本論文探討臺灣原生藥用植物-高氏柴胡(Bupleurum kaoi Liu, Chao et Chuang)利用組織培養技術建立種苗大量繁殖生產系統，及細胞培養生產柴胡皂苷(saikosaponin)之可行性。將田間植株腋芽培養於含1 mg/L 6-苄氨基嘌呤(benzyladenine, BA)配合0.1 mg/L奈乙酸(α-naphthalene- acetic acid, NAA)之半量Murashige and Skoog (1/2MS)培養基，可建立瓶苗之初代培養。無菌苗繼代培養於添加0.25 mg/L BA之1/2MS培養基可達到大量繁殖之目的，然而部份瓶苗卻呈現玻璃質化現象。利用雙層鋁箔紙(aluminum foil, AF)覆蓋培養容器瓶口培養2週，以三層藥包紙(dispense paper, DP)進行容器封口置換處理，可以達到抑制玻璃質化苗形成之效果。此外，利用液態培養或是固、液態培養基交替培養方式，並不利於高氏柴胡組培苗之生長與增殖。
組培苗培養於含有0.5 mg/L吲哆丁酸(indole-3-butyric acid, IBA)與0.1-0.2 mg/L NAA之1/2MS培養基，並且配合藥包紙封口置換處理，瓶苗生長發根良好，並具有較高之出瓶移植成活率；出瓶組培苗移植於已滅菌混合介質，藉由透明盒保濕處理，於22℃ (14 h光照) / 18℃ (10 h黑暗)條件進行馴化，存活率可達92%。掃描式電子顯微鏡(scanning electron microscopy, SEM)觀察結果顯示，利用三層藥包紙進行封口置換處理，可有效促進葉片蠟質累積與改善氣孔功能。本研究利用瓶內發根處理(改善培養基組成與容器封口)及瓶外馴化處理(保濕處理及生長箱溫控環境)之結果，可提昇高氏柴胡組培苗馴化存活率。利用高壓液相層析法(high performance liquid chromatography, HPLC)檢測各檢品柴胡皂苷a、c、d單位含量結果顯示，高氏柴胡田間栽培18個月後組培苗植株根部的SSa、SSc與SSd單位含量和(SSa+SSc+SSd)較高於種子苗，更分別為粗、細兩種柴胡市場品藥材SSa+SSc+SSd的2.02及1.57倍。
高氏柴胡無菌苗葉片培養於添加4 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D)之1/2MS培養基，誘導癒合組織形成之效率最高，而降低濃度至0.2 mg/L 2,4-D則較適於癒合組織之繼代培養。癒合組織因生長週期長短而致使柴胡皂苷累積有所變化，其SSa+SSc+SSd單位含量於培養後第8-10週達到最高。此外，癒合組織培養於不同MS鹽類濃度培養基添加0.2 mg/L 2,4-D、3%蔗糖、0.9%洋菜(Difco Bacto- agar)、500 mg/L蛋白胨(peptone)、500 mg/L水解酪蛋白(casein hydrolysate)與100 ml/L椰子汁(coconut water)之培養基，經4週培養後之1/2MS處理癒合組織可達最大鮮重與乾重，但是培養時間若超過6週，則癒合組織出現嚴重褐化現象；在上述四種MS鹽類處理試驗所得癒合組織經HPLC分析其柴胡皂苷含量結果顯示，全量MS鹽類處理所得癒合組織之SSa+SSc+SSd單位含量最高，因此後續繼代培養應以MS處理較適宜。
利用含有0.2 mg/L 2,4-D之1/2MS液態培養基可建立高氏柴胡細胞懸浮培養，適合之振搖速度與細胞接種密度分別為80 rpm與15% (6 mL細胞/40 mL培養液/250-mL三角瓶)。針對酵母抽出物(yeast extract, YE)茉莉酸(jasmonic acid, JA)與離層酸(abscisic acid, ABA)三種誘引劑測試其對於細胞增殖與柴胡皂苷含量的影響結果顯示，以酵母抽出物(YE)對細胞生長抑制作用較低，但離層酸(ABA)對柴胡皂苷累積有較大促進效果，若就細胞生長量與柴胡皂苷單位含量之相乘效果而言，則YE與ABA兩處理之效果相似；另一YE試驗結果顯示以1 mL 500 mg/L YE溶液處理培養3週後之懸浮培養細胞，經2週處理後其SSa+SSc+SSd達對照組之2.78倍，顯示施用酵母抽出物有利於懸浮細胞利用兩階段培養(two-stage culture process)策略大量生產柴胡皂苷。現階段懸浮細胞之柴胡皂苷單位含量雖較低，但可藉由調整培養條件與選擇高產細胞系增進生產效率，因培養系統不受環境影響且具有週年生產之優點，應可有助於利用細胞懸浮培養生產柴胡皂苷系統於製藥產業應用之實用性。The aims of this study were to establish a micropropagation and cell suspension culture system for seedling and saikosaponins production of Bupleurum kaoi Liu, Chao et Chuang, an endangered medicinal herb native to Taiwan. In vitro micropropagation of B. kaoi was established by culturing the axillary buds on a half-strength Murashige and Skoog (1/2MS) medium supplemented with 1 mg/L benzyladenine (BA) and 0.1 mg/L α-naphthaleneacetic acid (NAA). Maximum shoot multiplication rate was achieved in a medium containing 0.25 mg/L BA however many hyper- hydric shoots were also produced after 6 weeks of culture. Improvement of ventilation by using 3 layers of dispense paper (DP) as container closure was able to overcome the hyperhydric disorder and to increase the quality of in vitro shoots. In addition, liquid and liquid-solid culture alternating systems were proved not beneficial for in vitro culture of B. kaoi.
A protocol for producing high quality rooted plantlets with high efficient acclimatization rate was established in this study. Leaves of in vitro rooted plantlets were partial trimmed before transplanting into the sterile mixed substrate inside a transparent plastic box with cover for maintaining a higher relative humidity. Boxes were put into a growth chamber under 22℃ (14 h in light) / 18℃ (10 h in darkness) environmental condition. A survival rate of 92% after 4 weeks of acclimation was obtained from plantlets previously cultured on a 1/2MS basal medium containing 0.5 mg/L indole-3-butyric acid (IBA) and 0.1-0.2 mg/L NAA, in combination with changing aluminum foil (AF) with 3 layers of DP as container closure after 2 weeks culturing. Scanning electron microscope (SEM) showed that the leaf with the DP container closure treatment had functional stomata and the better epicuticular wax accumulation.
The bioactive ingredients of saikosaponin a, c and d (SSa, SSc, SSd) were analyzed using high performance liquid chromatography (HPLC). Root of 18-month-old field-grown plants derived in vitro shoots was higher total content of SSa, SSc and SSd (SSa+SSc+SSd) than that of plants derived from seeds. Moreover the SSa+SSc+SSd of plant derived from in vitro was 2.02- and 1.57-fold than that from the coarse and thinner crude drug from market (Radix Bupleuri- roots of B. chinensis), respectively. Our results suggested that the in vitro micropropagation of B. kaoi may be beneficial both on cultivation and conservation for this endangered medicinal plant in Taiwan.
Callus induction was obtained from leaf segments cultured on a 1/2MS basal medium supplemented with 4 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) in darkness however the concentration of 0.2 mg/L 2,4-D was more suitable for callus maintenance. The SSa+SSc+SSd contents from callus culture reached the highest amount after 8-10 weeks culturing however it is relative low while comparing with other derived samples. Medium contained various strength MS basal salts in combination with 3% sucrose, 0.9% Difco Bacto-agar, 500 mg/L casein hydrolysate, 500 mg/L peptone, and 100 mL/L coconut water were tested for callus proliferation and saikosaponins content analysis. Among 4 concentrations of MS salt strength, 1/2MS treatment had the highest fresh and dry weight after 4 weeks of culturing however browning callus was observed at week six. Although callus weight derived from 1/2MS treatment was higher than that of from MS treatment, callus derived from the full-strength MS medium had the highest SSa+SSc+SSd content.
Cell suspension culture of B. kaoi was established using minced callus cultured in a liquid 1/2MS basal medium with 15% cell density (6 mL cells, 40 mL liquid medium, 250-mL Elementary flask) and 80 rpm. Effects of elicitors of yeast extract (YE), abscisic acid (ABA) and jasmonic acid (JA) on biomasses and saikosaponin contents on B. kaoi suspension cells were investigated. No effect of fresh and dry weight by treating with yeast extract (YE), furthermore YE and abscisic acid (ABA) treatments both increased saikosaponins content after 1 week of treating. Overall the combinative efficiency of cell production and saikosaponins content, no significant difference was found between YE and ABA treatments. The results of another YE elicitor experiment, accumulation of SSa+SSc+SSd of YE treated cell was 2.78-fold higher than autoclaved water (control) after 2 weeks of treating. It is suggested that a two-stage culture system may be profitable for saikosaponins production using fast growth cells in combination with proper elicitor cultured for certain time.
It is thought that cell suspension culture in a long run has a great potential and advantage to produce secondary metabolites therefore more research efforts should develop for pharmaceutical utilization. Although the total amount of SSa, SSc, and SSd of the cultured cell was low in this stage comparing with other sources of tested samples in this study, it is thought that by modifying several important factors for suspension culture of B. kaoi cells could increase this system efficiency greatly.中文摘要 …………………………………………………………….….i 英文摘要 ……………………………………………………………….iii
表目次 …………………………………………………………………vii
圖目次 ….………………………………………………………………ix
附錄目次 ………………………………………………………………xi
第一章 前言 ……………………………………………………………1
第二章 利用高氏柴胡腋芽培養建立瓶苗大量繁殖系統之研究……..3
第三章 液態培養與容器封口對高氏柴胡增殖與玻璃質化苗之影響..18
第四章 鹽類濃度、蔗糖、生長素及培養容器封口對高氏柴胡組培苗發根與馴化之影響 ………………………………………………………..33
第五章 2,4-D與培養基組成分對高氏柴胡葉片癒合組織誘導與增殖之影響 ……………………………………………………………….……57
第六章 培養時間、振盪速度、細胞接種密度與誘引劑對高氏柴胡懸浮培養細胞生長之影響 …………………………………………………..80
第七章 柴胡柴胡市場品藥材、高氏柴胡原植物與組織培養品柴胡皂苷含量分析 …………….…………………………………………………..99
第八章 綜合討論 ……………………………………………………..118
引用文獻 ……………………………………………………………….124
附錄 …………………………………………………………………….14
