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標題: 臺農25、臺農64與臺農66品種甘藷玻璃化法超低溫冷凍保存前處理流程之探討
Investigation of pretreatments protocol on the cryopreservation of sweet potato (Impomoea batatas(L.)Lam.) treated by vitrification
作者: 蔡叔芬
Tsai, Shu-Fen
關鍵字: cryopreservation;超低溫冷凍保存;vitrification;preculture;osmotic stress;cotransporter;玻璃化法;預培養;滲透逆境;共同運輸蛋白
出版社: 生命科學系
甘藷(Ipomoea batatas (L.) Lam.)為熱帶作物,在冷馴化期間對低溫敏感且對PVS2之脫水忍受度低,故較不易利用玻璃化法進行冷凍保存。為達到成功的超低溫冷凍保存,以高濃度蔗糖(0.3-1.0M)進行預培養3天,針對不同甘藷品種,選擇較佳的滲透逆境,並誘導甘藷莖頂產生對PVS2滲透與冷凍之忍受能力。
以不同的高濃度蔗糖預培養3天之莖頂,進行相對含水量、滲透潛勢、可溶性醣類與可溶性蛋白質含量之測定。發現三供試品種皆因蔗糖濃度處理而增高可溶性醣類含量,進而影響滲透潛勢變化。尤其臺農25號必須在較高濃度蔗糖(0.6-0.8M)時,可溶性醣類含量才明顯增高,滲透潛勢亦大幅下降。三供試品種經高濃度蔗糖之滲透逆境處理,也造成可溶性蛋白質含量增加,以SDS-PAGE電泳分析,發現分子量26.2kDa、25.3kDa及23.8kDa之蛋白質明顯增加,尤以分子量26.2kDa蛋白質累積最明顯。進一步進行蛋白質N端定序,並進行相似性蛋白質產物比對分析,得知此蛋白質為cation-chloride cotransporter。
本實驗以不同濃度蔗糖進行前處理,讓材料處於適當的滲透逆境,誘導cation-chloride cotransporter大量累積,推測此離子運輸蛋白選擇性地運輸離子並維持生理狀態所需之離子濃度,使組織能在逆境下存活。因此玻璃化法冷凍保存流程之前處理中,以高濃度蔗糖預培養與適當時間之冷凍保護劑處理,為甘藷超低溫冷凍保存成功關鍵之兩步驟。

Sweet potato (Ipomoea batatas (L.) Lam.) is a tropical plant which it has sensitivity to low temperature during cold-hardening and it has low tolerance to dehydration with PVS2. Cryopreservation of sweet potato using vitrification has been shown difficult. For successful cryopreservation by vitrification of this plant, we have precultured for 3 days on different high concentrations sucrose (0.3-1.0M) media and investigated suitable osmotic stress induced the osmotolerance to PVS2 solution and tolerance to subsequent freezing by different species of sweet potato.
The shoot tips from in vitro-grown plantlets of sweet potato TN25, TN64 and TN66 (Ipomoea batatas (L.) Lam. cv. Tainung No. 25, 64 and 66) were precultured for 3 days on hormone-free 1/2MS medium containing different sucrose concentrations. The precultured shoot tips were treated with a loading solution (LS) for 60 minutes and then dehydrated with a vitrification solution (PVS2) for 75 minutes, and following cryopreservation. The best survival rates of 50%, 76.6% and 86.6% were obtained when shoot tips were precultured on 0.8M, 0.5M and 0.4M sucrose for sweet potato TN25, TN64 and TN66, respectively.
After preculturing three species of sweet potato shoot tips on different sucrose concentrations for 3 days, we analyzed the hydric and biochemical modification. The results indicated that sucrose concentration affected the accumulation of soluble sugar, and indirect affected osmotic potential change. Especially in sweet potato TN25 that the soluble sugar content and osmotic potential were significantly increased when were precultuered on 0.6-0.8M sucrose. We obtained that proteins content increase in the different high sucrose concentrations, and accumulation of the 26.2kDa, 25.3 kDa and 23.8 kDa protein increased as detected by SDS-PAGE protein analysis, especially the 26.2 kDa protein was significantly accumulated. The N-terminal amino acid sequence of the 26.2 kDa protein was determined and exhibited 83% homology to the cation-chloride cotransporter.
This study pretreatment samples on the optimal high concentration sucrose which showed increase the cation-chloride cotransporter accumulated was induced by osmotic stress. Ion transporters selectively transport ions and maintain them at physiologically relevant concentrations, permitting plant survival and growth under osmotic stress. Thus preculture with high concentration sucrose and pretreatment with cryoprotectant time were achieved through investigating two key steps of the vitrification protocol.
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