臺灣博碩士論文加值系統

This study investigates the cryopreservation of the in-vitro grown axillary buds of Taiwan native medicinal herb Bupleurum kaoi Liu, Chao et Chuang（B. kaoi）using the vitrification procedure. Factors influencing the success of B. kaoi cryopreservation include the duration of exposure to the cryoprotectant solution and the composition of the post-cryostorage recovery medium. The plantlets obtained from 45-day shoot cultures were precultured for 3 days on solidified Murashige and Skoog (MS) medium supplemented with 0.2 M sucrose. The 2–3 mm excised stem segments with 1-2 axillary buds were treated for 60 min at room temperature with a loading solution (LS, 2 M glycerol + 0.4 M sucrose) and dehydrated with PVS2 (plant vitrification solution 2) for 120 min at 0℃ before rapid immersion in liquid nitrogen (LN). After 1 week in liquid nitrogen, rapid rewarming at 40℃ for 30 sec in water bath was followed by unloading in 1.2 M enriched sucrose solution for 35 min. The axillary buds were plated on petri dishes containing 1/2 MS medium as recovery medium for 2 weeks and transfered to conical flask containing 1/2 MS medium supplemented with 0.1 mg/L benzyladenine (BA) and 0.1 mg/L α-naphthaleneactic acid (NAA) for 2 month. This protocol achieved 59.63% buds recovery rates and 73.33% regeneration rate. The morphology of the plantlets after cryopreservation were indistinguishable to the normal ones. Analysis the contents of saikosaponins found that the main accumulation was in the aboveground. After cryopreservation, the subculture derived from aboveground and root of regeneration plantlet were the same as the non-cryopreserved plantlet. In conclusion, the protocol established in this study could be adapted to the cryopreservation of B. kaoi by vitrification.

目次
摘要………………………………………………i
Abstract………………………………ii
目次…………………………………………………iii
表目次……………………………………………v
圖目次……………………………………………………vi
縮寫表………………………………………………viii
一、前言………………………………………………………1
二、前人研究……………………………………………………4
三、材料與方法
(一) 試驗材料建立………………………………8
（二）玻璃質化法（vitrification）之超低溫冷凍保存實驗步驟
1. 植株預培養處理……………………………………………8
2. 冷凍保護劑PVS2（plant vitrivication solution 2）處理……8
3. 回溫及回復生長……………………………8
4. 回復培養基測試
（1）第一階段回復培養（培養皿）…………………………9
（2）第二階段回復培養（450 mL培養瓶）……………9
（三）生理分析測定項目之方法
1. 相對含水量（relative water content）……………………9
2. 水分潛勢（water potential,Ψw）、滲透潛勢（osmotic potential,Ψπ）及膨壓（turgor pressure,Ψp）之測定……………………10
3. 可溶性蛋白質（soluble protein）含量…………………10
4. 可溶性糖（soluble sugar）含量………………………10
5. 離子滲漏（ion leakage）測定…………………………11
6. 可溶糖種類及含量測定…………………………11
7. 基本生物量（biomass）測定
（1）株高測定……………………………………12
（2）植株重量……………………………………12
（3）枯葉率……………………………………12
8. 高氏柴胡皂素-a、-b（saikosaponin-a、-d）之含量測定
（1）材料…………………………………………12
（2）分析方法………………………………12
（四）統計分析……………………………………13
四、結果
（一）高氏柴胡芽體玻璃質化法之超低溫冷凍保存前處理比較
1. 不同濃度蔗糖預培養天數……………………16
2. 不同處理預培養冷凍保存之存活率及再生率比較………16
3. 不同預培養處理之芽體對PVS2的忍受性……………17
(二) 不同濃度蔗糖預培養處理對高氏柴胡芽體內含物質之影響
1. 相對含水量（relative water content）變化…………………33
2. 水分潛勢（water potential）、滲透潛勢（osmotic potential）及膨壓 （turgor pressure）之變化…………………33
3. 可溶性糖類（soluble sugar）含量變化…………………33
4. 可溶糖種類及含量分析……………………………………34
5. 可溶性蛋白質（soluble protein）含量變化………………35
(三) 回溫後高氏柴胡芽體回復及再生長之比較
1. 回復培養基對存活率及再生率之影響…………………44
（1）第一階段回復培養………………………44
（2）第二階段回復培養……………………………44
2. 植株基本生物量（biomass）分析……………………45
（1）植株高度……………………………………45
（2）枯葉率………………………………………45
（3）鮮重及乾重…………………………………45
3. 高氏柴胡皂素含量分析………………………………46
（1）不同回復培養基植株之柴胡皂素-a、-d含量分析……46
（2）未經冷凍保存之植株與移至增殖培養基之再生植株皂素-a-、-d含量分析比較……………………46
五、討論
（一）不同預培養蔗糖濃度及天數對高氏柴胡培植體生長之影響…60
（二）蔗糖預培養對高氏柴胡冷凍保存後之存活率及植株再生之影響…61
（三）不同濃度蔗糖預培養對高氏柴胡芽體內含物質之影響………62
（四）冷凍保護劑對高氏柴胡玻璃質化法超低溫冷凍保存存活率及再生率之影響………………………65
（五）不同回復培養基對高氏柴胡超低溫冷凍保存植株再生之影響…67
六、結論………………………………………………………74
七、參考文獻 ………………………………75
附錄………………………………………………86

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