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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/56288完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 杜宜殷(Yi-Yin Do) | |
| dc.contributor.author | Chin-Wet Lim | en |
| dc.contributor.author | 林菁薇 | zh_TW |
| dc.date.accessioned | 2021-06-16T05:22:01Z | - |
| dc.date.available | 2014-09-03 | |
| dc.date.copyright | 2014-09-03 | |
| dc.date.issued | 2014 | |
| dc.date.submitted | 2014-08-15 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/56288 | - |
| dc.description.abstract | 沙巴蛇草 (Clinacanthus nutans L.) 為馬來西亞民俗藥用植物,具有預防癌症之潛力。本研究所建立之沙巴蛇草組織培養再生及轉殖系統，將有助於大量繁殖及代謝體學研究。以含 1 mg/L BA 及1 mg/L Kinetin 之MS培養基，誘導莖頂組織生成不定芽，可獲得最高增殖率。將不定芽移至僅含0.5 mg/L之MS培養基可順利抽長並發根。以各種生長素及細胞分裂素誘導葉圓片生成癒傷組織，並依形態進行分類，經選取黃色且緊密結實之癒傷組織，培養於含2,4-二氯苯氧乙酸 (2,4-dichlorophenoxyacetic acid) 之MS培養基，可生成具有胚性之細胞並分化為體胚。癒傷組織及芽體以濃度為OD600 0.5之含有pCRFG質體的農桿菌 LBA4404浸染1小時後，共培養72小時，經1 mg/L Hygomycin篩選，獲得轉殖之癒傷組織及芽體；將癒傷組織以45℃進行熱休克處理後，再與膿桿菌共培養，可獲最高效率GUS陽性反應。以超音波震盪前處理節間，所誘導之芽體伴隨農桿菌菌液再進行轉殖，可檢測到報導基因GFP表現，經聚合酶連鎖反應分析，結果顯示超音波處理5秒，可達90%轉殖率。 | zh_TW |
| dc.description.abstract | In Acanthaceae family, Clinacanthus nutans L. is a popular folk medicine and has potential in cancer prevention. Therefore, establishing in vitro regeneration and genetic transformation systems of C. nutans is important for mass production and metabolomics studies of this medicinal plant. Successful regeneration system from difference explant types of C. nutans was developed. The adventitious shoots induced from tissue mass formed from shoot tips gave high proliferation rate on MS medium containing 1 mg/L BA and 1mg/L kinetin. MS medium containing 0.5 mg/L kinetin promoted shoot elongation and rooting. For callus initiation, various types of auxins and cytokinins were tested on leaf laminas and types of callus were classified. Callus induced from leaf explants on MS medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) produced competent embryogenic cells. In addition, MS supplemented with 2 mg/L 2,4-D was found to be optimal in callus maintenance while 0.3 mg/L 2,4-D was optimal in the development of somatic embryos. Furthermore, Agrobacterium-mediated transformation system of C. nutans was established. The effects of bacterial concentration, infection time, co-cultivation, sonication period, heat shock and centrifugation were tested. Transgenic calli and shoots were obtained through selection with 1 mg/L hygromycin after transformation with Agrobacterium tumefaciens strain LBA4404 harboring plasmid pCRFG. The bacterial concentration of A600=0.5 with co-cultivation periods of 72 hours was optimal conditions for transformation. Callus treated with heat shock showed highest frequency of GUS positive tissues. GFP expression was observed in shoots induced from nodal explants with sonication treatment. PCR was carried out on hygromycin-resistant plants to identify possible transformants. Results showed optimal transformation efficiency for shoots were explants treated with 5 s of sonication. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T05:22:01Z (GMT). No. of bitstreams: 1 ntu-103-R00628140-1.pdf: 5586392 bytes, checksum: 47d417bd98bd86d9b663beedd4ff3683 (MD5) Previous issue date: 2014 | en |
| dc.description.tableofcontents | Table of Contents
Acknowledgement I 摘要 II Abstract III List of Tables VII List of Figures IX Abbreviations XI Chapter 1 Introduction 1 Chapter 2 Literature review 2 2.1 Traditional uses 2 2.2 Pre-clinical and clinical trial of C. nutans extract 2 (1) Antiviral and anti-inflammatory activities 2 (2) Antioxidant activity 3 (3) Anti-venom activity 4 2.3 Tumour and cancer inhibitor 4 2.4 Plant regeneration system 4 (1) Hormones tested for callogenesis and shoot proliferation of plants from Acanthaceae family. 5 2.5 Genetic transformation 7 Chapter 3 Materials and Methods 9 3.1 Materials 9 (1) Explant preparation 9 (2) Bacterial strain and plasmid 9 3.2 Methods 10 (1) Callus induction, callus maintenance and embryogenic callus formation 10 (2) Organogenesis and shoot elongation 12 (3) Rooting and acclimatization 12 (4) Hygromycin sensitivity test 13 (5) Genetic transformation of Clinacanthus nutans 13 (6) Confirmation for the presence of transgenes 16 (7) Statistical analysis 19 Chapter 4 Results 20 4.1 Primary callus induction 20 4.2 Callus maintenance and embryogenic callus formation 22 4.3 Organogenesis 23 4.4 Shoot elongation and rooting 24 4.5 Acclimatization 25 4.6 Hygromycin sensitivity test 25 4.7 Effect of different parameters on genetic transformation efficiency. 26 (1) OD level 26 (2) Infection time 26 (3) Co-cultivation period 27 (4) Sonication period 28 (5) Heat shock and centrifugation assisted Agrobacterium-mediated transformation (CAAT) 28 Chapter 5 Discussions 64 5.1 Induction of embryogenic callus cells 64 5.2 Shoot induction 64 5.3 Shoot elongation and rooting 66 5.4 Effect of hygromycin 66 5.5 Effect of OD level, infection time and co-cultivation period 67 5.6 Effect of sonication 69 5.7 Heat Shock and CAAT 69 Chapter 6 Conclusions 72 References 75 | |
| dc.language.iso | en | |
| dc.subject | 胚性細胞 | zh_TW |
| dc.subject | 熱休克 | zh_TW |
| dc.subject | 農桿菌 | zh_TW |
| dc.subject | 超音波震盪輔助農桿菌媒介。 | zh_TW |
| dc.subject | 不定芽誘導 | zh_TW |
| dc.subject | 植株再生 | zh_TW |
| dc.subject | adventitious shoot induction | en |
| dc.subject | sonication assisted Agrobacterium-mediated transformatio. | en |
| dc.subject | heat shock | en |
| dc.subject | plant regeneration | en |
| dc.subject | Agrobacterium tumefaciens | en |
| dc.subject | embryogenic cells | en |
| dc.title | 沙巴蛇草之再生與轉殖系统之建立 | zh_TW |
| dc.title | Establishment of In vitro Regeneration and Genetic Transformation systems of Clinacanthus nutans L. (Acanthaceae) | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 102-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.coadvisor | 黃鵬林(Pung-Ling Huang) | |
| dc.contributor.oralexamcommittee | 何錦玟(Chin-Wen Ho),李昆達(Kung-Ta Lee) | |
| dc.subject.keyword | 植株再生,不定芽誘導,胚性細胞,農桿菌,熱休克,超音波震盪輔助農桿菌媒介。, | zh_TW |
| dc.subject.keyword | plant regeneration,adventitious shoot induction,embryogenic cells,Agrobacterium tumefaciens,heat shock,sonication assisted Agrobacterium-mediated transformatio., | en |
| dc.relation.page | 81 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2014-08-15 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 園藝暨景觀學系 | zh_TW |
| 顯示於系所單位： | 園藝暨景觀學系 | |
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