臺灣博碩士論文加值系統

本研究以2年生及4年生台灣杉之莖段，放入含有1 mg/L BA + 0.1 mg/L NAA + 0.1 mg/L KIN的MMS培養基中可成功誘導出芽體。每個莖段約可誘導約7個芽體，移入發根培養基中，以MMS 添加0.05 mg/L NAA最適合台灣杉發根，待根長至約2 cm後，放入無荷爾蒙的MMS培養基，即可成功建立台灣杉之微體繁殖。另一方面，台灣杉苗木癒傷組織誘導則是以2年生及4年生台灣杉幼苗、嫩芽及莖等為材料，培養於不同荷爾蒙濃度試驗中，研究結果顯示，以3 mg/L 2,4-D + 0.1 mg/L BA可誘導出較多量且鬆散的癒傷組織，而若培養基中添加50 mg/L ascorbic acid則可以減少台灣杉的癒傷組織褐化。除此之外，為了研究癒傷組織之調控基因，利用扣除雜交技術，收集3 mg/L 2,4-D + 0.1 mg/L BA荷爾蒙處理1、2和3週培植體，以經荷爾蒙處理組扣除沒有荷爾蒙處理組的組織，選殖出dirigent like protein (DIR)，命名為TcDIR1，TcDIR1的轉譯區共有585個核苷酸，轉譯出21.5 kDa的蛋白質，pI為9.57，屬於鹼性蛋白，利用反轉錄多聚合鏈反應定量分析，TcDIR1於處理2週的培植體癒傷組織表現量較高，證明此基因在台灣杉癒傷組織之誘導及生成扮演一個重要角色。此外，透過農桿菌媒介並應用真空滲透進行培植體感染，共培養2天後放入含抗生素MMS培養基，所誘導出的癒傷組織，以組織化學法分析GUS活性，獲得轉殖的證據。

Two-year-old and four-year-old stem explants of Taiwania cryptomerioides were used to induce shoot multiplication that in vitro cultured in MMS medium containing 1 mg/L BA + 0.1 mg/L NAA + 0.1 mg/L KIN. Each stem of T. cyptomerioides could induce seven shoots. The experimental results indicated that MMS medium supplemented 0.05 mg/L NAA is a well medium for inducting Taiwania root. When root growth about 2 cm long, it was transferred to MMS medium without hormone. In addition, the callus inducing was performed by cutting 0.3 - 0.5 cm stem, shoot tip and leaf of two-year-old and four-year-old T. cryptimerioides. Among different hormone concentrations of callus induction that 3 mg/L 2, 4-D + 0.1 mg/L BA is optimal condition for inducing amount friable callus. To study the gene regulation of callus formation, the PCR-select cDNA subtraction hybridization technology was used. A dirigent like protein named TcDIR1 were cloned from callus in induction medium. There are 585 nucleotides (195 amino acids) with molecular weigh of 21.5 kDa pI 9.57. TcDIR1 expresed highest quality in 2 weeks using by reverse-transcription polymerase chain reaction (RT-PCR) analysis. It indicated that TcDIR1 might play an important role in callus formation. Furthermore, the Agrobacterium-mediated transformation conjugated vacuum infiltration to infect 2 month seedling of T. cryptomerioides. Cultivation in 3 mg/L 2, 4-D + 0.1 mg/L BA antibiotic MMS plate for two month. GUS enzyme activity was estimated to demonstrate the efficiency of genetic transformation.

口試委員會審定書 ................................................................................................. i
誌謝........................................................................................................................... ii
中文摘要................................................................................................................... iii
Abstract..................................................................................................................... iv
縮寫(Abbreviation).................................................................................................. v
一、前言.................................................................................................................. 1
二、前人研究.......................................................................................................... 3
2. 1無性繁殖. ...................................................................................................... 3
2. 1. 1 微體繁殖................................................................................................ 3
2. 1. 2 癒傷組織之誘導.................................................................................... 3
2. 2 扣除雜交....................................................................................................... 5
2. 2. 1 扣除雜交................................................................................................ 5
2. 3 轉殖系統....................................................................................................... 5
2. 3. 1 基因轉殖................................................................................................ 6
2. 3. 2 基因轉殖系統建立之必要性及其應用性............................................ 6
2. 3. 3 影響農桿菌媒介轉殖轉殖法效率的可能因子.................................... 7
三、材料與方法...................................................................................................... 10
3. 1無性繁殖...................................................................................................... 10
3. 1. 1培植體消毒............................................................................................. 10
3. 1. 2 培養基.................................................................................................. 10
3. 1. 3 培養環境.............................................................................................. 10
3. 1. 4多芽體誘導及發根............................................................................... 10
3. 1. 5 癒傷組織誘導........................................................................................ 11
3. 2扣除雜交........................................................................................................... 11
3. 2. 1 材料........................................................................................................ 11
3. 2. 2 總量RNA及mRNA之抽取與分析.................................................... 11
3. 2. 3 扣除雜交及序列分析............................................................................ 11
3. 2. 4 RACE (Rapid Ampilfy of cDNA Ends) 進行基因選殖..................... 12
3. 2. 5 核酸序列比對及分析............................................................................ 12
3. 2. 6 反轉錄多聚合酶連鎖反應.................................................................... 13
3. 3 農桿菌媒介轉殖系統之建立....................................................................... 13
3. 3. 1農桿菌轉型............................................................................................. 13
3. 3. 2 培植體感染................................................................................................. 13
3. 3. 2. 1不同濃度AS感染培殖體............................................................... 14
3. 3. 2. 2應用真空滲透感染培植體.............................................................. 15
3. 3. 3 GUS活性組織化學染色法.................................................................... 15
四、結果.................................................................................................................. 16
4. 1 微體繁殖....................................................................................................... 16
4. 2 癒傷組織誘導............................................................................................... 16
4. 3 扣除雜交....................................................................................................... 18
4. 4 農桿菌媒介轉殖........................................................................................... 19
4. 4. 1偶型載體轉型到農桿菌......................................................................... 19
4. 4. 2不同濃度AS農桿菌感染...................................................................... 19
4. 4. 3 農桿菌媒介感染應用真空滲透............................................................ 19
五、討論................................................................................................................... 21
5.1微體繁殖......................................................................................................... 21
5.2癒傷組織之誘導............................................................................................. 21
5.3 扣除雜交........................................................................................................ 22
5.4 基因轉殖………............................................................................................ 24
六、結論.................................................................................................................. 26
七、參考文獻........................................................................................................... 27
八、附錄................................................................................................................... 49

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