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研究生:江孟縈
研究生(外文):Meng Ying Jiang
論文名稱:培養虎杖毛狀根生產白藜蘆醇
論文名稱(外文):Production of trans-resveratrol in hairy root cultures of Polygonum cuspidatum Sieb. et Zucc
指導教授:廖宇賡古國隆
指導教授(外文):Yue Ken LiaoKuo Lung Ku
學位類別:碩士
校院名稱:國立嘉義大學
系所名稱:森林暨自然資源學系研究所
學門:農業科學學門
學類:林業學類
論文種類:學術論文
畢業學年度:100
語文別:中文
論文頁數:50
中文關鍵詞:農桿根群菌幾丁聚醣毛狀根虎杖白藜蘆醇紫外線
外文關鍵詞:Agrobacterium rhizogeneschitosanhairy rootsPolygonum cuspidatumtrans-resveratrolultraviolet
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虎杖(Polygonum cuspidatum Sieb. et Zucc.)中的白藜蘆醇(trans-resveratrol;t-Res)具有極佳的藥用價值,本試驗取虎杖無菌播種苗之葉子做為培殖體,並將其以6-benzylaminopurine (BAP)預培養,利用農桿根群菌(Agrobacterium rhizogenes) BCRC 15011感染後移置於1/2 MS基礎培養基上,可成功誘導出毛狀根(hairy root)。以高效液相層析法(high performance liquid chromatography;HPLC)進行t-Res含量分析。毛狀根以烘乾萃取做為前處理者其內含的t-Res量會較新鮮萃取者高。將毛狀根根群1-11及1-13培養於1/2 MS或B5培養基中,其生長量並無顯著差異但均優於1/2 B5培養基。三種培養基中又以1/2 MS所培養的毛狀根內含的t-Res較高。在誘引因子的試驗中以紫外線(ultraviolet)照射處理後之毛狀根,t-Res的含量可提高到原來的兩倍;在幾丁聚醣(chitosan)試驗中,以10 mg/L chitosan加入至1/2 MS液體培養基內黑暗共培養48h,毛狀根(1-11)內t-Res的含量可提高到26.62±0.85 µg/g-dw,約為未處理的四倍。
Trans-resveratrol (t-Res), a valuable medicinal compound, is available and can be extracted from Polygonum cuspidatum Sieb. et Zucc. In this study, the leaf explants of P. cuspidatum seedlings grown in vitro were pre-cultured with 6-benzylaminopurine (BAP) and inoculated by Agrobacterium rhizogenes strain BCRC 15011 to induce hairy roots growing on 1/2 MS basal medium. The high performance liquid chromatography (HPLC) analysis demonstrated that extracts derived from oven-dried hairy root material contains greater t-Res than those extracted from fresh cultures. Hairy root lines 1-11 and 1-13 performed similar biomass increase on 1/2 MS and B5 media; both were superior to 1/2 B5 medium. However, the t-Res concentration was detected, significantly higher in root cultures incubated on 1/2 MS medium. Under elicitation treatments, the ultraviolet (UV) radiation stimulated and doubled the t-Res content in the illuminated cultures. Furthermore, the root cultures (line 1-11) increased t-Res content by 4-fold (26.62 ± 0.85 μg/g-dw) when dark incubated with 10 mg / L chitosan on 1/2 MS liquid medium.
目錄 Ⅰ
圖表目錄 Ⅳ
摘要 Ⅴ
Abstract Ⅵ
Ⅰ、前言 1
Ⅱ、前人研究 2
(Ⅰ) 虎杖藥用價值 2
一、虎杖特性 2
二、t-Res之概述 3
(II)、農桿根菌(Agrobacterium rhizogenes)的生理特性及致病機制 5
一、農桿根群菌之簡介 5
二、農桿根群菌之致病機制 5
三、質體與T-DNA之調控 7
(Ⅲ) 、毛狀根之培養 8
一、毛狀根之特性 8
二、毛狀根生產二次代謝物 9
三、誘引因子(elicitor) 10
(Ⅳ)、虎杖之組織培養 12
Ⅲ、材料與方法 13
(Ⅰ)、植物材料準備 13
(Ⅱ)、農桿根群菌株之培養 14
(Ⅲ)、毛狀根誘導 15
一、農桿根群菌感染流程 15
二、BAP(6-benzylaminopurine)預培養對毛狀根轉殖率之影響 15
(Ⅳ)、毛狀根培養 16
一、毛狀根離体培養 16
二、毛狀根之液體培養 16
(Ⅴ)、毛狀根之遺傳鑑定 16一、DNA之萃取 16
二、PCR (polymerase chain reaction) 17
(Ⅵ)、萃取前處理對毛狀根t-Res含量之影響 18
(Ⅶ)、選擇最適毛狀根生長及生產t-Res之培養基 19
(Ⅷ)、誘引因子誘導毛狀根生產t-Res含量之測定 19
一、UV照射毛狀根 19
二、以chitosan處理毛狀根 20
(Ⅸ)、HPLC分析條件及標準曲線之繪製 20
(Ⅹ)、統計分析 21
(Ⅺ)、藥品 22
IV、結果 23
(Ⅰ)、虎杖無菌苗之培育 23
(Ⅱ)、BAP預培養對毛狀根接種率之影響 23
(Ⅲ)、虎杖毛狀根之轉殖基因鑑定 25
(Ⅳ)、以HPLC檢測毛狀根內t-Res 26
(Ⅴ)、毛狀根萃取前處理對t-Res含量之影響 27
(Ⅵ)、選擇最適毛狀根生長及生產t-Res含量之培養基 28
(Ⅶ)、誘引因子誘導毛狀根內t-Res之生產 29
一、UV照射 29
二、chitosan處理 30
Ⅴ、討論 31
VI、參考文獻 37
VII 附錄 47

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