Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22294
標題: 巨峰與蜜紅葡萄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 grapevine(Vitis vinifera L.) treated by vitrification
作者: 陳瓊均
Chen, Chiung-Chun
關鍵字: cryopreservation
冷凍保存
出版社: 生命科學系所
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摘要: 摘要 本實驗以巨峰葡萄和蜜紅葡萄組織培養苗莖頂為材料,探討高濃度蔗糖預培養及冷凍保護前處理對葡萄超低溫冷凍保存之影響,同時為瞭解在高濃度蔗糖預培養下,影響葡萄超低溫冷凍保存存活率之主要因素,進行莖頂相對含水量、水分潛勢、滲透潛勢、膨壓、可溶性糖類與可溶性蛋白質含量之測定。 巨峰葡萄和蜜紅葡萄經不同高濃度蔗糖預培養3天後,以LS處理60分鐘,PVS2冷凍保護劑脫水115分鐘,進行超低溫冷凍保存。巨峰葡萄經0.6M高濃度蔗糖可達60.8%之存活率;蜜紅葡萄經0.3M高濃度蔗糖可達58.9%之存活率。二供試品種在最適預培養濃度下, LS與PVS2處理不同時間其存活率會有異,處理時間太短,無法誘導組織適當脫水,太長則易造成細胞產生過度滲透衝擊及毒性。 經不同的高濃度蔗糖預培養3天後,巨峰和蜜紅葡萄之水分潛勢、滲透潛勢均有下降的現象,蜜紅葡萄在0.4M高糖處理時,膨壓就會大幅下降,巨峰葡萄在0.7M高糖處理下,膨壓才大幅下降。二供試品種經高濃度蔗糖之滲透逆境處理,皆會造成可溶性蛋白含量增加。可溶糖含量的累積會造成滲透潛勢改變。而在滲透逆境下,累積的可溶性糖類主要為雙糖,且累積量足夠時即可達到較高之超低溫冷凍保存存活率。經TTC活性染色後,顯示太高濃度蔗糖預培養會導致細胞喪失活性。 因此,玻璃化法超低溫冷凍保存流程之前處理中,高濃度蔗糖預培養與冷凍保護劑處理時間,為葡萄超低溫冷凍保存成功關鍵。
Abstract Effects of preculture in high concentration sucrose and cryoprotectant pretreatment on two cultivars of grapevine (Vitis vinifera L.) named cv.‘Kyoho'and cv.‘Honey Red'during cryopreservation were investigated. To understand the effects of high concentration of sucrose in preculture medium to the survival of cryopreservation from grape shoot tips, we analyzed the hydric and biochemical modification. Precultured shoot tips with different sucrose concentrations for 3 days were loaded with LS for 60 min and dehydrated with PVS2 for 115 min at 00C before immersion into LN. The best survival rate of 60.8% and 58.9% were obtained when shoot tips were precultured on 0.6M and 0.3M sucrose for grape of cv.‘Kyoho'and cv.‘Honey Red', respectively. In the adaptable preculture median, cryoprotectant pretreatment is important, when pretreatment duration too short could led to tissue without adaptable dehydration, pretreatment duration too long could have a severely osmotic stress and toxicity. After preculturing two cultivars of grape shoot tips on different sucrose concentrations for 3 days, the water potential and osmotic potential decreased. In cv. ‘Honey Red', the turgor pressure was significantly decreased when were precultured on 0.4M sucrose. However, cv. ‘Kyoho 'that turgor pressure was significantly decreased when were precultured on 0.7M sucrose. Preculturing with sugar-rich medium induced changes in the level of total soluble proteins. Sucrose concentration affected the accumulation of soluble sugar, and indirectly affected osmotic potential change. What kinds of soluble sugars accumulate after osmotic stress is important. Accumulation of disaccharide can enhance survival of cryopreserved shoot tips. The TTC reduced in stained shoot tips, indicated that preculturing with high concentration sucrose led to tissue vitality loss. Thus, preculture with high concentration sucrose and the duration for cryoprotectant pretreatment play two key roles for investigating grape vitrification.
URI: http://hdl.handle.net/11455/22294
其他識別: U0005-0408200623245600
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-0408200623245600
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