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研究生:林宇萱
研究生(外文):Yu-Hsuan Lin
論文名稱:玉山金絲桃(Hypericum nagasawai Hayta)植物組織培養及次級代謝產物分析
論文名稱(外文):Plant tissue culture and secondary metabolites analysis of Hypericum nagasawai Hayta
指導教授:何錦玟
指導教授(外文):Chin-wen Ho
口試委員:何錦玟
口試委員(外文):Chin-wen Ho
口試日期:2020-01-20
學位類別:碩士
校院名稱:大同大學
系所名稱:化學工程與生物科技學系(所)
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2020
畢業學年度:108
語文別:中文
論文頁數:77
中文關鍵詞:玉山金絲桃不定根培養植物組織培養金絲桃素
外文關鍵詞:adventitious rootplant tissue culturehypericinHypericum nagasawai Hayta
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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
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