Influence of 2,4-dichlorophenoxyacetic Acid on Leaf Callus Induction, Proliferation and Saikosaponin Formation of in vitro Bupleurum kaoi Liu, Chao et Chuang

本研究利用臺灣原生藥用植物-高氏柴胡 (Bupleurum kaoi) 組培苗葉片片段，培養於添M 0.5-4 mg L-1 2,4- dichlorophenoxy- acetic acid (2,4-D)之 1/2 Murashige and Skoog (MS) 培養基均可誘導癒合組織形成，其中以 4 mg L-1 2,4-D 處理之誘導率達 75%最佳。選擇生長快速、淡黃色且質地綿 細之癒合組織進行試驗後發現，較低濃度 2,4-D (0.1-0.2 mg L-1 )較適於癒合組織之生長與增殖，提高 2,4-D 濃度 (0.4-0.8 mg L -1 2,4-D)則降低癒合組織之鮮重與乾重。葉 片癒合組織培養於含有 0.2 mg L-1 2,4-D 之1/2 MS 培養基，結果顯示其鮮重在培養後第 1 至第 10 週呈直線上升，而後鮮重增M 減 緩，此時已達最大乾重，且外觀部分呈現褐化現象，因此繼代時間應早於培養後 10週為宜。高壓液相層析 (high performance liquid chromatography HPLC)分析結果顯示，柴胡皂苷 a、c、d (saikosaponin a, c, d; SSa, SSc, SSd) 的累積與癒合組織生長週期有密切關係，培養中期(第 4 週與第 6 週 )隨著癒合組織大量增殖，三種柴胡皂苷總合含量(SSa+SSc+SSd)濃度較 低，培養 8 至 10週之細胞成熟時 SSa+SSc+SSd 累積達最大量。惟培養後期 (第 12 週)細胞老化時，其 SSa+SSc+SSd 又再度下降。高氏柴胡葉片癒合組織經培養於含有 0.2 mg L-1 2,4-D 之1/2 MS 培養基 8 週 後，其SSa+SSc+SSd 為-1 1.12 mg g dry wt，單位產量雖然較柴胡市場品、田間高氏柴胡植株地上部與根部之含量低，但由於癒合組織具有生長週期短且 週年生產不受環境影響之諸多優 點，未來更 可藉由調整各項因子以提高細胞生長與柴胡皂苷含量，達到產業化應用之目標。 Effect of 2,4-dichlorophenoxyacetic acid (2,4-D) on leaf callus induction, proliferation and formation of saikosaponin along with callus culturing of Bupleurum kaoi, a native medicinal herb of Taiwan, have been investigated. A seventy-five percent primary callus induction rate was obtained from the explants cultured on half-strength Murashige and Skoog (MS) medium containing 4 mg L-1 2,4-D for 8 weeks in darkness. Among the varied 2,4-D concentrations (0 to 0.8 mg L-1) tested for callus proliferation, the highest callus weight was observed on half-strength MS medium containing lower concentration (0.1 to 0.2 mg L-1) of 2,4-D and increasing of 2,4-D concentration did not further improve callus proliferation. In addition, time course of callus growth and saikosaponins content were also established. There was linearly increasing in callus fresh weight from week 1 to 10 before a slowly growth occurred from week 10 to 12. The highest biomass of callus was obtained at week 10, where callus browning started. Therefore, the period for a proper growth of callus on a proliferation medium should be no longer than 10-week-incubation. Saikosaponin a, c and d (SSa, SSc, SSd) contents of callus were analyzed at two-week intervals in a total of 12 weeks span using a high performance liquid chromatography (HPLC). The total amount of SSa, SSc, and SSd (SSa+SSc+SSd), 1.12 mg g-1 dry wt, was founded reaching the peak at interval from week 8 to week 10 and diminished hereafter. The contents of SSa, SSc, and SSd in various sources of plant materials including the aerial parts and roots of eighteen-month-old field-grown plants of B. kaoi and market crude drug (Radix Bupleuri - roots of B. Chinense), were also determined by HPLC. Although the total amount of SSa, SSc, and SSd of the cultured callus was lower than other sources of tested samples, it is thought that by modifying several important factors for callus culture of B. kaoi could make this system more efficient. Cell suspension culture in a long run has a great potential and advantage to produce secondary metabolites stably year round comparing with the field-growth plants for pharmaceutical utilization

(68(2): 137-147)Effect of Explant Size, Sucrose and Activated Charcoal on In Vitro Plantlet Proliferation and Growth in Amaryllis

本研究以孤挺花「台農1 號」(Hippeastrum hybridum Hort. ‘Tainung No. 1’) 組培苗為材料，探討培植體大小、蔗糖及活性炭濃度對組培苗生長之影響。以鱗莖直徑約3、6、9 mm 之4 分縱切鱗莖作為培植體進行培養，結果顯示不同大小培植體之組培苗增殖率並無顯著差異。每個1/4 鱗莖於培養8 wk 後，約可形成1.1−1.3株組培苗。此外，9 mm 處理之培植體，在8 wk 培養後可獲得具有較大鱗莖之組培苗；然而，在延長培養時間至12 wk 後，9 mm 與6 mm 處理間已無顯著差異。利用鱗莖直徑約3−5 mm 之組培苗作為培植體，進行1.5−9.0% 蔗糖與0−2 g L-1 活性炭濃度組合之複因子試驗，結果顯示6.0% 蔗糖配合1.0−2.0 g L-1 活性炭處理，對於增加組培苗鱗莖直徑與鮮重較為有利，分別達到8 mm 與1.73−1.75 g；但是當蔗糖濃度達到9% 時，則會抑制組培苗之生長。就組培苗根數而言，在含有6% 蔗糖搭配0.5−2.0 g L-1 活性炭條件下，可形成8.8−9.6條根，均較高於其他處理。就組培苗葉數而言，於0.5−2.0 g L-1 活性炭濃度下，蔗糖濃度在1.5−6.0% 之間並無顯著差異。綜合而言，蔗糖濃度自1.5% 提高至6.0%，可增加組培苗之鱗莖直徑、鮮重及根數，若同時添加適量活性炭，則具有協同增效蔗糖之效果。 Factors of explant size, concentration of sucrose and activated charcoal in the culture medium were investigated on in vitro growth of Hippeastrum hybridum Hort. ‘Tainung No. 1’ plantlet in this study. One-quarter vertical cut bulb explants derived from 3, 6 and 9 mm diameters of bulb were evaluated after 8 wk and 12 wk of culture. Proliferation rate of 1.1−1.3 plantlets per one-quarter bulb explant was obtained after 8 wk of culture, and no significant difference was found among all treatments. Furthermore, plantlets with larger bulb were obtained from explants derived from explants of 9 mm treatment. However, no significant difference on bulb diameter of 6 mm and 9 mm treatments was found after extending culture time to 12 wk of culture. A factorial experiment using 1.5−9.0% sucrose in combination with 0−2.0 g L-1 activated charcoal was conducted using explants derived from plantlets with 3−5 mm diameter of bulb. The better results were obtained from 6% sucrose in combination with 1.0−2.0 g L-1 activated charcoal on increase of bulb diameter and fresh weight which were 8.0 mm bulb diameter and 1.73−1.75 g fresh weight per plantlet, respectively. It was also found that growth of plantlets was inhibited in the medium containing 9.0% sucrose. In terms of the root number of plantlet, the best results of 8.8−9.6 roots per plantlet were obtained from 6.0% sucrose in combination with 0.5−2.0 g L-1 activated charcoal, and higher than the other treatments. For the leaf number of plantlet, no significant difference was found by using 1.5−6.0% sucrose in combination with 0.5−2.0 g L-1 activated charcoal. In summary, raising sucrose concentration from 1.5% to 6.0% would increase bulb diameter, fresh weight and root number of plantlet, and adding proper concentration of activated charcoal would has a synergistic effect with sucrose

(66(4):286-297) Effect of Surface Disinfection, Explant Type, and Plant Growth Regulators on Establishment of In Vitro Bulblet Proliferation of Amaryllis

本研究以孤挺花 (Hippeastrum hybridum Hort.)「紅獅」(‘Red Lion’)、「千禧之星」(‘Blossom Peacock’) 及「台農1 號-紅粉佳人」(‘Tainung No.1-Pink Lady’) 等3 個品種之鱗莖為材料，探討消毒方法、培植體部位及生長調節劑組合對消毒效率與小鱗莖增殖之影響。結果顯示，「紅獅」短縮莖培植體 (2 cm × 1 cm × 1 cm) 以1.2% NaOCl 處理15 min 之消毒效果較佳，且以靜置隔夜處理相對於直接處理的汙染率較低。比較「台農1 號」上半部與下半部短縮莖培植體 (6 mm × 6 mm × 2 mm) 之消毒結果顯示，上半部培植體不僅汙染率低且小鱗莖誘導率較高。利用「千禧之星」小鱗莖 (周徑約2.5 cm) 之1/2 縱切或1/4 縱切鱗莖作為培植體進行試驗，1/4 鱗莖處理之每個鱗莖可形成5.2 個小鱗莖，顯著高於1/2 鱗莖處理之2.0 個小鱗莖。「紅獅」1/4 鱗莖培養於1–4 mg L-1 苯甲基腺嘌呤 (benzylaminopurine; BA) 或0.1–0.4 mg L-1 賽本隆 (thidiazuron; TDZ) 配合0.2 mg L-1 奈乙酸 (α-naphthalene acetic acid; NAA) 之MS 培養基，結果顯示0.1 mg L-1 TDZ 處理可獲得較高之小鱗莖增殖率，經8 wk 培養後共獲得11.6 個小鱗莖。本研究結果可提供新品種孤挺花在種苗量化參考。 Field-grown bulbs of Amaryllis (Hippeastrum hybridum) ‘Red Lion’ (about 25 cm circumference), ‘Blossom Peacock’, and ‘Tainung No. 1-Pink Lady’ (about 12 cm circumference) were used as culture materials. Effects of surface disinfection, explant type, and plant growth regulators combinations on establishment in vitro bulblet proliferation were investigated. Stem explants (2 cm × 1 cm × 1 cm) of ‘Red Lion’ left on a filter paper overnight before using 1.2% sodium hypochlorite solution for 15 min resulted in the least contamination. Stem explants (6 mm × 6 mm × 2 mm) cut from the upper or lower part of ‘Tainung No. 1-Pink Lady’ were compared. Although explants derived from different positions of the stem had the same proliferation rates of 1.2 bulblets per explant, the upper stem explants had a significant lower contamination rate of 3% than that of 37% from the lower part explants. Comparison of proliferation efficiencies was conducted using half- and quarter-bulb explants of ‘Blossom Peacock’ derived from an in vitro bulb with 2.5 cm circumference in size, and no significant difference was found on bulblet formation between both treatments. However, a higher proliferation rate of 5.2 bulblets per original bulb was calculated by using quarter-bulb explants than that of 2.0 bulblets from the half-bulb explants. The highest bulblet proliferation rate with 11.6 bulblets from per 2.5 cm circumference in ‘Red Lion’ in vitro bulb was obtained by culturing quarter-bulb explants on the MS medium supplemented with 0.1 mg L-1 thidiazuron (TDZ) and 0.2 mg L-1 α-naphthalene acetic acid (NAA) for 8 wk. Results of this study could provide useful information on bulblet proliferation for new varieties of Amaryllis

Establishment of High-Quality Plantlet Production System for Chinese Cymbidium

小花蕙蘭 (國蘭) 每年外銷金額可達2~3 億元新臺幣，是金額僅次於蝴蝶蘭之外銷蘭花，商品以平價蘭為主，主要銷往韓國、日本、新加坡及香港等地。近年來國蘭栽培面積雖快速擴充，但達外銷標準之產品率偏低，以致於出口貨源不足。探究其原因可為下列幾點：1. 慣行栽培以分芽繁殖為主，繁殖倍率低，大量栽種時蘭苗供應不敷使用；2. 蕙蘭嵌紋病毒 (Cymbidium mosaic virus，CymMV)及齒舌蘭輪斑病毒 (Odontoglossum ringspot virus，ORSV) 為小花蕙蘭主要之病毒，以及具寄主專一性-尖鐮孢菌 (Fusarium oxysporum)所引起之假球莖腐敗病，此三種病害傳播途徑均為帶菌之種苗，而分芽繁殖方式導致病毒病及假球莖腐敗病迅速擴散，以致栽培管理不易。3. 目前多數蘭農採用雙層遮蔭網室，此種設施由於無法防雨，因此導致病害難以控制。上述小花蕙蘭繁殖方法與栽培的缺失必須加以克服，才能提供生育整齊、生理一致與栽培管理便利達到外銷需求標準之商品，因此建立小花蕙蘭健康種苗供應系統與開發病害檢測方法以利種苗健康管理實屬必要。本團隊針對四季類 (Cymbidium ensifolium) 與報歲類(Cymbidium sinense) 小花蕙蘭種苗進行組織培養大量繁殖、出瓶馴化與肥培管理技術目前已有初步成果；針對兩種主要病毒病害亦已研發血清檢測與生物晶片兩種偵測方法，以及利用RT-PCR 方式檢測尖鐮孢菌之感染。試驗結果顯示，利用健康種苗進行栽培能大幅度降低種苗帶病之機率，主要病害檢測方法之建立則有助於健康栽培管理。本團隊後續擬以(1)組織培養種苗生產及其肥培管理；(2)設施栽培管理與病害控制；(3)建構國蘭健康種苗供應中心等三項主題為主要研究目標。利用整合組培苗繁殖栽培技術、病害檢測技術與管理策略，並配合育種家、蘭農與貿易商的觀點選擇適當商業栽培品種，建立高效率小花蕙蘭生產模式，大量生產符合外銷需求之高品質小花蕙蘭成株商品。 Chinese Cymbidium has reached annual production of 2-3 hundred million NTD ranked only after the Phalanopsis flower industry. The main exporting countries are South Korea, Japan, Singapore and Hong Kong with low-price products. Although Chinese Cymbidium productivity has been fast expanded in recent years, selling products were insufficient due to lower acceptable rates. Three reasons were concluded for the upper mentioned problem, 1. Plantlet propagation rate through shoot dividing method was quite low. 2. Two major virus, Cymbidium mosaic virus (CymMV), Odontoglossum ringspot virus (ORSV), and one fungus pathogen-pseudobulb decayed disease (Fusarium oxysporum), are found in Chinese Cymbidium which could spread with reproduced plantlets and leading to serious cultivation problems. 3. Ordinarily, Chinese Cymbidium is cultivated under the double-layers sunshade net system which may cause active fungus disease because of lacking of rainfall prevention. In order to overcome these drawbacks and to obtain healthy cloned plants for industry cultivation and for exporting high quality products, it is very essential to establish the in vitro masspropagation system to provide disease-free plantlets and to have disease detection techniques for Chinese Cymbidium industry. So for, our team has developed in vitro micropropagation and ex vitro acclimation system as well as fertilization applications on Cymbidium ensifolium and Cymbidium sinense plantlet production. In addition to ELISA and biochip detection methods for CymMV and ORSV, a RT-PCR method for detecting F. oxysporum were also established. Our results showed that using healthy plantlets derived from micropropagation could reduce disease spreading and methods for disease detection are very beneficial for Chinese Cymbidium industry. Subjects to follow up are: 1) The techniques of micropropagation and plantlet cultivation system, 2) Improvement of cultivation and disease management, 3) The healthy plantlet production and supplement system. The optimal goal for this study is to combine with the unique vision of breeders, farmers and traders to establish a new and efficient operation system for production of healthy plantlets. The entire system will not only supply healthy plantlets for farmers but also provide cultivation system to produce high quality Chinese Cymbidium plants for exporting purpose

(67(1):44-53)Effects of Two-Stage Culture and Paclobutrazol on In Vitro Bulblet Growth and Rooting of Hippeastrum hybridum

本研究利用孤挺花 (Hippeastrum hybridum Hort.)「台農1 號-紅粉佳人」(‘Tainung No.1-Pink Lady’)、「紅獅」(‘Red Lion’) 及「千禧之星」(‘Blossom Peacock’) 組培苗為材料，探討兩階段培養及巴克素對鱗莖生長與發根之影響。「台農1 號」鱗莖 (直徑約5 mm) 於含有0.1 mg L-1 苯甲基腺嘌呤 (benzylaminopurine; BA) 與0.1 mg L-1 奈乙酸 (α-naphthalene acetic acid; NAA) 之液態或固態MS培養基中培養8 wk (第一階段)，再接種至相同組成之固態培養基8 wk 後 (第二階段)。結果顯示，液-固態兩階段培養具有顯著增加組培苗鮮重、葉數、葉長及根數之效果。利用「紅獅」與「千禧之星」鱗莖 (直徑約10 mm) 進行巴克素 (Paclobutrazol; PBZ) 液態培養試驗，結果顯示，對照組鱗莖培養至8 wk 時，已有部分組培苗出現嚴重褐化的現象，建議繼代培養間隔時間不宜超過8 wk。「紅獅」組培鱗莖在50–75 mg L-1 PBZ 培養條件下，PBZ 處理之根數顯著較多於對照組，但PBZ 處理間並無顯著差異。然而，「千禧之星」鱗莖在25–75 mg L-1 PBZ 處理時根系生長受到抑制，且濃度越高時根數就越少，而75 mg L-1 PBZ 處理則較對照組具有顯著較短與較寬的葉片；將PBZ 濃度降低至5 mg L-1，則有增大鱗莖與促進發根之效果。 An improved method for in vitro bulblet growth and rooting of Hippeastrum hybridum was established in this study. In vitro bulblets of Hippeastrum hybridum ‘Tainung No. 1’, ‘Red Lion’, and ‘Blossom Peacock’ were used as materials to conduct the liquid-solid or solid-solid two-stage culture, and additional paclobutrazol was added into culture medium to enhance growth and rooting of in vitro Amaryllis bulblets. In vitro bulbs (5 mm diameter) of ‘Tainung No. 1’ were cultured in liquid or solid MS medium containing 1 mg L-1 BA and 0.1 mg L-1 NAA for 8 wk of culture before transferring to a solid MS medium with the same components for another 8 wk of culture. The results showed that liquid-solid culture was superior to solid-solid culture on fresh weight of bulblets, leaf number, leaf length, and root number. In vitro bulblets of ‘Red Lion’ and ‘Blossom Peacock’ showed severe browning or death after 8 wk of culture on the control medium. Therefore, it is suggested that the subculture interval should not exceed 8 wks. The root number of ‘Red Lion’ bulblets by 50–75 mg L-1 PBZ treatment was found significantly higher than that of control. No significant difference among PBZ treatments was found. However, root numbers of ‘Blossom Peacock’ were reduced treated with 25–75 mg L-1 PBZ concentrations. Leaf with shorter and wider shape was observed in 75 mg L-1 PBZ treatment. However, a lower concentration of 5 mg L-1 PBZ was found beneficially to bulb diameter, root number and root length

Masspropagation and Saikosaponins Analysis from In Vitro Cultures of Bupleurum kaoi Liu, Chao et Chuang

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

Influence of ventilation closures on plant growth parameters, acclimation and anatomy of leaf surface in Scrophularia yoshimurae Yamazaki - a medicinal plant native to Taiwan

Different ventilation closures, including aluminum foil (AF) and a varying number of dispense papers (DP) had different effects on leaf growth parameters, in vitro rooting, survival rate and the anatomical features of the leaf surface of in vitro and ex vitro acclimated plants of Scrophularia yoshimurae—an important medicinal plant. The lowest plant growth parameters and ex vitro acclimation rate (<7.0%) were obtained using AF as ventilation closure. A scanning electron microscopy (SEM) study of leaf surfaces of plants derived from different ventilation closure treatments showed that parameters—including density and size of epidermal cell and stomata, size of guard cells, and stomata aperture-differed significantly among various treatments, and this in turn affected plant survival rate. Leaves derived from AF treatment had higher epidermal cell (15094 cells/mm2) and stomata (38/mm2) densities than DP treatments. Well-ventilated container closures, such as with DP, improved the morphological characteristics of leaves and in turn enhanced the survival rate during ex vitro acclimation (maximum rate being 66.7%). The present study not only provides an improved micropropagation method of S. yoshimurae but also gives scientific reasons for the different acclimation rates obtained with various container closures

(65(4):384-394) Influence of Explant, Plant Growth Regulator and Illumination on Adventitious Shoot Induction of In Vitro Cultured Salvia miltiorrhiza

本研究利用丹參 (Salvia miltiorrhiza) 組織培養苗之葉柄和葉片培植體進行不定芽的誘導試驗。葉柄培植體於含有1 mg L-1 芐腺嘌呤 (N6-benzyladenine; BA) 與0.5 mg L-1 奈乙酸 (α-naphthaleneacetic acid; NAA) 之MS (Murashige & Skoog 1962) 培養基中培養6 wk 後不定芽形成率達100%，平均每個培植體可形成3.7 個不定芽。將葉柄和葉片培植體預培養於含有1–2 mg L-1 2,4-二氯苯氧乙酸 (2,4-dichlorophenoxyacetic acid; 2,4-D) 之MS培養基2 或3 wk 後，再分別繼代培養於不含植物生長調節劑之MS 培養基中培養4 或3 wk，可誘導癒合組織和不定根形成。葉片癒合組織於含有0.25 mg L-1 BA 與0.2 mg L-1 2,4-D 之MS 培養基於黑暗中培養8 wk，顯示除了癒合組織增殖外，亦可形成少數不定芽。將癒合組織繼代培養於含有相同2,4-D 濃度但不同BA 濃度之培養基中，於光照或黑暗環境培養8 wk 後，其中以2 mg L-1 BA 配合光照處理可產生最多不定芽，每接種0.2 g 癒合組織平均可形成14.1 個不定芽。本研究利用丹參組織培養苗建立直接與間接不定芽再生大量繁殖體系，除可供生產丹參種苗所需外，亦可應用於誘變與轉基因之研究。 Petiole and leaf explants derived from in vitro Salvia miltiorrhiza were used for shoot regeneration in this study. The highest adventitious shoot formation rate of 100% with 3.7 shoots/explant in average was obtained from petiole segments cultured on Murashige and Skoog’s (MS) medium containing 1 mg L-1 N6-benzyladenine (BA) and 0.5 mg L-1 α-naphthaleneacetic acid (NAA) for 6 wk of culture. Callus and adventitious roots were induced from petiole and leaf segments pre-cultured on the MS medium supplemented with 1–2 mg L-1 2,4-dichlorophenoxyacetic acid (2,4-D) for 2–3 wk followed by transferring on a hormone-free MS basal medium for a total 6 wk of culture. Calli were proliferated on the MS basal medium containing 0.25 mg L-1 BA and 0.2 mg L-1 2,4-D under darkness for 8 wk of culture along with few adventitious shoots were found. Proliferated calli were subcultured to the medium containing with same concentration of 2,4-D in combination with various BA concentration under light and dark condition for shoot regeneration. The highest induction number of adventitious shoots was 14.1 shoots 0.2 g-1 callus from the medium containing 2 mg L-1 BA under light condition. An efficient micropropagation system of Salvia miltiorrhiza by direct and indirect adventitious shoot regeneration systems were established in this study which would not only supply for plantlet production but also apply on mutation and genetic transformation studies

Explant Types Derived from Flower Stalk Culture and 6-Benzyladenine Concentrations Affect Shoot Differentiation of Phalaenopsis Hybrid in Subculture

Phalaenopsis (Taisuco Snow × Wataboushi) ‘T343’在可見第一朵花蕾分化時期之花梗節為培植體，進行初代花梗芽誘導，再利用初代花梗節誘導長出之營養芽的短縮莖、葉片、根，以及切除營養芽後保留約2-3 mm 莖基組織之原花梗節，作為繼代培養芽體誘導之材料，接種於含不同濃度(0.0、1.25、2.5、5.0、10.0 及 15.0mg•L-1)苯基腺嘌呤(6benzyladenine, BA)與0.01 mg•L-1 萘乙酸(α-naphthalene aceticacid, NAA)之半量Murashige & Skoog 基本鹽類培養基中8 週。試驗結果顯示，保留少量莖基組織之原花梗節培植體，其繼代培植體分化率在71%-100%之間，隨著BA濃度的提升，平均最高可誘導4.8 個芽體；BA 濃度除對增殖芽數有顯著影響外，對芽體分化之型態亦有顯著影響，誘導產生之芽體型態可分為單一營芽、叢生營養芽、芽體與擬原球體(protocorm-like bodies, PLBs)共存，以及芽體與綠色團塊類癒合組織共存等四種。以營養芽去除葉片及根部後之短縮莖作為繼代培養之培植體，於0.0-1.25 mg•L-1 BA 中，有較高的根誘導率，為50%-70%，隨著BA 濃度提升至5.0-10.0mg•L-1，分化型態以芽體為主，平均誘導之芽體數可達3.3-3.5 個。以營養芽上長出之葉與根作為培植體，在0.0-10.0 mg•L-1 BA 的濃度下，葉片可誘導出PLBs，根可誘導出癒傷組織(callus)之分化型態，誘導率於10.0 mg•L-1 BA 之培養基中達最高，其PLBs 與callus 誘導率分別為20%與8%。 Flower-stalk nodes of Phalaenopsis (Taisuco Snow × Wataboushi) ‘T343’, taken at the developmental stage when the first flower bud was visible, were used as explants in tissue culture. Four types of explants were subsequently taken from the induced buds of the initial flower stalk culture. They were leaves, roots,leafless-basal-stems of the new buds, and the original flower-stalk nodes containing 2-3 mm basal stems after removing the new buds. Explants were then cultured on a 1/2MS basal salt medium containing 0.01 mg•L-1 NAA and various concentrations of BA (0.0, 1.25, 2.5, 5.0, 10.0 and 15.0 mg•L-1) for 8 weeks. Those explants of original flower nodes with 2-3 mm basal stems had 70% to 100% survival rates after the subculture. The number of shoots produced from each explant increased with BA concentration increase, and 4.8 shoots per explant was the highest. Besides the effect on shoot number, BA concentration also had a significant influence on the type of shoot differentiation. There were four differentiation types, namely: single vegetative shoot, multiple vegetative shoots, shoot plus PLBs, and shoot plus callus-like tissue. For the explants of leafless-basal-stems of the new buds, the highest root induction rate was 50% to 70%, which was at 0.0 or 1.25 mg•L-1 BA level. At higher concentrations of BA, more adventitious shoots were produced. They had 3.5 shoots per explant at 10.0 mg•L-1 BA. When leaves and roots were used as explants, PLBs and callus were developed respectively. The former had 20% and the latter had 8% of induction rate at 10.0 mg•L-1 BA

Dehydroepiandrosterone Ameliorates Abnormal Mitochondrial Dynamics and Mitophagy of Cumulus Cells in Poor Ovarian Responders

Mitochondrial dysfunction is related to reproductive decline in humans, with consequences for in vitro fertilization (IVF). We assessed whether dehydroepiandrosterone (DHEA) could regulate mitochondrial homeostasis and mitophagy of cumulus cells (CCs) in poor ovarian responders (PORs). A total of 66 women who underwent IVF treatment at the Reproductive Medicine Center of Kaohsiung Veterans General Hospital were included in this study. Twenty-eight normal ovarian responders (NOR) and 38 PORs were enrolled. PORs were assigned to receive DHEA supplementation (n = 19) or not (n = 19) before IVF cycles. DHEA prevents mitochondrial dysfunction by decreasing the activation of DNM1L and MFF, and increasing MFN1 expression. Downregulation of PINK1 and PRKN occurred after DHEA treatment, along with increased lysosome formation. DHEA not only promoted mitochondrial mass but also improved mitochondrial homeostasis and dynamics in the CCs of POR. We also observed effects of alterations in mRNAs known to regulate mitochondrial dynamics and mitophagy in the CCs of POR. DHEA may prevent mitochondrial dysfunction through regulating mitochondrial homeostasis and mitophagy