台灣欒樹種子超低溫冷凍保存技術之研究

A Study on Cryopreservation of Koelreuteria formosana Seeds

陳睿家(Jui-Chia Chen)

指導教授 ： 李魁鵬

國立臺北科技大學/機電學院/能源與冷凍空調工程系碩士班/碩士(2008年)

摘要

存放於低溫狀態下的生物體，其生理變化能被抑制，而為保存生物體，將其置入液態氮中，便長期保存生物體。本研究經由田口品質方法評量植物種子在超低溫物種保存預處理及過程中，以不同冷凍保護劑種類、劑量濃度、浸泡平衡時間、降溫程序及復溫程序為控制因子，搭配田口方法之直交表表進行試驗，並依田口品質分析法分析超低溫保存對植物種子發芽的影響，探討在超低溫冷凍過程之最佳參數組合。
結果顯示，本試驗設計較佳參數組合為『20%DMSO冷凍保護劑 + -50℃/min的降溫速率 + 20min浸泡時間 + 直接30℃水浴的復溫程序』及『20%甘油冷凍保護劑 + -50℃/min的降溫速率 + 30min浸泡時間 + 直接30℃水浴的復溫程序』；以本試驗設計較佳參數組合進行台灣欒樹種子超低溫冷凍保存，DMSO試驗組種子發芽率可達到82%，甘油試驗組種子發芽率可達78%。研究利用變異數分析試驗，發現影響DMSO或甘油試驗組之重要因子為降溫速率因子。
結果證明採取適當且合理的冷凍前處理、降溫程序及復溫程序，並輔以適合的冷凍保護劑、濃度及並依適當的保護劑浸泡時間，可使種子經由超低溫保存避免冰晶所造成細胞的傷害。

關鍵字

超低溫冷凍保存 ； 生物多樣性 ； 田口方法

並列摘要

Organism’s physiological changes can be inhibited under low temperature environment, and long-term preservation of organisms will be actualized by keeping them in liquid nitrogen. This research brought Taguchi Methods into the treatment and process of plant seeds in cryopreservation, and adopted different kinds of cryoprotectants, dose concentration, immersion balance time, cooling procedure, and re-warming procedure as controlling factors. Then, the research conducted experiments by matching Taguchi Methods Orthogonal. Besides, the research analyzed the impacts of cryopreservation on germination of plant seed according to Taguchi analysis Method, and discussed the best parameter combination in process of cryopreservation.
The result indicated the relatively excellent parameter combination designed by this experiment as being “20%DMSO cryoprotectant + cooling rate of -50℃/min + re-warming procedure with direct 30℃ water bath”, as well as “20% glycerol cryoprotectant + 30min immersion time + re-warming procedure with direct 30℃ water bath”; The experiment used the relatively excellent parameter combination designed by this experiment to carry out the cryopreservation of goldenrain tree seed. The germination rate of the seeds from the DMSO experiment group could reach to 82%, while the germination rate of the seeds from the glycerol experiment group could reach to 78%. This research discovered the cooling rate was an important factor impacting the DMSO or glycerol experiment group, by utilizing analysis of variance (ANOVA).
The result proved that the harm to seed cells caused by ice crystal during cryopreservation could be prevented by adopting proper and appropriate pre-freezing treatment, cooling procedure, and re-warming procedure, together with proper cryoprotectant, concentration, and appropriate time of cryoprotectant immersion.

並列關鍵字

Cryopreservation ； Biodiversity ； Taguchi Methods

參考文獻

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