺的共生關係，因此共生藻成為出於珊瑚保育為目的的低溫冷凍保存研究焦點。共生藻的低溫冷凍保存在空間以及經濟上具有長期的效益，不僅減少培養的污染及遺傳漂變，更可協助育種及保種。本研究利用本實驗室先前已發展之冷凍保存條件以及專利設計之兩段式冷凍保存法進行五種共生藻冷凍保存後，以穿透式電子顯微鏡探討共生藻的超微結構變化。本研究結果顯示，共生藻經由冷凍保存後其超微結構不論是從白鞭珊瑚分離的Gerakladium或是培養的Symbiodinium、Breviolum、Cladocopium與Durusdinium的共生藻其葉綠體皆出現約30~70%的破壞，葉綠體作為光合作用唯一場所參與許多其他代謝途徑，因此葉綠體被破壞會影響相關能量、脂質、色素、植物激素和許多其他代謝產物的合成；另外，以乙二醇作為抗凍劑的Symbiodinium與Cladocopium以及甲醇混合蔗糖作為抗凍劑的Gerakladiu經由冷凍保存後觀察到約5~16%細胞壁受損，推論細胞壁的破壞可能由抗凍劑、細胞內外冰晶或是冷凍時細胞密度過大所導致。此外，除了Symbiodinium以外的四種共生藻在解凍後有4~7%不規則的小泡顯著增生。但脂質、澱粉、澱粉核大小、細胞大小在經由冷凍保存後並無顯著差異，推測胞器在解凍後並不會立即出現型態上之變化，因此冷凍傷害對這些胞器是否產生影響需要經由解凍後的長期培養或是分子層次的測定來觀察。在超微結構的觀察下，新鮮分離的共生藻細胞與脂質的大小皆大於培養的共生藻，可能與珊瑚共生的共生藻受到宿主所影響，因此內部的能量會高於培養的共生藻。本實驗結果顯示Symbiodinium以甲醇、乙二醇；Breviolum以甲醇；Cladocopium以甲醇；Durusdinium以丙二醇、乙二醇配合兩段式冷凍法有著較佳的結果。本研究對未來共生藻冷凍保存的發展有實質上的幫助。

There is an inseparable symbiosis relationship between symbiotic algae and coral, therefore, symbiotic algae have become the focus of cryopreservation research for coral conservation. The cryopreservation of symbiotic algae has long-term benefits in terms of space and economy as it not only just reduces the pollution of the culture being preserved, but also genetic drifts within the culture. Additionally, it can assist in breeding and develop cryobank. In this study, five species of symbiotic algae were preserved using the cryopreservation conditions of symbiotic algae previously developed and the two-step freezing method patented by our lab. Following by observation of ultrastructural changes of the symbiotic algae were investigated using a transmission electron microscope (TEM). The result showed that the ultrastructure of symbiotic algae whether it was the fresh isolated, Gerakladium, separated from Junceella fragilis, or the cultured, Symbiodinium, Breviolum, Cladocopium, and Durusdinium all had 30 to 70% destruction in the chloroplast after freezing. As the organelle responsible for photosynthesis and the chloroplast participates in many other metabolic pathways, the destruction of chloroplasts in Symbiodiniaceae will affect related energy, lipids, pigment, hormone, and the synthesis of other metabolites. In addition, using ethylene glycol as a cryoprotectant for Symbiodinium and Cladocopium, and using methanol mixed with sucrose as a cryoprotectant for Gerakladium, after cryopreservation, it was discovered that around 5 to 16% of the cell wall was damaged. It is inferred that the damage to the cell wall might be caused by the cryoprotectant, intracellular and extracellular ice crystals, or excessive cell density while freezing. In addition, the four symbiotic algae other than Symbiodinium have 4 to 7% of irregular vesicles significantly proliferating after thawing. However, there is no significant difference in the morphology and size of lipid, starch, pyrenoid, and cell after cryopreservation. It is speculated that the organelle will not change in shape immediately after thawing. Accordingly, whether freezing damage affect these organelles will require long-term culture. Under the observation of the ultrastructural, the sizes of both freshly isolated symbiotic algae cells and lipids are larger than the cultured ones. It is possible the higher internal energy provided by the host. The results of showed that using methanol and ethylene glycol for Symbiodinium; methanol for Breviolum; methanol for Cladocopium; propylene glycol and ethylene glycol for Durusdinium as cryoprotectants resulted in the most successful cryopreservation. The results from this study will support future development of Symbiodiniaceae cryopreservation.

摘要 I
Abstract III
目錄 V
圖目錄 VII
表目錄 IX
第一章 前言 1
1.1 珊瑚危機 1
1.2 共生藻與珊瑚的關係 1
1.3 共生藻細胞週期 3
1.4低溫生物學冷凍保存 4
1.5共生藻冷凍保存 6
1.6共生藻超微結構之研究現況 8
1.7藻類冷凍保存超微結構研究 13
1.8研究目的 16
第二章 材料與方法 17
2.1珊瑚採集 17
2.2珊瑚共生藻分離 18
2.3共生藻鑑種 18
2.4人工海水配置 18
2.5共生藻培養 19
2.6兩段式冷凍保存共生藻 20
2.7穿透式電子顯微鏡Transmission electron microscope (TEM) 21
2.7.1固定 21
2.7.2脫水 22
2.7.3滲透 22
2.7.4切片 22
2.7.5染片 22
2.7.6上機 23
2.8數據統計 25
第三章 結果與討論 27
3.1前言 27
3.2結果 29
3.3討論 32
3.4結論 35
參考文獻 47

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