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研究生:朱庭慧
研究生(外文):Tin-Hui Chu
論文名稱:巴西鳶尾及射干根莖組織液體培養及其鳶尾異黃酮、總黃酮含量分析之研究
論文名稱(外文):THE RHIZOME TISSUE MUTIPLACATION, TECTORIGENIN AND TOTAL FLAVONOID ANALYSIS OF Neomarica gracilis AND Belamcanda chinensis BY LIQUID CULTURE
指導教授:何錦玟
指導教授(外文):Chin-Wen Ho
學位類別:碩士
校院名稱:大同大學
系所名稱:生物工程學系(所)
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:英文
論文頁數:57
中文關鍵詞:射干巴西鳶尾黃酮
外文關鍵詞:total flavonoidIris
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巴西鳶尾(Neomarica gracilis)液體培養方面:(1) Flask culture最佳接種量:根莖組織以modified MS液體培養基繼代,3 %( g fresh weight/100 ml)接種量,在培養3週後增加約5倍鮮重。(2)生長調節劑組合測試:3 %接種量在IAA-Kinetin與2,4-D-NAA組合之培養基培養8週,其中以添加0.1 mgl-1 NAA培養基,根莖鮮重可從1.5 g增至12.88±2.96 g,並含有55±0.67 mg/ kg D.W. tectorigenin (鳶尾異黃酮)為最佳,比較各週生長速率以第4週為最高,且total flavonoid(總黃酮)由起始的195.1±9.76 mg cinnamic acid eq./g D.W.提升到759.58±71.38 mg cinnamic acid eq./g D.W.,故培養週期以4週最佳。(3)潮汐式培養(Temporary Immersion System):將10個平均1.5 g根莖組織,置於含200 ml的modified MS培養基之潮汐式培養裝置,進行放大規模培養,4週後根莖組織鮮重平均增加至約40 g,且含有tectorigenin 37.6±4.9 mg/ kg D.W.及total flavonoid 885±46.69 mg cinnamic acid eq./g D.W.。2.射干(Belamcanda chinensis)液體培養方面:使用巴西鳶尾flask culture之培養條件,射干根狀癒合組織,以NAA 0.5 mgl-1培養基培養4週,可得到鮮重約2.41倍之細胞團與不定根,所有生長調節劑組合培養中,細胞皆測不到tectorigenin,但可測得高量的total flavonoid,且在不含生長調節劑之MS培養基培養的細胞團,可測得最高total flavonoid 2713±88.15 mg cinnamic acid eq./g D.W.。比較巴西鳶尾與射干在田間與液體培養中根莖之成分:(1)tectorigenin:射干田間根莖 (100.4±1.56 mg/ kg D.W.)>巴西鳶尾液體培養根莖(55.5±0.67 mg/ kg D.W.),巴西鳶尾田間根莖與射干癒合組織皆測不到。(2)total flavonoid:以flask culture之射干根狀癒合組織最高(2713±88.15 mg cinnamic acid eq./g D.W.),最低則是田間的射干根莖(337.7±15.05 mg cinnamic acid eq./g D.W.),顯示巴西鳶尾及射干的根莖液體培養,具有生長快速和生產tectorigenin及相關黃酮類的極佳潛力。
Abstract
In flask culture of Neomarica gracilis, the best inoculum was 3 % (g fresh weight/100 ml) which the rhizome tissue proliferated up to 5 folds in modified MS medium within 3 weeks culture. In the test of combination of IAA-Kinetin and 2,4-D-NAA, the best multiplication of rhizome tissue was about 8.5 folds when it cultured in the MS medium with 0.1 mgl-1 NAA after 8 weeks culture, and the content of tectorigenin was 55±0.67 mg/ kg D.W. in the rhizome tissue. The highest growth rate appeared at the 4th week culture in cell growth curve, and the total flavonoid accumulated from 195.1±9.76 mg cinnamic acid eq./g D.W. to 759.58±71.38 mg cinnamic acid eq./g D.W. Thus, the best culture period was 4 weeks. Temporary Immersion System (T.I.S.) was used for scale up culture. In this system, the container added 200 ml modified MS medium and 10 rhizome tissues (average 1.5 g) were placed in the container. After 4 weeks culture,the average of biomass increase was about 40 g (2.67 folds)of the rhizome tissues, and the content of tectorigenin and total flavonoid in the tissue were 37.6±4.9mg/ kg D.W. and 885±46.69 mg cinnamic acid eq./g D.W. respectively. In the test of combination of 2,4-D and NAA of Belamcanda chinensis liquid culture, the best biomass increase of root-like aggregates approached to 2.41 folds when cultured in modified MS medium with 0.5 mgl-1 NAA for 4 weeks. However, it showed high total flavonoid, but no tectorigenin could be detected in all of the proliferated aggregates of B. chinensis.
Comparison with the flavonoid in in vitro and wild rhizomes of N. gracilis and B. chinensis: (1) the content of tectorigenin in wild rhizome of B. chinensis was 100.4±1.56 mg/ kg D.W. higher than the rhizome of liquid culture N. gracilis(55.5±0.67 mg/ kg D.W.),while it could not be found in the wild rhizome of N. gracilis and callus of B. chinensis. (2) The highest total flavonoid (2713±88.15 mg cinnamic acid eq./g D.W.) was showed in the callus of B. chinensis by flask culture, and the lowest (337.7±15.05 mg cinnamic acid eq./g D.W.) was the wild rhizome of B. chinensis. It appeared that fast multiplication of the rhizome tissue of N. gracilis and B. chinensis by liquid culture had the great potential in production of tectorigenin and related flavonoid.
Content
Chinese Abstract………………………………………………………...i
Abstract..……………………………………………………………….iii
Content…………………………………………………………………. v
List of tables………………………………………………….……….viii
List of figures…………………………………………………………...x
Abbreviation…………………………………………………………...xiii
1.Introduction…………………………..……………………………….1
1.1Introduction of Neomarica gracilis and Belamcanda chinensis………1
1.2the components of rhizome tissue of Iridaceae and research of plant tissue culture……………………………………………………………..2
1.3 The cell and animal experiments of total flavonoid and tectorigenin in rhizome of Iridaceae………..…………………………………………….7
1.4 The source of tectorigenin……………………………………………8
1.5 Introduction of Temporary Immersion System………………………9
1.6 Aim of research ……………………………………………………..10
2.Material and method…………………………………………………..10
2.1 Plant material…………………………………………………….….11
2.2 Media……………………………………………………….……….12
2.3Neomaric gracilis………………………………………………...….14
2.3.1Flask culture……………………………………………………….14
(1) Neomaric gracilis….………………………………………..………14
(2) Effect of inoculum and plant growth regulators to biomass increase and tectorigenin of N. gracilis……………………………………..14
(3)Growth curve of N. gracilis and variation of tectorigenin and total flavonoid……………………………………………………………..….14
(4)Inhibition of shoots of N. gracilis by adding ancymidol and effect of tectorigenin………………………………………………………..17
2.3.2 T.I.S. culture……………………………………………………....17
(1) Effect of inoculum to biomass increase of N. gracilis……………...17
(2) Effect of inoculum and plant growth regulators to biomass increase and tectorigenin of N. gracilis……………………………………..17
2.4. Belamcanda chinensis..……………………………………………..19
2.4.1.Callus induction and somatic embryogenesis..…………………...19
2.4.2.Flask culture...….…………………………………………………19
2.5. Extraction of tectorigenin and total flavonoid...……………………22
2.6. Measurement of total flavonoid..…………..………………….…...22
2.7. Analysis of tectorigenin by HPLC.………..………………………23
2.8. Statistic analysis…………………………..…………………………23
2.9.Photographic methood………..……………………………………23
3.Result………………………………………………...………………..24
3.1. Neomaric gracilis………………………………………….………24
3.1.1.Flask culture………………………………………………………24
(1) Effect of inoculum to biomass increase of N. gracilis………………24
(2) Effect of plant growth regulators to biomass increase and tectorigenin of N. gracilis..……………………………………………………..24
(3) Growth curve of N. gracilis and variation of tectorigenin and total flavonoid……...……………………………………….…………….30
(4) Inhibition of shoots of N. gracilis by adding ancymidol and effect of tectorigenin………………………………………………………..33
3.1.2. T.I.S. culture………………………………………………...…....36
(1) Effect of inoculum to biomass increase of N. gracilis……………...36
(2) Effect of plant growth regulators to biomass increase and tectorigenin of N. gracilis….……………………………………………………………36
3.2. Belamcanda chinensis.……………………………………………..36
3.2.1. Callus induction and somatic embryogenesis…………………….39
(1) Establishment of in vitro plant….…………………………………...39
(2) Induction of callus of different part and somatic embryogenesis…...41
3.2.2.Flask culture..….………………………………………………….43
3.3.Comparision of in vitro and wild rhizome tissues of N.gracilis and B.chinensis….………………………………………………….….44
4.Discussion…………………………..…………………………………47
4.1.Neomarica gracilis.…………………………………………………47
4.1.1.Flask culture…………………………………………………..…..47
(1) Effect of inoculum to biomass increase of N. gracilis………………47
(2) Effect of plant growth regulators to biomass increase and tectorigenin of N. gracilis……………………………………………………………..47
(3) Growth curve of N. gracilis and variation of tectorigenin and total flavonoid……………………………………………….……….……47
(4) Inhibition of shoots of N. gracilis by adding ancymidol and effect of tectorigenin………………………………………………………..47
4.1.2. T.I.S. culture…………………………………………….……....49
(1) Effect of inoculum to biomass increase of N. gracilis…………….49
(2) Effect of plant growth regulators to biomass increase and tectorigenin of N. gracilis…………………………………….…………………49
4.2. Belamcanda chinensis…………………………….…………………49
4.2.1. Callus induction and somatic embryogenesis……………………49
4.2.2. Flask culture…….……………………………….……………….50
4.3. Comparision of in vitro and wild rhizome tissues of N.gracilis and B.chinensis………………………………………………..…………….51
5、Conclusion…………………………………………………..…….52
6、Refferences…………………..………………………….………....54
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