珊瑚礁生態系為海洋中生產力及生物多樣性最高的生態系，而現今海洋環境在人爲污染及全球暖化的影響下，導致了珊瑚礁受到破壞，因此珊瑚礁的復育及保育刻不容緩，而珊瑚細胞的冷凍保種正是方法之一。本研究的目的是發展珊瑚細胞的低溫冷凍保存條件，並建立其冷凍基因庫，同時比較野生及養殖珊瑚物種細胞在生理、生化、結構上的不同，分析在二段式冷凍保存後細胞在抗凍劑適用性、存活率、所需平衡時間、萃取時間及共生藻含量的差異。從本研究中發現珊瑚細胞有支持細胞(Supporting cell)、腺細胞(Gland cell)、表皮刺絲囊(Epidermal nematocyst)、共生藻(Symbiodiniaceae)和共生藻內胚層細胞(Symbiotic Endoderm Cell)，其中大部分的珊瑚以紡綞細胞(Spindle cell)和簇細胞(Cluster cell)爲主，胃皮層刺絲囊(Gastrodermal nematocyst)最不常見的。本研究的結果顯示，珊瑚細胞冷凍保存的條件會根據細胞大小、形狀、脂質含量、抗凍劑影響、冷凍敏感性和冰晶的形成而有所不同，雖然從26種珊瑚細胞中可以識別出9種細胞，但這些細胞會因黏液和季節變化等因素而對冷凍保存產生影響。另外，從本研究結果發現一半以上的測試珊瑚細胞適用於使用DMSO作為冷凍保護劑進行冷凍保存，而使用MeOH和EG作為抗凍劑可以成功冷凍保存另一半約40種的珊瑚內的細胞，其中珊瑚A. tenuis、C. ocellina、P. clavus、P. cactus、P. lobata、P. lutea、P. pini、T. peltata、T. stellulata、M. valenciennesi和M. colemani適用於所有本研究中測試的冷凍條件，與此同時，我們也發現共生藻平均佔總珊瑚細胞的6%。另一方面，相同種類的野生和養殖珊瑚細胞在大小、形狀、抗凍劑的適用性、平衡和萃取時間方面，並沒有顯著差異。相比五種養殖珊瑚，80%的野生珊瑚物種都擁有共生藻內胚層細胞，而大多數珊瑚的細胞種類沒有顯著差異。此外，比較同種但不同環境的珊瑚，本研究發現支持細胞在野生H. coerulea和培養的H. coerulea、P. cactus、P. damicornis含量較多。野生E. lamellosa中的共生藻和野生P. damicornis中的支持細胞以及野生S. caliendrum中的蟲黃藻內胚層細胞與其養殖同類相比具有顯著更高的含量。共生藻也普遍大於所有珊瑚細胞，並且在野生和養殖珊瑚之間的支持細胞和共生藻大小沒有顯著差異。除了養殖珊瑚中的共生藻外，大多數珊瑚除了養殖的珊瑚E. lamellosa和S. caliendrum明顯比它們的野生同類大，上述的結果推測和野外及養殖珊瑚的細胞發育環境、細胞反應和細胞結構而導致細微差異。最後，在我們所建立的珊瑚細胞冷凍基因庫中，每種珊瑚至少低溫冷凍保存了14組冷凍麥管的珊瑚細胞，共超過1884組冷凍麥管（0.5毫升/組），平均濃度為每毫升6.4x106。本研究對於珊瑚冷凍保存技術、珊瑚冷凍基因庫建立及未來珊瑚的保育都具有實質上的益助。

As coral reefs have suffered as a result of global pollution and climate change, they have been deteriorated and require the assistance of scientists, in which case research into suitable cryopreservation procedures and the physiological differences between wild and cultured corals becomes necessary. This, however, has yet to be done. The main objective of this study was to develop a protocol that examines the suitability of each coral species to freezing in order to establish the first-ever coral cell cryobank and compare the differences between wild and cultured coral species in terms of cryopreservation on factors such as CPA suitability, viability rate, equilibrium time, extraction time, type of cell identification, and Symbiodiniaceae concentration. Coral cells including supporting and gland cells, epidermal nematocysts, Symbiodiniaceae and symbiotic endoderm cells (SEC) were found from the extracted protocol. Approximately half of the corals from the experimental corals consisted spindle and cluster cells. Gastrodermal nematocysts were the least common. The freezing protocol conducted in this study was executed with specific compatibility depending on size, shape, lipid content, CPA impact, chilling sensitivity, and ice formation. Although, 9 types of cell functions out of 26 types of cells were able to be identified, it was clear that limiting variables like mucus and seasonal change might have an impact on the effectiveness of cryopreservation. The result showed that freezing using DMSO as a cryoprotectant was suitable for over half of the tested coral, while the other half could be successfully frozen using MeOH and EG as CPAs. A. tenuis, C. ocellina, P. clavus, P. cactus, P. lobata, P. lutea, P. pini, T. peltata, T. stellulata, M. valenciennesi, and M. colemani could be cryopreserved with all types of CPAs. The identified Symbiodiniaceae accounted for 6% of the average from total cell concentration. On the other hand, the same species of wild and cultured coral cells had no significant difference in terms of size, shape, suitability of CPAs, equilibration and extraction time for majority of the coral species examined. Eight out of ten wild coral species consisted of Symbiotic Endoderm Cell (SEC) compared to five cultured corals. Also, there was no significant different in the cell type abundance for most of the corals as supporting cells were the dominant cells except for wild H. coerulea and cultured H. coerulea, P. cactus, P. damicornis. Additionally, the Symbiodiniaceae in wild E. lamellosa, and supporting cells in wild P. damicornis, as well as SEC in wild S. caliendrum have significantly higher abundance compared to their culture counterparts. The cell diameter of symbiodiniaceae was generally larger than the supporting cells in all coral species and there were no significant differences observed in cell diameter of supporting cells and Symbiodiniaceae between wild and cultured corals for most of the corals with the exception of the Symbiodiniaceae in cultured corals E. lamellosa and S. caliendrum which were noticeably larger than theirs wild counterparts. The minor differences found could be related to the cell development environment, cell responses, and cell structures. This study has cryo-banked coral cells from at least 14 straws from each species, accounting for more than 1884 straws (0.5 mL) with an average concentration of 6.4x106 per ml and the findings will undoubtedly aid in coral cryobanking and conservation in the future.

ACKNOWLEDGEMENT I
中文摘要 III
ABSTRACT V
TABLE OF CONTENTS VII
LIST OF TABLES XI
LIST OF FIGURES XIII
LIST OF APPENDICES XVII
CHAPTER 1: INTRODUCTION 1
1.1. Coral reefs 1
1.1.1. Importance of coral reefs 5
1.1.2. Coral metabolism 5
1.1.3. Relationships between Symbiodiniaceae and coral 5
1.1.4. Coral cell identification 7
1.2. Coral reef crisis 7
1.2.1. Climate changes 7
1.2.2. Stress on coral 8
1.2.3. Coral diseases 8
1.2.4. Coral bleaching 9
1.3. Global effort on reduction of coral reefs crisis 9
1.4. Cryopreservation 10
1.4.1. An update on the study of coral cryopreservation 11
1.5. Coral cell cultures 14
1.5.1. Types of coral cell extraction 17
1.5.2. Methods for measuring cultured cell viability 18
1.5.3. Challenges in cell culture 18
1.5.4. Application of cultured cell 19
1.6. Cryobanking 19
1.6.1. Cryobanking procedure 23
1.6.2. Database management of a cryobank 24
1.6.3. Problem of cryobanking for coral and other marine invertebrates 24
1.6.4. Aim of the research 26
CHAPTER 2: MATERIALS AND METHODS 27
2.1. Coral collection 27
2.2. Coral identification 27
2.3. Host cell and Symbiodiniaceae extraction 27
2.4. Cryopreservation 28
2.5. Viability assay 28
2.6. Coral cryobanking 29
2.7. Classification of cell size abundance 29
2.8. Statistical analysis 30
CHAPTER 3: CRYOPRESERVATION AND CRYOBANKING OF
CORAL CELLS 33
3.1. Introduction 33
3.2. Results 36
3.3. Discussion 51
3.3.1. Coral cell type and function 53
3.3.2. ATP assay 55
3.3.3. Limitation of successful cryopreservation 55
3.4. Conclusion 56
CHAPTER 4: CORAL CELL TYPE, SIZE, AND DENSITY BETWEEN WILD AND CULTURED CORALS 57
4.1. Introduction 57
4.2. Results 59
4.3. Discussion 77
4.3.1. Cryoprotectant suitability between wild and cultured corals 77
4.3.2. Coral cellulation and differentiation 78
4.3.3. Coral cells density and formation 79
4.4. Conclusion 82
REFERENCES 83
APPENDICES 111

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