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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 王俊能(Chun-Neng Wang) | |
dc.contributor.author | Zuo-Hao Chan | en |
dc.contributor.author | 曾佐豪 | zh_TW |
dc.date.accessioned | 2021-06-16T17:44:07Z | - |
dc.date.available | 2015-08-16 | |
dc.date.copyright | 2012-08-16 | |
dc.date.issued | 2012 | |
dc.date.submitted | 2012-08-14 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/64384 | - |
dc.description.abstract | 大岩桐(Sinningia speciosa)是今日廣為栽培的觀賞植物。其栽培歷史悠久,故品種眾多,各品種之間也不難找到一些變異,花形的變異更是明顯,例如花市常見的輻射對稱品系便是現在最普遍的。因此大岩桐可以作為我們探究控制花部對稱性候選基因的材料。一旦建立了有效的農桿菌轉殖系統,這些基因的功能則會漸漸浮出水面。雖然先前已經有學者針對大岩桐的轉殖發表過文章,但是該文獻著重的是基因功能分析,因此有需要進一步針對大岩桐轉殖的部分作探討。我們先將無菌組培的大岩桐葉片切成0.5平方公分大小的葉片,在使用農桿菌菌株LB4404進行感染。感染之後以潮黴素(Hygromycin) 5 mg/L進行篩選。使用的載體是pCAMBIA1301,其中的beta-glucuronidase(GUS)帶有內含子。除此之外,我們又發現,在含MS、2 ppm BA和0.2 ppm NAA的培養基上,大岩桐的葉子切片能透過這樣的組培方式在兩個星期後分化出小型的愈傷組織,並且在一個禮拜後分化成不定芽,並長根繼續生長,再將這些不定芽移植至於菌環境以外的培養土上,仍能繼續生長。運用同一個轉殖系統轉殖後,在120片外體植片中,分化出14棵不定芽,經gDNA PCR檢測GUS基因,證實其中五株確定為轉殖株。另外RT-PCR結果又顯示在這五株轉植株中,全都表現GUS基因,轉殖成功率為4.16%。這是繼貓臉苦苣苔(Kohleria sp.)後,第二種苦苣苔科植物的轉殖系統,可作為往後研究計畫的基礎技術,進而得以在演化發育的研究領域中進行花部控制、發育或是逆境反應基因的功能性研究。 | zh_TW |
dc.description.abstract | Sinningia speciosa is an ornamental plant species that is widely cultivated. Many cultivars, however, generated a range of floral mutations such as Peloria (actinomorphy). It thus allows us to examine candidate genes that control floral symmetry. These functions should be able to be clarified if a suitable transformation system is established. Although there was a transformation protocol published on S. speciosa, yet it mainly focused on genetic analysis. Due to this reason, a more detailed transformation should be clarified. We therefore chose Agrobacterium, strand LBA4404, with medium Hygromycin as a selective agent, to infect leaf explants after they were sectioned into the size of 0.5 cm2. We also found out that S. speciosa has the capability to regenerate vigorously through vegetative cuttings after being placed on MS medium containing 2 ppm of BA and 0.2 ppm of NAA. So the major optimizing factor is on Agrobacterium infections. New plantlets induced from shoots were beta-glucuronidase(GUS)-stained and confirmed with gDNA PCR and RT-PCR in order to predict the rate of success. Out of all 120 explants, 14 regenerants were collected and five of which was successfully transformed as confirmed by PCR using GUS primers, while all of which showed GUS expression as confirmed by RT-PCR, resulting in a transformation rate of 4.16%. This project shall serve as an important mile stone in building a reliable transformation protocol in Gesneriaceae, allowing functional validations of several candidate floral developmental genes in evo-devo, thus assists any possible future project which could be brought out by our laboratory. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T17:44:07Z (GMT). No. of bitstreams: 1 ntu-101-R98b44019-1.pdf: 1355069 bytes, checksum: 3d85c67aea933522e486ca720db68ddd (MD5) Previous issue date: 2012 | en |
dc.description.tableofcontents | INDEX
口試委員會審定書 # ACKNOWLEDGEMENT I 中文摘要 III ABSTRACT IV INDEX VI LIST OF FIGURES IX LIST OF TABLES X Chapter 1 Introduction 1 Overview 1 Agrobacterium-mediated transformation 3 Possible applications of Agrobacterium-mediated transformation in members of Gesneriace 7 Infection of Agrobacterium on Sinningia speciosa 9 Aim of study 11 Chapter 2 Materials and Methods 13 Plant material and tissue culture 13 Hygromycin resistance of Sinningia speciosa 16 Plasmid preparation 16 Preparation of competent cells of Agrobacterium tumefaciens 17 Vector transformation into Agrobacterium tumefaciens through electroporation 18 Agrobacterium-mediated transformation 18 Verification of transformed regenerants by genomic DNA PCR 20 Verification of transformed regenerants by RT-PCR 23 GUS-staining 26 Chapter 3 Results 28 Tissue culture: callus induction and plant regeneration 28 Hygromycin resistance of S. speciosa and its working concentration 31 Agrobacterium-mediated transformation 32 DNA extraction 36 PCR using GUS-F and GUS-R as primers 37 RT-PCR 38 GUS-staining 39 Chapter 4 Discussion 41 Tissue culture in Sinningia speciosa 41 Selection of Hygromycin resistance in Sinningia speciosa 42 Duration of immersion plays a crucial role in survival of leaf explants 42 Future prospectives 46 References . 52 Appendix 58 | |
dc.language.iso | en | |
dc.title | 大岩桐(Sinningia speciosa)農桿菌轉殖系統之建立 | zh_TW |
dc.title | Agrobacterium-mediated transformation in Darwin’s Gloxinia, Sinningia speciosa | en |
dc.type | Thesis | |
dc.date.schoolyear | 100-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 洪傳揚(Chwan-Yang Hong),陳仁治(Jen-Chih Chen),鄭秋萍(Chiu-Ping Cheng) | |
dc.subject.keyword | 大岩桐,農桿菌轉殖,苦苣苔科,GUS染色,組織培養, | zh_TW |
dc.subject.keyword | Agrobacterium-mediated transformation,Sinningia speciosa,Gesneriaceae,GUS staining,Tissue culture, | en |
dc.relation.page | 61 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2012-08-14 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生態學與演化生物學研究所 | zh_TW |
顯示於系所單位: | 生態學與演化生物學研究所 |
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