台灣蒲公英大量繁殖、毛狀根誘導與咖啡酸含量分析之研究

Establishment of in vitro propagation and hairy root transformation system and determination of caffeic acid contents of Taraxacum formosanum Kitamura

謝宗禧(Tsung-Hsi Hsieh)

指導教授 ： 蔡新聲 ； 張宏祺

朝陽科技大學/理工學院/生物技術與化學工程研究所碩士班/碩士(2010年)

摘要

台灣蒲公英(Taraxacum formosanum Kitamura)為台灣原生種菊科植物，是常用中草藥，具有治療乳腺腫瘤、扁桃腺炎及膽囊炎等功效。近年來由於土地開發、農藥濫用、外來種蒲公英競爭及在自然環境下較難自花授粉，已被列為瀕臨滅絕之保育級物種。因此本試驗利用植物組織培養技術進行大量繁殖以提供優良生藥來源，及建立本品毛狀根系統作為未來生產大量活性物質之基礎。結果發現臺灣蒲公英葉片培養於1/2 X MS（Murashige & Skoog, 1962）基本鹽類、3 % 蔗糖及0.3 mg/L Benzyl aminopurine（BA）固態培養基中三週後可獲得大量叢生芽體。將小芽移到1/4 X MS基本鹽類、3 % 蔗糖及0.9 % agar的固態培養基中可誘發良好根系，瓶苗馴化並移植於土壤中，成活率可達95 ％以上；台灣蒲公英根部經農桿根群菌（Agrobacterium rhizogenes,BCRC 15011）感染之毛狀根品系共有七個，依生長速率挑選其中四個品系，做後續成分測試。毛狀根繼代培養基為WPM (Lloyd and McCown, 1980)基本鹽類、3 % 蔗糖之固態培養基；分析溫室組培苗、癒合組織、毛狀根以及市售藥材之咖啡酸含量顯示差異極大，經種植一個月之溫室植株咖啡酸含量與市售藥材（生長約一年）含量差一倍，毛狀根及癒合組織則較少。

關鍵字

台灣蒲公英 ； 癒合組織 ； 毛狀根 ； 咖啡酸 ； 植物組織培養

並列摘要

Pugongying (Taraxacum sp.) is well known genus of flowering plants, belongs to the family Asteraceae. Pugonying were first recorded in Shen-Nung-Pen-Tsao-Ching from China. Some species of the genus Taraxacum are being use as folk medicine. Pugongying are mainly used in treatment of breast cancer, tonsillitis and cholecystitis. Modern research showed that it was used for the treatment of osteoporosis in menopausal women. However due to limited geographical distribution and indiscriminate collection, T. formosanum is now rarely found in its natural habitat. Over development of land for many uses and introduction of alien species (T. officinale) in its natural habitat make it difficult for T. formosanum to self pollinate, thus Taraxacum formosanum has been declared as endangered species in Taiwan. So to meet the increasing demands of plants for different medicinal uses and its conservation can be overcome by plant tissue culture methodologies. Thus, plant regeneration via in vitro culture of T. formosanum would be valuable tool for mass propagation of this endemic species. It was found that the multiple shoots were induced from mature leaf cultured on half-strength MS (Murashige and Skoog, 1962) medium supplemented with 0.3 mg/L N6-benzylaminopurine (BA), 0.9 % Difco agar, and 3 % (w/v) sucrose for 3 weeks. Rooting was achieved upon transferring the regenerated shoots on 1/4 X MS medium supplemented with 0.1 mg/L NAA, 0.9 % Difco agar, and 3 % (w/v) sucrose. Micropropagated plantlets were acclimatized with 95 % of survival rate and successfully grown on soil.
Roots of T. formosanum were used for Agrobacerium rhizogenes (BCRC 15011) mediated transformation. The infected roots were transferred on MS basal salt media for hairy root induction. Four best lines were selected depending on the higher hairy root growth rate. The maximum hairy root was achieved on plant growth regulators (PGRs) free WPM (Lloyd and McCown, 1980) solid media supplemented with 3 % sucrose and 0.9 % Difco agar. Caffeic acid content in the hairy root was analyzed using HPLC and their data was compared with the green house grown plantlet, callus and commercially available medicine. It was found that hairy root and callus accumulate less caffeic acid as compared to green house grown plantlet and commercially available medicine, however green house grown plant (1 month old) and commercially available medicine accumulate same amount of caffeic acid.

並列關鍵字

plant tissue culture ； caffeic acid ； Taraxacum formosanum ； hairy root ； callus

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