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研究生:楊忠祐
研究生(外文):Chung-Yu Yang
論文名稱:台農69號品種甘藷之超低溫冷凍保存研究
論文名稱(外文):Cryopreservation of Tai-nung 69 sweet potato(Ipomoea batatas [L.] Lam)
指導教授:廖松淵
指導教授(外文):Song-Iuan Liaw
學位類別:碩士
校院名稱:國立中興大學
系所名稱:植物學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:55
中文關鍵詞:超低溫冷凍保存甘藷冷凍保護劑玻璃質化法前處理
外文關鍵詞:cryopreservationsweet potatoPVS2vitrificationpretreatment
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本實驗以甘藷(Ipomoea batatas (L.) Lam)台農69號組織培養苗莖頂為材料,探討高濃度蔗糖培養基預培養及冷凍保護前處理對甘藷超低溫冷凍保存之貢獻,同時探討高濃度蔗糖預培養下莖頂可溶性糖類與蛋白質累積情形,以了解溶質累積與超低溫冷凍保存存活率間的關係。
台農69號甘藷經0.3 M高濃度蔗糖預培養7天後,分別以LS處理20-60分鐘、PVS2冷凍保護劑脫水10-80分鐘後直接置入液態氮中保存可得到存活成果。以LS處理20分鐘,PVS2脫水70分鐘可達80﹪存活。以相同冷凍保護流程進行徐薯超低溫冷凍保存,結果以LS處理40分鐘,配合PVS2脫水70分鐘可達最高存活率25﹪,明顯低於台農69號。
經0.3 M高濃度蔗糖預培養之台農69號甘藷與徐藷滲透潛勢均有下降的現象,以台農69號甘藷為例,發現滲透潛勢下降原因為:可溶性醣類累積量增加了3.6倍、可溶性蛋白質增加了3倍;經由SDS-PAGE電泳分析發現蛋白質大量累積的現象,以小分子蛋白質:13.1、8.8及7.7 kDa之蛋白質累積情形較為特殊。
經超低溫冷凍保存後之莖頂明顯發生冷凍保護劑毒害及冰晶形成之物理性傷害。因此,高濃度蔗糖培養基與冷凍保護劑處理時間為甘藷超低溫冷凍保存成功與否的決定關鍵。
This study investigates effects of preculturing high concentration of sucrose and effects of cryoprotectant pretreatment on sweet potato (Ipomoea batatas (L.) Lam) cryopreservation, and changes of solute content after precultured in high concentration sucrose medium in order to reveal the importance of solutes in cryopreservation.
After 7 days precultured in 0.3 M sucrose medium, shoot tips of TN69 sweet potato pretreated with LS for 20 to 60 minutes, followed by PVS2 dehydration for 10 to 80 minutes, and immersed in liquid nitrogen directly. 80% survival rate was obtained by pretreatment with LS 20 minutes and PVS2 70 minutes. When the same methods were applied to different clones, the survival rates were obviously much lower than that of TN69.
The osmotic potential decreased after preculturing with 0.3 M sucrose in TN69. It was because the soluble sugars increased 3.6 fold, and soluble proteins increased 3 fold than those of untreated ones. Using SDS-PAGE electrophoresis, we find that proteins contents increase tremendously, especially in low molecular weight proteins with MW of 13.1, 8.8 and 7.7 kDa.
Obviously, shoot tips were suffered from cryoprotectant toxicity and ice crystal injury that made meristems can’t be recovered well after cryopreservation.
The conclusion is that preculture with high concentration of sucrose, time of cryoprotectant pretreatment are key factors leading to successful cryopreservation.
表目錄…………………………………………………………………………I
圖目錄………………………………………………………………………II
縮寫表…………………………………………………………………………1
摘要……………………………………………………………………………2
英文摘要……………………………………………………………………….3
一、前言………………………………………………………………………4
二、材料與方法………………………………………………………………9
(一)供試材料………………………………………………………………9(二)甘藷莖頂之超低溫冷凍保存步驟……………………………………9
1.植株預培養馴化處理…………………………………………………9
(1) 高濃度蔗糖預培養…………………………………………………..9
(2) 低溫冷馴化…………………………………………………………..9
2.莖頂處理…………………………………………………………………9
(1) 高濃度蔗糖液體培養基預培養……………………………………...9
(2) 高濃度蔗糖固體培養基預培養…………………………………….10
3.冷凍保護處理…………………………………………………………..10
(1) 矽膠(silica gel)乾燥劑脫水………………………………………10
(2) 藻膠包埋(encapsulation)處理……………………………………10
(3) 冷凍保護劑(cryoprotectant)及玻璃質化(vitrification)處理...10
(4) 高濃度蔗糖處理…………………………………………………….11
(5) 無菌操作檯風乾(laminar flow)脫水…………………………….11
4.超低溫冷凍處理………………………………………………………..11
5.回溫與回復生長處理…………………………………………………..11
6.回復生長後之存活率計算……………………………………………..12
(三)測定項目及方法……………………………………………………12
1.滲透潛勢(osmotic potential)………………………………..12
2.可溶性蛋白質(soluble protein)含量………………………………12
3.可溶性蛋白質之電泳分析……………………………………………..12
(1) 樣品製備…………………………………………………………….12
(2) 膠體之製備………………………………………………………….13
(3) 電泳分析…………………………………………………………….13
(4) 染色及退染………………………………………………………….13
4.可溶性糖類(soluble sugars)含量測定………………………………14
5.解剖觀察(光學顯微鏡之石蠟切片法)……………………………..14
三、結果與討論………………………………………………………………16
(一)以不同冷凍保護前處理進行之甘藷超低溫冷凍保存法之比較……16
(二)高濃度蔗糖培養基預培養……………………………………………22
(三)冷凍保護劑脫水處理與超低溫冷凍保存……………………………24
(四)滲透潛勢………………………………………………………………30
(五)可溶性糖類累積………………………………………………………32
(六)可溶性蛋白質累積與蛋白質SDS-PAGE電泳分析…………………35
(七)莖頂組織切片觀察……………………………………………………37
(八)回復生長期之外觀變化………………………………………………40
四、結論………………………………………………………………………47
參考文獻………………………………………………………………………48
附錄一…………………………………………………………………………52
附錄二…………………………………………………………………………53
附錄三…………………………………………………………………………54
附錄四…………………………………………………………………………55
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