春石斛蘭(Nobile type Dendrobium)為蘭科石斛蘭屬節生石斛節(Dendrobium section Dendrobium)、以金釵石斛(Den. nobile)為基本種所雜交選育以及改良出之品種群的總稱。其擁有花色繁多豔麗、大且多之花朵、花期長及香氣濃郁等特色，主要作為贈禮盆花，為全球花卉市場的新興盆花。台灣具有多個石斛蘭原種，也是春石斛蘭的重要親本金釵石斛的分佈範圍之一，加上台灣處亞熱帶氣候，氣候上適合進行春石斛蘭的栽培與生產，因此春石斛蘭也為台灣具經濟栽培及種苗外銷潛力的新興蘭科作物。然台灣在新穎及恰當的外銷品種、長的幼年期以及低溫催花等技術上尚待開發及改進。
最近的研究顯示春石斛蘭可能有二個春化調控途徑：1. 依賴類似FLC途徑 (FLC like dependent pathway)：春化作用誘導VRN1 (AP1同源基因)表現，進而活化VRN3 (FT 同源基因)的表現之，VRN3也誘導更多VRN1大量表現，進而調控開花整合基因SOC1和FT，誘導開花；2. 不依賴FLC途徑 (FLC independent pathway)：春化作用誘導VIN3的大量表現，進一步誘導AGL19表現，最終活化LFY及AP1基因，啟動開花。因此本研究嘗試將SOC1、FT、VRN1、AGL19、VIN3及AP1等基因所構築的農桿菌轉殖載體，以單獨或混合不同載體的方式轉殖到春石斛蘭。本研究目的為以基因轉殖技術創新出調控植株花期的技術，開發出可調控花期之高品質轉殖春石斛蘭，促進台灣花卉產業技術升級。
本研究已完成：1. 共同轉殖p1304-35S-VRN1及p1304-35S-AGL19、2. 轉殖p1301-35S-VIN3、3. 共同轉殖pMLBART AlcA-AlcR-PaFT及pMLBART AlcA-AlcR-PaSOC1、4. 轉殖p1301-Ubi-AP1、5. 共同轉殖p1304-35S-FT和p1301-Ubi-GA2ox6等五種組合之農桿菌感染春石斛PLB、篩選、再生、增殖、健化、出瓶。擬轉殖植株葉片之PCR、RT-PCR分析的結果顯示，轉殖之DnAGL19、VRN1、VIN3、SOC1、FT、AP1及GA2ox6等基因已存在於轉殖葉片之基因組，並表現其mRNA。調查春石斛蘭苗株外表型顯示，擬轉殖株及未轉殖株之葉型、葉色、植株形態及生長狀態等園藝外表型性狀均無差異。但是受限於幼年期的因素，目前並未有開花的現象。

Nobile type Dendrobium is a section of Dendrobium of Orchidaceae which includes many related species. Nobile type hybrids are so named because they contain the species Den nobile. The potted nobile type Dendrobium is one of potential flower industries in global flowers market because of it has diverse beautiful color, gorgeous and elegant flower shape, long flowering period, and rich fragrance. Nobile type Dendrobium also has the great potential to be developed as an economic cultivation in Taiwan. But still have some bottleneck to be overcome that includes lake of new and suit for export varieties, long juvenile stage, and stable and effective flowering forcing technology.
Two types of vernalization pathway in nobile type Dendrobium have been proposed. 1. FLC like dependent pathway: VRN1 (AP1 ortholog) expression is induced by low-temperature vernalization, followed by activating VRN3 expression (FT ortholog), and then integrator genes are turn on such as FT and SOC1, which both rapidly promote floral development. 2. FLC independent pathway: Overexpression of VRN3 is induced by low-temperature vernalization, followed by activating AGL19 expression, and then markedly accelerates flowering is achieved by activation of LFY and AP1 expression. In this study, SOC1, FT, VRN1, DnAGL19, VRN3, VIN3 genes are attempted to be engineered into nobile type Dendrobium. The objective of this study is to develop the innovative biotechnologies for regulating flowering time in nobile type Dendrobium with early flowering time, and high marketing value via the art of gene transformation. Hopefully, these efforts could contribute to the sustainable development of Taiwan’s floral industry.
Five sets of combination of Agrobacterium mediated transformation were included in this study including: 1. Co-transformation of p1304-35S-VRN1 and p1304-35S-AGL19; 2. Transformation of p1301-35S-VIN3; 3. Co-transformation of pMLBART AlcA-AlcR-PaFT and pMLBART AlcA-AlcR-PaSOC1; 4. Transformation of p1301-Ubi-AP1; 5. Co-transformation of p1304-35S-FT and p1301-Ubi-GA2ox6. Constructed genes had been transformed into the PLB of the nobile type Dendrobium by Agrobacterium-mediated transformation. Regenerated plantlets were selected by antibiotics, induction of multiple shoots and roots formation, proliferation, hardiness, and finally transplanted into 2-inch pot and grown in greenhouse. The results of PCR and RT-PCR analysis of putative transformed plants indicated that the transformed genes were presented in the genome of transformed plants, and expressed its mRNA. There were no differences in leaf shape, leaf color, plant morphology, and growth status between non-transformed and transformed nobile type Dendrobium. The seedlings are in their juvenile stage, and there are no flowers produced at this time.

摘要………………………………………………………………………………………i
Abstract…………………………………………………………………………………ii
目次……………………………………………………………………………………iv
圖目次…………………………………………………………………………………v
表目次…………………………………………………………………………………vii
前言……………………………………………………………………………………1
前人研究………………………………………………………………………………3
一、春石斛簡介及春化處理………………………………………………………3
二、石斛蘭屬植物之基因轉殖概況………………………………………………5
三、植物開花調控途徑………………………………………………………7
四、蘭花開花調控途徑及相關基因………………………………………………8
五、SOC1基因……………………………………………………………………10
六、FT基因………………………………………………………………………11
七、AP1/MADS14基因…………………………………………………………12
八、VIN3、VRN1及AGL19基因………………………………………………13
九、GA2ox6基因…………………………………………………………………14
材料與方法……………………………………………………………………………17
結果……………………………………………………………………………………24
一、 共同轉殖p1304-35S-VRN1和p1304-35S-AGL19之擬轉殖植株………24
二、轉殖p1301-35S-VIN3之擬轉殖植株……………………………………25
三、共同轉殖pMLBART AlcA-AlcR-PaFT和
pMLBART AlcA-AlcR-PaSOC1之擬轉殖植株……………………………25
四、轉殖p1301-Ubi-AP1之擬轉殖植株……………………………………27
五、共同轉殖p1304-35S-FT和p1301-Ubi-GA2ox6之擬轉殖植株…………28
討論……………………………………………………………………………………49
參考文獻………………………………………………………………………………55
附錄……………………………………………………………………………………63

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