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)

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黴素磷酸轉移酶（neomycinphosphotransferase II；NPTII）基因和β-葡萄糖醛酸苷酶（β-glucuronidase；GUS）基因。北蕉癒合組織與含質體pBI121的農桿菌LBA4404品系共培養後，先以50 mg•L^(-1)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^(-1)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^(-1)玉米素（zeatin）、0.2 mg•L^(-1)異戊烯基腺嘌呤（isopentenyladenine；2iP）、0.1 mg•L^(-1)激動素（kinetin）、0.2 mg•L^(-1)萘乙酸（α-naphthalene acetic acid；NAA）和110 mg•L^(-1)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^(-1)苄基腺嘌呤（benzyladenine；BA）、0.1 mg•L^(-1)激勃酸（gibberellic acid；GA3）和30 g•L^(-1)蔗糖之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，可證實外來基因之表現與穩定轉殖。 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. Theembryogenic calli were co-cultivated with Agrobacterium tumefaciens strain LBA4404, harbouring thebinary plasmid pBI121. The vector carried NPTII (neomycin phosphotransferase) and GUS (β-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 subsequently transferred to theselection 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, theantibiotic-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 wereconfirmed by GUS assay, polymerase chain reaction (PCR), and genomic Southern hybridization analyses.