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標題: 番木瓜玻璃質化法超低溫冷凍保存流程之探討
Investigation of Protocol on the Cryopreservation of Papaya (Carica papaya L.) Treated by Vitrification
作者: 羅文陽
Ro, Wen-Yang
關鍵字: papaya;番木瓜;cryoperservation;vitrification;PVS2;超低溫冷凍保存;玻璃質化法;抗凍保護劑
出版社: 生命科學系所
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本研究以台農二號的番木瓜(Carica papaya L.)組織培養苗為材料,成功地進行玻璃質化法超低溫冷凍保存。將株齡6~8週大的番木瓜以0.09 M蔗糖預培養3天,誘導產生脫水及冷凍的耐受性,切取莖頂組織後,分別處理LS (loading solution) 60分鐘和PVS2 (plant vitrification solution 2) 60分鐘(0℃),接著置入液態氮中保存。保存的莖頂在回溫後,約二到四星期即可觀察到明顯的生長,二個月後即長成完整植株,存活率可高達96%。本流程也成功地在其他六個品種的番木瓜上得到穩定的存活率,大約介於85~96%,有效地達成種原長期保存的目的。
實驗中進行生理分析和切片觀察,探討玻璃質化法流程中預培養對冷凍保存存活率之影響。結果發現番木瓜隨著預培養基中蔗糖濃度增加,組織內會累積可溶糖,造成相對含水量、水分潛勢及滲透潛勢的下降。在低濃度(0.09 M)的蔗糖預培養下,冷凍保存的存活率最佳,蔗糖濃度太高(0.5~0.6 M)反而會導致嚴重的滲透逆境,冷凍保存存活率因此降低。顯示番木瓜不適合高糖預培養,提供適當濃度的蔗糖預培養加上前處理條件的配合,才是成功進行番木瓜玻璃質化法超低溫冷凍保存的關鍵。

Shoot tips of in vitro-grown plantlets of papaya (Carica papaya L. cv. Tai-nung No.2) from seven cultivars were successfully used for cryopreservation by vitrification. To induce the osmotolerance and tolerance to subsequent freezing, 6 to 8-week-old papaya plantlets were precultured in 0.09M sucrose for 3 days. After preculture, excised shoot tips were pretreated with LS (loading solution) for 60 min and dehydrated with PVS2 (plant vitrification solution 2) for 60 min at 0℃ and then were directly immersed into liquid nitrogen. Cryopreserved shoot tips after being warmed resumed growth between 2 and 4 weeks and developed plantlets after 2 months. The recovery percentages of shoot tips were about 96%. This study has successfully developed a simple and effective protocol for the cryopreservation of papaya shoot tips from other six cultivars and the recovery percentages were steady between 85~96%.
To investigate effects of the preculture protocol by vitrification on the survival of papaya, we analyzed the physiological responses and observed histological sections. Soluble sugar accumulated in papaya shoot tips with increase of the sucrose concentration, resulting in decrease of relative water content, water potential and osmotic potential. The best survival was obtained when papaya was precultured in a low sucrose concentration (0.09M). Preculturing with high sucrose concentrations (0.5~0.6M) could cause excessive osmotic stress, resulting in decrease of the survival of cryopreservation. The result indicated preculture in a high sucrose concentration was not suitable for papaya. It suggested preculture with a suitable sucrose concentration and pretreated durations were achieved through investigating two key steps of the vitrification protocol.
其他識別: U0005-1508200717492700
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