這篇論文主要是在討論如何冷凍保存埃及藍睡蓮(Nymphaea caerulea)的種子。由於缺乏對乾燥以及低溫的容忍力，埃及藍睡蓮被認為是很難用傳統方法長期保存的異儲型種子(recalcitrant seed)。冷凍保存(cryopreservation)是一種有效率、經濟的保存異儲型種子的方法。然而種子不能直接冷凍保存，需要先透過植物抗凍配方(Plant vitrification solution)的處理來達到玻璃化(vitrification)的狀態來防止冰晶的形成。然而最常被廣泛使用的植物玻璃化配方PVS3對埃及藍睡蓮的冷凍保存效果並不佳。對於PVS3的效果不佳我提出了兩個假設，冰晶形成以及自由基造成的程序性細胞死亡(ROS-related programmed cell death)。冰晶形成的溫度區間為攝氏零度以下到玻璃化溫度(glass transition temperature)以上。因此發展出玻璃化溫度較高的植物抗凍配方可以縮小冰晶形成的溫度區間進而減少冰晶形成。然而經由玻璃化溫度較高的植物抗凍配方處理的藍睡蓮種子並沒有回到一般水準。PVS+，一種添加了穀胱甘肽(glutathione)的植物抗凍配方能夠抑制因冷凍保存所產生的自由基累積並使處理過種子的發芽率能回復到一般水準。之前的共識認為種子直接冷凍保存會失敗是由於冰晶形成，但我的發現認為先前的共識過於草率、簡略。我的發現認為埃及藍睡蓮種子直接冷凍保存會失敗的理由是因為低溫造成自由基的累積進而傷害種子，而抗氧化劑可以避免這些傷害。

This thesis discussed how to cryopreserve blue waterlily (Nymphaea caerulea) seeds. Due to lack of desiccation and low-temperature tolerance, N. caerulea is considered as recalcitrant, which hard to long-term storage by conventional method. Cryopreservation is an effective and economical method to recalcitrant seeds preservation. Seeds need to be vitrified using the plant vitrification solution (PVS) before cryopreservation. PVS3, a widely used PVS, was not effective in N. caerulea seeds cryopreservation. I hypothesized two reasons that caused PVS3 inefficiency, ice crystals forming and reactive oxygen species (ROS) - related programmed cell death (PCD). Ice crystals form at temperature from below 0℃ to glass transition temperature (Tg). Therefore, developing a PVS with higher Tg can narrow the temperature range, therefore producing less ice crystals forming. Germination rate of seeds treated with higher Tg PVSs were not increased to the normal level. However, PVS+, a glutathione (GSH) adding PVS, decreased ROS accumulation induced by cryopreservation and increased the germination rate to normal level. Previous consensus asuggested that ice crystals forming may be the main reason for cryopreservation inefficiency but my findings indicated that this consensus is oversimplified. It also suggested that ROS accumulation harmed seeds after cryopreservation and antioxidants may prevent the damage.

Abstract………………………………………………………..1
摘要……………………………………………………………2
Key words………………………………………………………3
Abbreviation…………………………………………………...4
Introduction
Freshwater aquatic plants are facing seriously danger of extinction and need to be protected……………...………………………………………………...………5
It is important to conserve Nymphaea caerulea…………………………………..6
Diverse maneuvers to conserve freshwater aquatic plants………………………8
Different methods to store seeds base on seed storage characteristics…………..9
Store recalcitrant seeds by cryopreservation…………………………………...10
PVSs are good but there is still room for improvement………………………...11
Glass transition temperature (Tg) is a key point to ice crystals forming…….12
ROS-induced PCD causes failure of existed PVSs cryopreservation………….12
Methods and Materials………………………………………14
Results
The seed of N. caerulea is recalcitrant…………………………………………..20
Cryopreservation of N. caerulea seeds by PVS3 is not effective enough……...21
New PVS with high Tg might not be an alternative option to cryopreservation…………………………………………………………………22
PVS+ can increase germination rate in cryopreservation……………………..23
Amount of H2O2 truly increased after cryopreservation and decreased after adding GSH………………………………………………………………………24
Discussion…………………………………………………….26
Flow chart……………………….……………………………31
Tables and Figures…………………………………………...33
Supplementary data………………………………………….41
Appendix……………………………………………………...44
References…………………………………………………….69

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