小花睡蓮(Nymphaea minuta)與四色睡蓮(Nymphaea micrantha)是廣熱帶睡蓮亞屬(Brachyceras)中的其中兩種，而這兩種睡蓮的種子都無法承受低溫與乾燥環境，也不能利用傳統儲存方法進行長期保存。因此這兩種種子被歸類為非正儲型(non-orthodox)種子,需要藉由超低溫保存(cryopreservation)的方式來保存。在本研究中，使用植物玻璃化抗凍配方3(plant vitrification solution 3, PVS3)的處理，以避免在種子中形成致命性的冰晶，來進行以玻璃化法為基礎的超低溫保存。兩個小時和三個小時的PVS3前處理分別對四色睡蓮與小花睡蓮的種子超低溫保存是最適合的處理時間。為了進一步提升超低溫保存的效果，嘗試先對種子部分脫水以提高讓細胞玻璃化的效率，並使用了抗氧化劑如穀胱甘肽(glutathione, GSH)，添加到PVS3中以保護種子在一系列超低溫保存的處理過程中免於自由基產生的氧化傷害與進一步造成的細胞凋亡。本研究接著探討在經過不同前處理後超低溫保存的種子中，表觀基因(epigenetic)層級產生的變化。在兩種睡蓮種子中，進行超低溫保存後總體DNA甲基化(methylation)的比率普遍上升，因此推測改變DNA甲基化的比率或許可以改善超低溫保存後種子的活力。於是將超低溫保存後的四色睡蓮種子培養在含有去甲基化分子——5-氮胞咁的水溶液後，種子發芽率獲得提升。

Nymphaea minuta and Nymphaea micrantha are two species of waterlilies in Brachyceras subgenus. Both seeds are freezing and desiccation intolerant, and could not be stored by conventional method for long-term. Therefore, they were considered as non-orthodox seeds, which have to be conserved by cryopreservation. In this study, vitrification-based cryopreservation method was applied by using plant vitrification solution 3 (PVS3) to avoid lethal ice crystals formation. The 2hr and 3hr PVS3 pretreatment were most suitable for cryopreserved seeds of N. micrantha and N. minuta, separately. To further promote the efficacy of cryopreservation, partial dehydration was applied for enhance the efficiency of vitrification, and the antioxidant, glutathione (GSH) was added in PVS3 to prevent cryopreserved seeds from the oxidative damage and programmed cell death which was induced by reactive oxygen species (ROS). The changes of epigenetic states in cryopreserved seeds with different pretreatment were next investigated. The global DNA methylation rate of seeds increased after cryopreservation in both species. Therefore, it was considered that changing the DNA methylation rate might improve the viability of cryopreserved seeds. Then, the cryopreserved seeds of N. micrantha were treated with 5-azacytidine, a DNA-hypomethylating molecule, in recovery stage, and the germination rate increased.

摘要 ... II
Abstract ... III
誌謝 ... IV
Contents ... V
Abbreviations ... VII
Introduction
Current status of freshwater ecosystems and Nymphaea species ... 1
Characteristics of N. minuta and N. micrantha ... 2
Ex situ conservation strategy ... 4
Cryopreservation could be an effective way to store recalcitrant seeds ... 5
Reactive oxygen species and antioxidants are vital factors ... 8
Epigenetic changes may occur during cryopreservation ... 10
Materials and methods ... 12
Results
The storage behavior of N. minuta and N. micrantha seeds ... 17
Cryopreservation of N. minuta and N. micrantha with PVS3 treatment ... 18
The addition of antioxidants in PVS3 for cryopreservation ... 19
Cryopreservation of N. minuta and N. micrantha for medium- to long-term...20
Changes in global DNA methylation levels ... 21
Discussion ... 23
Conclusion ... 29
Figures and Tables ... 30
Supplementary data ... 43
Reference ... 46
Appendix ... 50

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