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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 王俊能(Chun-Neng Wang) | |
dc.contributor.author | Yu-Ling Hung | en |
dc.contributor.author | 洪育翎 | zh_TW |
dc.date.accessioned | 2021-05-13T09:20:42Z | - |
dc.date.available | 2016-11-02 | |
dc.date.available | 2021-05-13T09:20:42Z | - |
dc.date.copyright | 2016-11-02 | |
dc.date.issued | 2016 | |
dc.date.submitted | 2016-08-19 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/4098 | - |
dc.description.abstract | 大岩桐是十分著名的園藝植物,因其花色及花型上的多變而受到大眾喜愛。大岩桐基因體小(300 Mbp)、短的生活史、自交親和性高及組織培養上容易再生等優點,皆支持其發展成研究花部發育機制的模式物種,為此有必要以基因轉殖之技術,輔佐了解相關調控花發育基因之功能性,但目前仍沒有明確的轉殖流程可遵循,因此本研究之目的為優化農桿菌及基因槍的轉殖條件。農桿菌轉殖中 GUS 訊號顯示以 1 ppm BA 前處理大岩桐三週大幼苗三天並與農桿菌菌株 EHA 105 共配養五天的處理方式下可得到暫時性的轉殖率 78.3 %,同樣條件下可獲得 17.2 %的再生率及 2.1 %的轉殖率。GUS 報導基因顯示大岩桐四週大幼苗在基因槍氦氣壓力 900 psi 下,6 公分及 9 公分的距離可分別獲得 58.1 % 與 21.6 % 的暫時性轉殖率。兩者轉殖的效率顯示農桿菌轉殖策略優於基因槍。另一方面,癒傷組織因生長快速容易再生適合做為轉殖的材料,本研究也成功從大岩桐葉片中以0.1 ppm 2,4-D 和 1 或 2 ppm BA 搭配 25 mM 或 50 mM 的山梨糖醇誘導出胚性癒傷組織(embryogenic callus)。本研究優化農桿菌介導的轉殖條件將有助於大岩桐調控花部發育基因功能性分析研究。 | zh_TW |
dc.description.abstract | Sinningia speciosa is a popular houseplant because of its big flower with a remarkable diversity in colors, patterns and shapes. S. speciosa has a small genome size (300 Mb), short life cycle, self-compatible, easily propagated in tissue culture therefore is emerging as a model plant for flower development studies. However, a reliable genetic transformation system is not available in S. speciosa. To this end, the Agrobacterium mediated transformation and particle bombardment transformation were tested in this study. Transient GUS expression assay showed that 3 days pre-culture of three weeks old seedlings on medium supplied with 1 ppm BA and co-culture for 5 days with Agrobacterium strain EHA105 achieved an overall transient transformation rate of 78.3%. Under these optimized conditions, the regeneration rate is 17.2 % and the transformation rate is up to 2.1 %. Another approach is particle bombardment transformation for optimizing genetic transformation system. In GUS transient assay, it was found that under helium pressure 900 psi, at distance 6 and 9 cm displayed the transient transformation rate of 58.1 % and 21.6 % respectively. The transformation efficiency of two approaches demonstrated that Agrobacterium-mediated transformation is better than particle bombardment transformation. Because callus grows rapidly and regenerate easily, it serves as a good material for transformation. I also successfully induced embryogenic callus with 0.1 ppm 2, 4-D and 2 ppm BA plus 25 or 50 mM sorbitol in the medium. Callus transformation rate will be tested further. This study optimized the transformation protocol for studying gene regulation and gene function in S. speciosa. | en |
dc.description.provenance | Made available in DSpace on 2021-05-13T09:20:42Z (GMT). No. of bitstreams: 1 ntu-105-R03b21014-1.pdf: 10000163 bytes, checksum: ea7c7297301fd98c6a206d6276edf78b (MD5) Previous issue date: 2016 | en |
dc.description.tableofcontents | Content 致謝................................................................................................................................ I 中文摘要....................................................................................................................... II Abstract ........................................................................................................................ III Content ......................................................................................................................... IV Index of Tables and Figures ...................................................................................... VIII Abbreviations ............................................................................................................... XI Introduction .................................................................................................................... 1 1. Plant genetic transformation .......................................................................... 1 1.1. Agrobacterium-mediated transformation ........................................... 1 1.2. Particle bombardment transformation ................................................ 3 2. The high diversity in angiosperms (flowering plants) ................................... 4 3. Sinningia speciosa, a good material to study floral symmetry and flower shape ..................................................................................................................... 4 4. Genetic transformation in S. speciosa ............................................................ 7 4.1. Agrobacterium-mediated transformation ........................................... 7 4.2. Particle bombardment transformation .............................................. 12 5. Callus as a novel substitute for genetic transformation in S. speciosa ........ 13 Aim of this study ................................................................................................ 14 Materials and Methods ................................................................................................. 15 1. Plant material and growth conditions ............................................................. 15 2. Transient transformation by Agrobacterium-mediated transformation for Sinningia speciosa .............................................................................................. 15 2.1. Plasmid and Agrobacterium strains ................................................... 15 2.2. Preparing aseptic seedlings for Agrobacterium-mediated transformation ........................................................................................... 16 2.3. Agrobacterium-mediated transformation ........................................... 17 2.4. Paraffin section ................................................................................... 22 3. Transgenic plant selection .............................................................................. 22 3.1. Agrobacterium washing ..................................................................... 23 3.2. Selection of transformed shoots ......................................................... 23 3.3. Calculation of regeneration rate ......................................................... 25 3.4. Vitrification of transgenic plants ........................................................ 26 3.4.1. GUS activity assay .......................................................................... 26 3.4.2. RT-PCR of GUS and NPTII ........................................................... 26 3.4.3. Southern blotting ............................................................................. 29 3.5. Calculation of transformation rate...................................................... 30 4. Callus induction and regeneration of S. speciosa ........................................... 30 4.1. Callus induction from mature leaf ...................................................... 30 Results .......................................................................................................................... 38 1. Optimization of Agrobacterium-mediated transformation .......................... 38 1.1. Optimization of co-culture time ....................................................... 38 1.2. Optimization of developmental stage of S. speciosa and Agrobacterium strain ................................................................................. 42 1.3. Optimization of pre-culture time ..................................................... 44 1.4. T-DNA insertion confirmation ........................................................ 48 2. Establishment of particle bombardment transformation for genetic transformation in S. speciosa: ............................................................................. 56 2.1. Optimization of physical parameters of hilum pressure and target distance ...................................................................................................... 56 3. Callus as an excellent alternative explant for genetic transformation ......... 60 3.1. Callus induction ............................................................................... 60 3.2. Shoot regeneration from callus ........................................................ 72 3.3. Callus transformation ....................................................................... 75 Discussion .................................................................................................................... 79 1. The conditions of Agrobacterium infection in S. speciosa .......................... 79 1.1. The effects of photoperiod and temperature during co-culture on transformation ........................................................................................... 79 1.2. The extended co-culture time needed for S. speciosa transformation. ……………………………………………………………………..80 1.3. The genotype-dependent susceptibility to Agrobacterium EHA 105 ……………………………………………………………………..81 2. Factors that cause long regeneration time in transformation of S. speciosa 82 2.1. The effect of cefotaxime on callus formation and shoot regeneration ……………………………………………………………………..82 2.2. Dosage effect of kanamycin on transformation success .................. 83 3. Factors contribute to the unstable transformation system............................ 84 3.1. Unstable pressurization process by syringe ..................................... 84 3.2. The different cell type between transformed cell and regenerated cell ……………………………………………………………………..85 4. Callus as explants to facilitate transformation ............................................. 86 4.1. Optimization of other factors on callus transformation with Agrobacterium ........................................................................................... 86 4.2. Particle bombardment transformation .............................................. 87 Conclusion and future prospects .................................................................................. 89 References .................................................................................................................... 91 Appendixes .................................................................................................................. 99 | |
dc.language.iso | en | |
dc.title | 大岩桐基因轉殖系統之優化 | zh_TW |
dc.title | Optimization of the genetic transformation system for Sinningia speciosa | en |
dc.type | Thesis | |
dc.date.schoolyear | 104-2 | |
dc.description.degree | 碩士 | |
dc.contributor.coadvisor | 洪傳揚(Chwan-Yang Hong) | |
dc.contributor.oralexamcommittee | 陶建英(Jian-Ying Tao),蘇睿智(Jui-Chih Su),蔡新聲(Hsin-Sheng TSAI) | |
dc.subject.keyword | 大岩桐,基因轉殖,農桿菌,基因槍,癒傷組織, | zh_TW |
dc.subject.keyword | Sinningia speciosa,genetic transformation,Agrobacterium,particle bombardment,callus, | en |
dc.relation.page | 120 | |
dc.identifier.doi | 10.6342/NTU201602983 | |
dc.rights.note | 同意授權(全球公開) | |
dc.date.accepted | 2016-08-20 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生命科學系 | zh_TW |
顯示於系所單位: | 生命科學系 |
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