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題名 | 建立臺灣栽培種香蕉之農桿菌媒介轉殖系統=Establishment of Agrobacterium-mediated Transformation System in a Cultivated Banana (Musa ‘Pei Chiao’, AAA Group) |
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作者 | 徐善德; 廖玉琬; 黃鵬林; | 書刊名 | 臺灣園藝 |
卷期 | 54:2 2008.06[民97.06] |
頁次 | 頁173-181 |
分類號 | 435.371 |
關鍵詞 | 癒合組織; 報導基因; GUS活性分析; 聚合酶連鎖反應; 南方氏核酸雜交法; Callus; Reporter gene; GUS assay; Polymerase chain reaction; Southern hybridization; |
語文 | 中文(Chinese) |
中文摘要 | 本試驗以臺灣香蕉栽培種'北蕉' (Musa 'Pei Chiao ', AAA group)雄花序所誘導的胚性癒合組織為材料,建立香蕉之農桿菌媒介法轉殖系統。試驗採用的轉殖質體為pBI121,其攜有轉殖篩選與報導所需之新徽素磷酸轉移酶(neomycin phosphotransferase II ; NPTII)基因和β葡萄糖醛酸苷酶。它lucuronidase ; GUS)基因。 北蕉癒合組織與含質體pBIl21的農桿菌LBA4404品系共培養後,先以50mg.L-l 抗生素geneticin(G418)進行篩選,獲得抗生素抗性癒合組織後,續以110 mg.L-l G418 持續篩選,具GUS活性表現的癒合組織系再移置含有0.05 mg.L-l玉米素(zeatin)、 0.2 mg.L-l異戊烯基腺嘌呤(isopentenyladenine ; 2iP)、0.1 mg.L-l激動素(kinetin)、0.2 mg.L-l蔡乙酸(α-naphthalene acetic acid ; NAA)和110 mg.L-l G418的MS培養基上令其分化體胚。最後將體胚移至添加1 mg.L-l 芐基腺嘌呤(benzyladenine; BA)、0.1 mg.L-l激勃酸(gibberellic acid ; GAÙ和30 g.L.1蕪糖之MS培養基上發育並轉換成植株。篩選期間進行癒合組織之GUS活性表現、聚合酶連鎖反應與植株之GUS活性表現與南方氏核酸雜交分析,可證賞外來基因之表現與穩定轉殖。 |
英文摘要 | An Agrobacterium-mediated plant transformation system was developed for the commercial banana (Musa spp. AAA group) 'Pei Chiao' by using embryogenic calli induced from young male inflorescence. The embryogenic calli were co-cultivated with Agrobacterium tumefaciens strain LBA4404, harbouring the binary plasmid pBI121. The vector carried NPTII (neomycin phosphotransferase) and GUS (ß-glucuronidase) genes in the T-DNA. Putatively transformed calli were produced on a selection medium supplemented with 50 mg.L-1 geneticin (G418). The primary antibiotic-resistant calli were subsequent1y transferred to the selection medium containing 110 mg.L-1 G418. After selective cultures, the callus lines (putativehomogenous GUS-expression) were transferred to MS medium containing 0.05 mg.L-1 zeatin, 0.2 mg.L-1 2iP, 0.1 mg.L-1 kinetin, 0.2 mg.L-1 NAA and 110 mg.L-1 G418 for somatic embryogenesis. Finally, the antibiotic-resistant somatic embryos were transferred to MS medium containing 1 mg.L-1 BA, 0.1 mg.L-1 GA3 and 30 mg.L-1 sucrose for plant regeneration. Stable integration and expression of the transgenes were confirmed by GUS assay, polymerase chain reaction (PCR), and genomic Southern hybridization analyses. |
本系統之摘要資訊系依該期刊論文摘要之資訊為主。