台灣原生種白花蝴蝶蘭 (Phalaenopsis aphrodite subsp. formosana)，是屬於高經濟價值的花卉作物，且在蝴蝶蘭的商業化雜交育種中常使用作為親本。細胞分裂素 (CK) 為重要的植物賀爾蒙參與調控植物生長和發育，而細胞分裂素氧化酶 (CKX) 可調節細胞分裂素的濃度。所以利用阿拉伯芥 CKX 基因比對中研院蝴蝶蘭基因資料庫(Orchidstra)，鑑定出白花蝴蝶蘭至少有三個 CKX 基因，而且在不同組織有特異性的表現。在親緣關係演化分析可將植物 CKX 基因分成3大群。利用基因剔除系統 (CRISPR / Cas9)，嘗試剔除蝴蝶蘭中的單個或多個 CKX 基因，因此建構4個不同的 Cas9 / gRNA 細胞核表現載體。利用農桿菌轉殖花粉再行授粉的方式，將外源基因導入蝴蝶蘭。經 MSO (L-methionine sulfoximine) 篩選，分別自 pCas9-CKX1、pCas9-CKX2 和 pCas9-CKX3 轉殖品系得到18，15和3個具 MSO 抗性的幼苗。抽樣選取各品系的2株植株進行 PCR 分析確認含有外源基因之存在。但是定序分析 Cas9作用位點，則未發現有序列變異情形，雖然在其他位置有發現轉殖植物與對照組之序列有差異。

P. aphrodite subsp. formosana is an endemic moth orchid in Taiwan, and is one of the most commonly used parental strains in breeding programs. Cytokinins (CKs) play a role in plant development and growth. The cytokinin oxidases (CKXs) are important regulators of active cytokinin levels. In moth orchid, three CKXs genes were identified by blast the corresponding genes of Arabidopsis against Orchidstra database. The CRISPR / Cas9 editing system was applied to knock out single or multiple CKX function in moth orchid. Four Cas9/gRNA nuclear expression vectors carrying bar gene as selection marker were constructed. Transgenes were introduced into moth orchid by pollination through Agrobacteria-mediated transformation of pollinia. Subsequently, the seeds were germinated and then selected with L-methionine sulfoximine (MSO). In total, 18, 15 and 3 MSO-resistant seedlings were obtained for pCas9-CKX1, pCas9-CKX2 and pCas9-CKX3 transgenic lines, respectively. At lease two seedlings from each transgenic line were conformed with integration of transgene into nuclear genome by PCR. Sequencing analysis to detect the mutational status in CKX genes, no difference was observed in target sites among wild type and transgenic plants, although some sequence mutation was observed in other part of CKX genes.

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