Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22664
標題: 大蒜玻璃化法超低溫冷凍保存流程之探討
Study of Protocols on the Cryopreservation of Garlic(Allium sativum L. cv. Great Black Leaf) Treated by Vitrification
作者: 洪永倫
Hung, Yung-Luen
關鍵字: garlic;大蒜
出版社: 生命科學系所
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摘要: 
本試驗的目的,在於如何讓大蒜頂芽經過冷凍保存試驗流程後,有最佳的存活率與生長狀況。試驗結果顯示,將蒜頭置於10 ℃低溫處理一週至四週,配合LS 60分鐘、PVS2 100分鐘、unloading solution 20分鐘,結果以低溫處理一週最佳,存活率為71.33%。預培養試驗以低溫處理一週、含0.1 M至0.7 M蔗糖MS培養基預培養一週、LS處理60分鐘、PVS2處理100分鐘、unloading solution處理20分鐘,結果以含0.1 M蔗糖MS培養基預培養最好,存活率為79.81%。
冷凍保護劑試驗中,若以PVS2為vitrification solution,低溫處理一週、LS處理0至100分鐘、PVS2 100分鐘、unloading solution 20分鐘,結果以LS處理60分鐘最好,存活率為75.35%。PVS2處理時間測定方面,低溫處理一週、LS處理60分鐘、PVS2處理0至120分鐘、unloading solution 20分鐘,結果以PVS2處理100分鐘最優,存活率為74.61%。Unloading solution方面,低溫處理一週、LS處理60分鐘、PVS2處理100分鐘、unloading solution 0至80分鐘,結果以unloading solution處理20分鐘最適合,存活率為72.88%。
若以PVS3為vitrification solution,低溫處理一週、LS處理0至90分鐘、PVS3處理150分鐘、unloading solution處理40分鐘,結果以LS處理50分鐘為最佳,存活率為75.96%。PVS3處理時間測定方面,低溫處理一週、LS處理50分鐘、PVS3處理0至190分鐘、unloading solution處理40分鐘,結果以PVS3處理150分鐘最好,存活率為52.86%。Unloading solution處理測定:低溫處理一週、LS處理50分鐘、PVS3處理150分鐘、unloading solution處理0至80分鐘,結果以unloading solution處理40分鐘最優,存活率為66.01%。
回復生長基試驗中,回復生長培養基A與B對於大蒜之存活率與再生率皆無顯著差異。
分析經低溫處理以及預培養之莖頂可溶性糖類及可溶性蛋白含量變化。結果顯示低溫處理後的大蒜莖頂,可溶性糖類累積並沒有顯著改變;可溶性蛋白總量則隨著低溫處理時間增加而略為上升,但低溫處理二週後之存活率開始下降,可溶性蛋白含量卻持續增加,因此可推測可溶性蛋白之累積與低溫處理之存活率並無明顯相關。預培養試驗之結果則顯示,隨著預培養基中蔗糖濃度提高,大蒜莖頂內可溶性糖類也跟著提高,顯示組織的滲透濃度也跟著蔗糖濃度而上升,但冷凍保存之存活率並沒有因此增加,顯示以高濃度蔗糖培養基,在 25℃下預培養一週,並無法提高大蒜莖頂冷凍保存之存活率。

The main purpose of our experiment is how to obtain the best survival rate and grew situation of the shoot tip of garlic after cryopreservation study process. The result indicated after the study that when the garlic was treated under low temperature such as 10℃ for one-four weeks, then LS for 60 minutes, PVS2 for 100 minutes and treated with the unloading solution for 20 minutes, the best treatment was the one week group and the survival rate was 71.33 %. In the preculture study, it treated the garlic under low temperature for one week and precultivated in 0.1 M-0.7 M sucrose concentration MS medium for one week, then LS for 60 minutes, PVS2 for 100 minutes and treated with the unloading solution for 20 minutes, precultivated in 0.1 M sucrose concentration MS medium was the best treatment and the survival rate was 79.81%.
In the vitrification study, it took PVS2 as the vitrification solution and treated the garlic under low temperature for one week, then LS for 0-100 minutes, PVS2 for 100 minutes and treated with the unloading solution for 20 minutes, the best treatment was LS for 60 minutes and the survival rate was 75.35 %. In the study of PVS2 treatment time, it was treated under low temperature for one week, then LS for 60 minutes, PVS2 for 0-120 minutes and treated with the unloading solution for 20 minutes, the best treatment was PVS2 for 100 minutes and the survival rate was 74.61 %. In the study of unloading solution treatment time, it was treated under low temperature for one week, then LS for 60 minutes, PVS2 for 100 minutes and treated with the unloading solution for 0-80 minutes, the best treatment was unloading solution for 20 minutes and the survival rate was 72.88 %.
In the study of PVS3, it treated the garlic under low temperature for one week, then LS for 0-90 minutes, PVS3 for 150 minutes and treated with the unloading solution for 40 minutes, the best treatment was LS for 50 minutes and the survival rate was 75.96 %. In the study of PVS3 treatment time, it was treated under low temperature for one week, then LS for 50 minutes, PVS3 for 0-190 minutes and treated with the unloading solution for 40 minutes, the best treatment was PVS3 for 150 minutes and the survival rate was 52.86 %. In the study of unloading solution treatment time, it was treated under low temperature for one week, then LS for 50 minutes, PVS3 for 150 minutes and treated with the unloading solution for 0-80 minutes, the best treatment was unloading solution for 40 minutes and the survival rate was 66.01%.
In the study of recovery medium, the survival rate and regeneration rate of garlic in recovery medium A and B were not significantly different.
The evaluation of soluble sugars and soluble proteins accumulation after low temperature treatment and preculture revealed that there was no significant change in accumulation of soluble sugar in garlic shoot tip under low temperature treatment, on the other hand, the total amount of soluble protein grew slightly as the low temperature treatment time increased. However, the survival rate decreased after low temperature treatment for two weeks, while the amount of soluble protein remained increasing. It can assume that there is no significant relationship between the accumulation of soluble protein and survival rate after low temperature treatment. The result indicate in the evaluation of preculture that as the concentration rose, the amount of soluble sugar in garlic shoot tip rose, too, showing that the osmosis concentration in the tissue rose with the sugar concentration, while the survival rate of cryopreservation did not increase consequently. The result showed that one week preculture with high sugar culture medium under 25℃ does not increase the survival rate of garlic shoot tip cryopreservation.
URI: http://hdl.handle.net/11455/22664
其他識別: U0005-1108200815021600
Appears in Collections:生命科學系所

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