生物多樣性種原之超低溫保存技術研究---以竹柏為例

Cryopreservation of Germplasm of Biodiversity－the Case Study of Nageia Nagi

李胤德(Yin-Te Lee)

指導教授 ： 李魁鵬

國立臺北科技大學/機電學院/冷凍空調工程系所/碩士(2005年)

https://doi.org/10.6841/NTUT.2005.00015

摘要

由於在低溫狀態能抑制生物體的生理變化，甚至在液氮溫度下，更能長期保存植物。本試驗以不同抗凍劑甘油濃度（無、0.2M、0.6M及1M）對種原竹柏進行不同降溫速率（-4℃/min、-10℃/min、-20℃/min、-30℃/min、-35℃/min、-40℃/min及-50℃/min）保存處理，探討其在超低溫冷凍過程中之最佳化處理程 序的生物多樣性種原超低溫保存技術。
種原竹柏進行超低溫冷凍保存後，經TTC檢測，結果顯示當降溫速率-30℃/min、抗凍劑 1M的條件具有較佳存活率74％；最低的為降溫速率-4℃/min 、抗凍劑0.2M為50％。本研究再將活與死種子藉由石蠟包埋切片法後，於光學顯微鏡下觀察，可發現死細胞比活細胞的澱粉粒明顯減少及細胞壁破損；而活細胞比原始細胞澱粉粒來得緊密。

關鍵字

超低溫保存 ； 種原 ； 生物多樣性

並列摘要

This research set up a precision and high speed temperature controlled biological cryofreezer. The germplasm preservation technique for biodiversity was developed through the analysis of TTC viability tests and observation of ultrastructural changes in cells brought about by low temperatures. This study used the seeds of Nageia Nagi as an example to find the optimal protocol of the freezing process.
The experiments were designed to determine the optimum freeze/thaw cycle for cryopreservation of Nageia Nagi and the optimum cryoprotectant agent (CPA) for the seeds of Nageia Nagi. The viability was analyzed by TTC viability tests after exposure to four concentrations of CPA (glycerol) treatments (0M, 0.2M,0.6M and 1M) and seven cooling rates（-4℃/min、-10℃/min、-20℃/min、-30℃/min、-35℃/min、-40℃/min and -50℃/min）while the thawing rate was kept constant as 20℃/ min.
The optimum freeze/thaw cycle for cryopreservation of the seeds of Nageia Nagi was the cooling rate of -30%℃/min and CPA of 1M and got the viability of 74%. The worst cycle was the cooling rate of -4%℃/min and CPA of 0M and got the viability of 50%. Through the observation of ultrastructural changes in cells brought about by low temperatures, the results reveal that the cell textures of died seeds were damaged.

並列關鍵字

Cryopreservation ； Germplasm ； Biodiversity

參考文獻

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