本研究建立一個玉山金絲桃(Hypericum nagasawai Hayta)組織培養及不定根液態培養的方法。節段培養於Murashige and Skoog (MS)基礎鹽類並添加0.5 mg/L 6-benzylaminopurine(BA)和1.0 mg/L naphthaleneacetic acid (NAA)之固態培養基，兩個月後誘導出側芽與癒傷組織。芽體增殖測試，再生芽體於MS加0.1、0.5、1.0 mg/L不同濃度的BA、thidiazuron (TDZ)、6-γ,γ-dimethylally laminopurine (2iP)、6-furfurylaminopurine(KN)共12種培養基組合，培養一個月後，在含TDZ培養基誘導出叢生芽體但玻璃質化，含0.1 mg/L BA培養之芽體，平均可增殖7個正常芽體。發根測試，再生枝條培養於MS 添加 0.1 mg/L NAA及0.22 % gelrite一個月後，生根率達77.8 %。再生植株直接出瓶存活率為0 %，以含1/5 MS液態培養基之介質培養，可增加存活率。不定根之最佳接種量為0.05 g F.W./10 mL，培養於MS 添加0.5 mg /L NAA液態培養基，一個月後增殖倍率達11.52倍。分析不定根甲醇萃取物總酚含量介於29.85 - 57.39 eq. gallic acid mg/g D.W.，高於野生植株根部含量(29.54 ± 0.25 eq. gallic acid mg/g DW)。金絲桃素含量分析，使用MS加0.5 mg /L TDZ培養一個月芽體可得5.76 mg/g D.W.為田間植株3.7倍；不定根中使用液態MS加0.1 mg/L NAA培養一個月可獲得最高含量(2.42 mg/g D.W.)，與田間植株根部(2.42 mg/g D.W.)含量相當。

In this study, an method for plant regeneration system and adventitious root liquid culture of Hypericum nagasawai Hayta was achieved. The nodal explants were cultured on Murashige and Skoog (MS) basal salts soild medium supplemented with 0.5 mg/L 6-benzyl aminopurine (BA) +1.0 mg/L naphthaleneacetic acid (NAA).Lateral bud emerged and callus formed from the base of nodal explant after two months. In shoot multiplication text, regeneration shoots were cultured on MS soild medium supplemented with the different 0.1、0.5 or 1.0 mg/L concentrations of BA、thidiazuron (TDZ)、6-γ,γ-dimethylallylaminopurine (2iP)、6-furfurylaminopurine(KN) for one month . The vitrificated multiple shoots produced when bud cultured on MS medium with TDZ. The average of normal shoots per bud explant was 7 when cultured on MS medium supplemented with 0.1 mg/L BA . For the rooting test, roots were induced when regenerated shoots cultured in MS supplemented with 0.1 mg/L NAA and 0.22 % gelrite after one month and the highest rooting frequency was 77.8 %. Plantlets expressed higher survival rate when acclimatized in 1/5 MS basal salts with medium. The optimal inoculum of adventitious roots was 0.05 g F.W./10 mL. The proliferation rate of adventitious roots was 11.52 times when adventitious roots cultured in MS supplemented with 0.5 mg/L NAA for one month. The total phenol content of methanol extract in adventitious roots was between 29.85 to 57.39 eq. gallic acid mg/g D.W. which higher than roots of wild plant (29.54 ± 0.25 eq. gallic acid mg/g DW). The extract of shoots from MS with 0.5 mg/L TDZ medium showed 5.76 mg/g DW of hypericin which was 3.7–fold higher than field plants. The highest hypericin content of adventitious roots was 2.42 mg/g D.W. when cultured in liquid MS medium with 0.1 mg/L NAA for one month which was equal to field plants of root.

中文摘要I
Abstract III
目錄V
表目錄X
圖目錄XI
縮寫字XIII
一、前人研究1
1.1玉山金絲桃植物特性1
1.2金絲桃屬植物之利用1
1.2.1台灣產金絲桃屬植物種類2
1.2.2金絲桃屬植物藥用成分3
1.2.3中藥製劑6
1.2.4抗菌7
1.3金絲桃植物組織培養研究7
1.3.1直接器官發生9
1.3.2癒傷組織誘導與間接器官發生9
1.3.3二次代謝產物生產10
1.4研究目的12
二、材料與方法14
2.1 實驗架構14
2.2植物材料來源14
2.3 培養基製備15
2.4 培養環境15
2.5無菌培養建立15
2.6芽體培養與增殖17
2.6.1 cytokinins對芽體增殖之影響17
2.6.2 BA與TDZ對不同培植體器官發生之影響17
2.7生根測試18
2.7.1 auxins對節段或莖頂培植體生根之影響18
2.7.2 NAA與不同凝結劑對枝條培植體生根之影響18
2.8再生植株馴化20
2.9不定根培養20
2.9.1不定根誘導與增殖20
2.9.2不定根生長曲線20
2.9.3不定根接種量測試21
2.9.4 auxins對不定根增殖之影響21
2.10二次代謝產物分析22
2.10.1甲醇萃取液製備22
2.10.2總酚含量分析22
2.10.3總黃酮含量分析23
2.10.4 HPLC分析指標性成分23
2.11統計分析所有實驗皆使用25
三、結果26
3.1 無菌培養建立26
3.3芽體培養與增殖28
3.3.1 cytokinins對芽體增殖之影響28
3.3.2 BA與TDZ對不同培植體芽體增殖之影響31
3.4生根測試35
3.4.1 auxins對節段或莖頂培植體生根之影響35
3.4.2 NAA與不同凝結劑對枝條生根之影響40
3.5再生植株馴化43
3.6不定根培養44
3.6.1不定根誘導與增44
3.6.2不定根生長曲線44
3.6.3不定根接種量測試45
3.6.4 auxins對不定根增殖培養之影響46
3.7二次代謝產物分析48
3.7.1總酚及總黃酮含量分析48
3.7.2 HPLC指標性產物分析52
四、討論56
4.1無菌培養建立56
4.2芽體培養與增殖57
4.3生根測試58
4.4再生植株馴化59
4.5不定根培養60
4.6二次代謝產物分析61
五、結論63
六、參考文獻65

王宇紅、趙洪慶、韓遠山、杜青、楊冬梅. 2019. 一種複方物的提取方法及其在治療焦慮性CN 109908216A
王昭君. 2000. 五種台灣金絲桃屬植物之pseudohypericin和hypericin含量分析與元寶草粗萃物之體內外抗氧化性以及對acetaminophen所誘發之大鼠急性肝、腎毒性之影響. 國立中興大學食品科學系碩士論文
王慧. 2010. 黃酮類化合物生物活性的研究進展. 食品與藥品 12(9):347-350
冉懋雄. 2004. 中藥組織培養實用技術p442. 科學技術文獻出版社. 北京.
任繼文、雷穎. 2010. 金絲桃組織培養再生體系建立. 西北林學院學報25(3):90-92
呂瓊瓊、劉巨源、楊俊、潘艷娟、路璐. 2012. 中藥貫葉金絲桃治療抑鬱症的進展. 國際神經精神科學雜誌1:22-26
李哖. 1984. 台灣一葉蘭之生長習性與生產. 台灣省農業試驗所特刊14:53-64
肖偉、商晶、常秀娟、王偉、李璐、楊一帆、楊彪、吳雲、王永香、王振中. 2018. 一種中藥組合物及其在製備治療心律失常藥物中的應用CN 109248201A
邢震、鄭維列. 2000. 多蕊金絲桃的組織培養. 江蘇農業研究21(1)45-47
尚火力、辛秀娟. 2019. 一種用於治療胰腺癌的組合藥物及應用CN 110327318A
林雨蓁. 2019. 培養因子對白花檵木(Loropetalum chinense(R.Br.) Oliv.)癒傷組織誘導、植株在生和二次代謝產物累積的影響. 大同大學碩士論文
邵文麗、姚佳、張雪青、邱士真、趙鐵軍、徐玲玲. 2016. 金絲桃素界導的光動力學療法在腫瘤治療中的應用研究. Journal of Anhui Agri. Sci.44(31)122-124,134
柯俊良. 2013. 金絲桃素抑制Ava．5細胞株中Ｃ型肝炎病毒複製之應用TW140065B
孫霞. 2019. 用於治療潰瘍性結腸炎的金絲海棠提取物的提取方法及應用CN106588827B
張定霖、洪進雄、吳昭祥. 2007. 香藥草植物圖鑑 p.62. 行政院農業委員會種苗改良繁殖場.台中
張聖顯、陳怡蓉. 2013. 金絲桃屬植物抗氧化能力比較分析.花蓮區農業改良場研究彙報31:21-32
張聖顯. 2008. 台灣金絲桃之栽培與利用. 花蓮區農業專訊66:15-17
陳怡璇. 2015. 數種草莓(Fragaria ╳ ananassa)栽培種為體繁殖及二次代謝物含量之研究. 大同大學碩士論文
陳傅杰. 2004. 台灣產金絲桃屬植物之分類研究.國立臺灣師範大學生命科學系碩士論文
陳銘田、陳秋明、郭長生. 1989. 短柄金絲桃之成分研究.技術學刊4(4):383-388
黃何興. 2019. 台灣金絲桃萃取物之用途 TW1646968B
劉燕、龍成昌、巫華美、周艷、周洪英. 2011. 貴州金絲桃組培快速繁育技術研究.種子30(10):40-42
劉興寬、郁建平、古練權. 2005. 貴州金絲桃中黃酮類成分研究. 中藥材28(10):34-35
冀祖恩、周寒利、楊艷、羅恒磊. 2018. 一種抗流感病毒的中藥組合物及其製備方法CN 108379329A
鮑力恆. 2009. 臨床常用科學中藥與西藥交互作用評估之Ⅱ. 中醫藥年報27(3):403-432
應紹舜. 1980. 臺灣的高山植物p.56. 台灣渡假出版社. 台北
繆劍華、喬柱、肖冬、韋坤華、朱艷霞、張占江、秦雙雙、肖培根、黃璐琦. 2018. 藥用植物引種馴化原理研究.廣西植物. 38(8):973-983
謝長奇、劉琳琳、陳珠亮、鄭登貴、歐柏榮、李維儒、劉羿欣、鄧至涵. 2016. 元寶草發酵產物之用途及其製備方法 TW1517856B
龔宇、周蕙禎、江泓、陳胡蘭. 2018. 金絲桃素藥理作用以及製備方法研究概況. 27(15)37-40
Azeez H.A. and K.M. Ibrahim. 2014. Hypericum Triquetrifolium callus cultures a potential source of phenolics and flavonoids. JZS -A 16:381-388
Bacila I., A. Coste, A. Halmagyi, and C. Deliu. 2010. Micropropagation of Hypericum maculatum Cranz an important medicinal plant. Rom. Biotech. Lett. 15(1):86-91
Baruah A., D. Sarma, J. Saud and R.S. Singh. 2001. In vitro regeneration of Hypericum patulum Thumb.-A medicinal plant. Indian J. Exp. Biol. 39:947-949

Chang C., M. Yang, H. Wen and J. Chern. 2002. Estimation of total flavonoid content in propolis by two complementary colorimetric methods. J. Food Drug. Analy. 10:178-182
Chen M.T., C.H. Wan, C.M. Chen and C.S. Kuoh.1988. Flavonoids from Hypericum nagasawai Hayta. J. Chin. Chem. Soc. 35:167-1 72.
Coste A., A. Halmagyi, A.L. Butiuc-Keul, C. Deliu, G. Coldea and B. Hurdu. 2012. In vitro propagation and cryopreservation of Romanian endemic and rare Hypericum species. Plant Cell Tiss. Organ. Cult. 110:213–226
Cui X.H., D. Chakrabarty, E.J. Lee and K.Y. Paek .2010. Production of adventitious roots and secondary metabolite by Hypericum perforatum L. in a bioreactor. Biosour. Technol. 101:4708–4716
Dall’Agnol R., A. Ferraz, A. P. Bernardi, D. Albring, C. Nör, L. Sarmento, L. Lamb, M. Hass, G. von Poser and E.E.S. Schapoval. 2003. Antimicrobial activity of some Hypericum species. Phytomedicine 10: 511–516
Duncan D.B.. 1955. Multiple range and multiple F-test. Biometrics 11:1-42.
El-Sawy A., M.A. Matter and N. D. Girgis. 2015. Effect of gelling agent on in vitro tuberization of potato. J. Agric. & Environ. Sci 15 (10): 1934-1939
Fenner R., M. Sortino, S.M. Kuze Rates, R. Dall’Agnol, A. Ferraz, A.P. Bernardi, D. Albring, C. Nör, G. von Poser, E. Schapoval and S. Zacchino. 2005. Antifungal activity of some Brazilian Hypericum species. Phytomedicine 12: 236–240
Gaid M., P. Hass,T. Beuerle, S.Scholl and L. Beerhues. 2016. Hyperforin production in Hypericum perforatum root cultures. J. Biotechnol. 222:47-55
Hopkins W. G. and N.P.A. Huner. 2008. Introduction to Plant Physiology 4ed. p346. John Wiley&Sons, Inc. Hoboken
Hostanska K., J. Reichling, S. Bommer, M. Weber and R.Saller. 2003. Hyperforin a constituent of St John's wort (Hypericum perforatum L.) extract induces apoptosis by triggering activation of caspases and with hypericin synergistically exerts cytotoxicity towards human malignant cell lines. Eur. J. Pharm. Biopharm. 56: 121-32
Huang L.F., Z.H. Wang, S.L.Chen. 2014.Hypericin :chemical synthesis and biosynthesis. Chin. J. Nat. Medicines 12(2): 0081−0088 DOI: 10.3724/SP.J.1009.2014.00081
Hudson J.B., I. Lopez-BazzocchF and G.H.N. Towers. 1991. Antiviral activities of hypericin. Antiviral Res. 15:101-112.
Kartnig T., I. Gobel and B.Heydel. 1996. Production of hypericin, pseudohypericin and flavonoids in cell cultures of various Hypericum species and their chemotypes. Planta Med. 62: 51–53
Khan S. A., P. Verma, A. Arbat, S. Gaikwad and V. A. Parasharami. 2018. Development of enhanced hypericin yielding transgenic plants and somaclones: High throughput direct organogenesis from leaf and callus explants of Hypericum perforatum. Ind. Crops Prod. 111:544–554
Kirakosyan A., H. Hayashi, K.Inoue, A. Charchoglyan and H. Vardapetyan. 2000. Stimulation of the production of hypericins by mannan in Hypericum perforatum shoot cultures. Phytochemisty 53:345-348
Kitanov G.M.. 2001. Hypericin and pseudohypericin in some Hypericum species. Biochem. Syst. Ecol. 29: 171-178
Mašković P.Z., J.D. Mladenović, M.S. Cvijović, G. Aćamović- Đoković, S.R. Solujić and M.M. Radojković. 2011. Phenolic content, antioxidant and antifungal activities of acetonic,ethanolic and petroleum ether extracts of Hypericum perforatum L. Hem. Ind. 65(2):159–164
McCoy A. and N.D. Camper. 2002. Development of a micropropagation protocol for St.John’s Wort(Hyperıcum perforatum L.). HortSci. 37(6):978-980
McDonald S., P.D. Prenzler, M. Autolovich and K. Robards. 2001. Phenolic content and antioxidant activity of olive extacts. Food Chem. 73:73-84
Moura M.. 1998. Conservation of Hypericum foliosum Alton, anendemic azorean species, by micropropagation. In Vitro Cell. Dev. BioL Plant 34:244-248
Murashige T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15(3): 473–497.
Murthy H.N., V.S.Dandin and K. Paek. 2016. Tools for biotechnological production of useful phytochemicals from adventitious root cultures.Phytochem Rev. 15:129-145
Nahrstedt A. and V. Butterweck. 1997. Biologically active and other chemical constituents of the herb of Hypericum perforatum L. Pharmacopsychiatry 30:129-134
Ng Q.X.,N.Venkatanarayanan and C.Y.Ho. 2017. Clinical use of Hypericum perforatum (St John's wort) in depression: A meta-analysis. J. Affect Disord. 210:211-221
Oluk E. A. and S. Orhan. 2009. Thidiazuron induced micropropagation of Hypericum triquetrifolium Turra. Afri. J. Biotechnol. 8(15): pp. 3506-3510
Oluk E. A., S. Orhan, O. Karaka, A. Çakır and A. Gönüz. 2010. High efficiency indirect shoot regeneration and hypericin content in embryogenic callus of Hyperıcum trıquetrıfolıum Turra. Afri. J. Biotechnol. 9(15):2229-2233
Ozel C. A., K. M.Khawarb and O.Arslana. 2008. A comparison of the gelling of isubgol, agar and gelrite on in vitro shoot regeneration and rooting of variety Samsun of tobacco (Nicotiana tabacum L.)Sci. Hortic. 117:174-181
Pasqua G., P. Avato, B. Monacelli, A.R. Santamaria and M.P. Argentieri. 2003. Metabolites in cell suspension cultures, calli, and in vitro regenerated organs of Hypericum perforatum cv. Topas. Plant Sci. 165:977-982
Santarem E.R. and L.V. Astarita. 2003. Multiple shoot formation in Hypericum perforatum L. and hypericin production. Braz. J. Plant Physiol. 15(1):43-47
Savio L.E.B., L.V. Astarita and E.R. Santare´m. 2011. Secondary metabolism in micropropagated Hypericum perforatum L. grown in non-aerated liquid medium. Plant Cell Tiss Organ Cult DOI 10.1007/s11240-011-0058-9
Shilpashree H.P. and R. Ravishankar. 2009. In vitro plant regeneration and accumulation of flavonoids in Hyperıcum mysorense. I.J.I.B. 8(1): 43-49
Sood H., K. Shitiz and N. Sharma. 2015. Rapid method for In vitro multiplication of hypericin rich shoots of Hypericum perforatum. J.Plant Sci. 3(5): 279-284
Tekin M.. 2017. Pharmacobotanical study of Hypericum thymopsis. Rev. Bras. Farmacogn. 27:143–152
Tocci N., G. Simonetti , F.D. D’Auria , S. Panella , A.T. Palamara , A. Valletta and G. Pasqua. 2011. Root cultures of Hypericum perforatum subsp. angustifolium elicited with chitosan and production of xanthone-rich extracts with antifungal activity. Appl. Microbiol. Biotechnol. 91:977–987
Wahle K.W.J., I. Brown, D. Rotondo and S.D. Heys. 2010. Plant phenolics in the prevention and treatment of cancer. Adv. Exp. Med. Biol.698:36-51
Wo´ jcik A. and A. Podstolski. 2007. Leaf explant response in in vitro culture of St. John’s wort (Hypericum perforatum L.). Acta. Physiol. Plant. 29:151–156
Wu. S.Q., X.K. Yu, M.L. Lian , S.Y. Park and X.C. Piao. 2014. Several factors affecting hypericin production of Hypericum perforatum during adventitious root culture in airlift bioreactors. Acta Physiol Plan 36:975–981. DOI 10.1007/s11738-013-1476-6
Zeliou K , N. I. Kontaxis , E. Margianni , C. Petrou and F. N. Lamari. 2017. Optimized and validated HPLC analysis of St. John's Wort extract and final products by simultaneous determination of major ingredients. J. Chromatogr. Sci. 55(8) 805–812