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

• Main Authors: Tsung-Hsi Hsieh, 謝宗禧
• Other Authors: Hsin-Sheng Tsay
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/99943461250245622561

碩士 === 朝陽科技大學 === 生物技術研究所 === 98 === 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.