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研究生:呂玉雯
研究生(外文):Yu-Wen Lu
論文名稱:沉香超低溫冷凍保存流程及蝴蝶蘭去病毒技術之探討
論文名稱(外文):Investigation on Protocol of Cryopreservation of Aquilaria agallocha Roxb. and Virus Elimination method of Phalaenopsis
指導教授:廖松淵
口試委員:范宗宸蔡智賢
口試日期:2015-07-28
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生命科學系所
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:2015
畢業學年度:103
語文別:中文
論文頁數:70
中文關鍵詞:沉香超低溫冷凍保存蝴蝶蘭去病毒
外文關鍵詞:Aquilaria agallochacryopreservationPhalaenopsisvirus elimination
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本試驗以沉香 (Aquilaria agallocha Roxb.) 及V3大白花蝴蝶蘭 (Phalaenopsis Sogo Yukidian ‘V3’)、蝴蝶蘭 ‘台大綠蘋果’ (Phalaenopsis Unimax Moonlight ‘Taida Green Apple’) 無菌組織培養苗為試驗材料,探討玻璃質化法冷凍保存流程與冷凍療法、頂端分生組織療法結合化學療法去病毒成效,同時探討沉香根部誘導、馴化復育之方法。
以培植於增殖培養基3 - 4個月之沉香植株進行試驗。玻璃質化法冷凍保存流程,分別測試預培養蔗糖濃度及回復生長培養基之影響,結果顯示,經0.2 M蔗糖培養基預培養7天,切取1 - 2 mm莖頂,以冷凍保護劑LS處理60 min,PVS2處理90 min,冷凍保存一週後回溫,以含有活性碳之增殖培養基回復生長存活率27.41%。根部誘導以1.0 mgL-1 IBA固態培養基預先處理10天,再移至不含IBA之培養基培養2個月有最高發根率可達92 %,馴化移植成功率94 %。
V3大白花蝴蝶蘭受CyMV感染,以培植於生長培養基3個月之植株進行試驗。冷凍療法結合化學療法去病毒,植株於含有Ribavirin之0.3 M蔗糖培養基預培養5天後,切取2 - 2.5 mm莖頂進行玻璃質化法超低溫冷凍保存,以含有Ribavirin之培養基回復生長存活率30.4 %,去病毒率 0 %。頂端分生組織化學療法直接切取莖頂培植於含有Ribavirin之培養基,培養3個月後之去病毒率達52.17 %;再次切取莖頂以Ribavirin培養基培植2 - 3個月,去病毒率達100 %。
蝴蝶蘭 ‘台大綠蘋果’ 受CyMV及ORSV複合感染,以培植於生長培養基3個月之植株進行試驗。植株預先於含有Ribavirin之培養基處理1個月後,以同樣含有Ribavirin之0.3 M蔗糖培養基預培養5天,切取莖頂進行玻璃質化法超低溫冷凍保存,回復生長存活率為92.5 %。然而無論以冷凍療法結合化學療法或頂端分生組織化學療法,皆無法成功去病毒。

In vitro grown of Aquilaria agallocha Roxb., Phalaenopsis Sogo Yukidian ‘V3’ and Phalaenopsis Unimax Moonlight ‘Taida Green Apple’ were used in this study for cryopreservation, rooting, acclimatization and the virus elimination.
Aquilaria agallocha Roxb. were cultured on proliferation medium 3 - 4 months for experiments. Explants were cryopreserved by vitrification. 1 - 2 mm shoot tips were excised from seedling after preculturing on different sucrose concentration medium for 7 day, treated with LS for 60 min, subjected to PVS2 for 90 min then immerged in liquid nitrogen directly for cryostorage, rewarmed after one week and recovery with different medium. The result demonstrated the explants precultured on 0.2 M sucrose medium for 7 days, treated with LS for 60 min and PVS2 for 90 min, recovery with medium containing activated charcoal, reached a survival rate of 27.41% after cryostorage. In addition, an micropropagation protocol for rooting and accimatizating was developed. Plantlets were rooted (92%) on 1/2 WPM medium after treated with 1.0 mg/L IBA for 10 days and acclimatized successfully (94%).
CyMV infected Phalaenopsis Sogo Yukidian ‘V3’ and CyMV, ORSV doubly infected Phalaenopsis Unimax Moonlight ‘Taida Green Apple’ cultured on growth medium for 3 months were applied for investigating the effects of meristem tissue culture, cryotherapy, and chemotherapy on virus elimination. 52.17 % of virus-free Phalaenopsis Sogo Yukidian ‘V3’ were obtained when excised shoot tips were cultured on medium containing Ribavirin for 3 months. Up to 100 % of virus-free Phalaenopsis Sogo Yukidian ‘V3’ were obtained when shoot tips excised from previous treated explants were cultured on Ribavirin medium again. On virtrification-cryotherapy, Phalaenopsis Sogo Yukidian ‘V3’ precultured and recoverd with medium containing Ribavirin, reached a survival rate of 30.4%. However, it failed to eliminate virus. For Phalaenopsis Unimax Moonlight ‘Taida Green Apple’, plantlets were treated with Ribavirin for one month then subjected to virtrification-cryotherapy, reached a survival rate of 92.5%. Unfortunately, neither cryotherapy nor meristem tissue culture combined with chemotherapy could achive virus elimination.

中文摘要 I
Abstract II
目次 III
圖目次 VII
縮寫表 IX
一、 前言 1
二、 前人研究 4
(一) 玻璃質化法超低溫冷凍保存 4
(二) 莖頂冷凍療法去病毒 6
三、 材料方法 8
(一) 沉香 8
1. 試驗材料 8
2. 莖頂玻璃質化法超低溫冷凍保存流程 8
(1) 預培養蔗糖濃度試驗 8
(2) 回溫與回復生長 8
3. 根誘導生長試驗 9
(1) IBA濃度對沉香發根率及根部生長之影響 9
(2) 高濃度IBA處理天數對沉香發根率及根部生長之影響 9
(3) 泥炭苔培養對沉香發根率及根部生長之影響 9
(4) 以增殖培養基Pc取代Arr培養基對沉香發根率及根部生長之影響 9
(二) 大白花蝴蝶蘭 12
1. 試驗材料 12
2. 化學療法結合玻璃質化法超低溫冷凍療法去病毒流程 12
3. 頂端分生組織療法結合化學療法去病毒流程 12
(1) 一次切芽 12
(2) 二次切芽 13
4. 培植體病毒分析 13
(三) 蝴蝶蘭 ‘台大綠蘋果’ 14
1. 試驗材料 14
2. 化學療法結合玻璃質化法超低溫冷凍療法去病毒流程 14
3. 頂端分生組織療法結合化學療法去病毒流程 14
4. 培植體病毒分析 15
(四) 統計分析 15
四、 結果 17
(一) 沉香 17
1. 莖頂玻璃質化法超低溫冷凍保存流程 17
(1) 預培蔗糖養濃度試驗 17
(2) 回溫與回復生長 17
2. 根誘導生長試驗 18
(1) IBA濃度培養對沉香發根率及根部生長之影響 18
(2) 高濃度IBA處理天數對沉香發根率及根部生長之影響 18
(3) 泥炭苔培養對沉香發根率及根部生長之影響 18
(4) 以增殖培養基Pc取代培養基Arr對沉香發根率及根部生長之影響 18
(二) 大白花蝴蝶蘭 36
1. 化學療法結合玻璃質化法超低溫冷凍療法去病毒 36
2. 頂端分生組織療法結合化學療法去病毒 36
(1) 一次切芽 36
(2) 二次切芽 36
(三) 蝴蝶蘭 ‘台大綠蘋果’ 41
1. 化學療法結合玻璃質化法超低溫冷凍療法去病毒 41
2. 頂端分生組織療法結合化學療法去病毒 41
五、 討論 45
(一) 沉香 45
1. 莖頂玻璃質化法超低溫冷凍保存 45
2. 根部誘導生長與馴化 46
(二) 蝴蝶蘭去病毒 47
1. 化學療法結合冷凍療法去病毒 47
2. 頂端分生組織療法結合化學療法去病毒 49
六、 結論 50
參考文獻 51
附錄 60


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