建立青蔥農桿菌媒介基因轉殖系統

Establishment of an Agrobacterium-mediated Gene Transformation System in Welsh Onion (Allium fistulosum L.)

為建立青蔥農桿菌媒介基因轉殖系統，本研究將構築有新黴素磷酸轉移酶（nptⅡ）基因及β-葡萄糖苷酸酶（gus）基因的pBI121質體，利用農桿菌（LBA4404）轉移至‘黑葉’青蔥實生苗莖盤誘得之癒合組織。在與農桿菌共培養後的枝梢再生篩選階段採取建那黴素（geneticin, G418）抗生素自低濃度（10 mg•L^(-1)）至高濃度（50或80 mg•L^(-1)）逐步提高篩選壓力策略，或以單一高濃度（25或50 mg•L^(-1)）策略進行篩選。結果，擬轉殖枝梢團形成率以逐步提高濃度策略（11.7%-22%）高於單一高濃度策略（2.9%-4.6%）。透過PCR與GUS組織化學染色法分析擬轉殖株，證實轉基因已成功插入基因組並可表現蛋白。本試驗共獲得11株獨立轉殖植株。成功的轉殖效率在兩種篩選策略上差異不大，但單一高濃度篩選策略可縮短再生時間至6個月。因此建立快速篩選並獲得大量轉殖株的流程為在共培養後利用含有10 mg•L^(-1)G418的癒合組織誘導培養基篩選21天，再以添加25 mg•L^(-1)G48的枝梢再生培養基篩選90天，最後培養在不含G48的發根培養基30-45天。 To establish an Agrobacterium tumefaciens-mediated gene transformation system, previously constructed pBI121 plasmids containing neomycin phosphotransferase (nptII) gene and β-glucuronidase (gus) gene were transformed to the calli developed from stem discs of Welsh onion (Allium fistulosum L.) variety 'Hak-ip' via A. tumefaciens LBA4404. After co-cultivation with A. tumefaciens, the callus explants were cultured either in a sequentially increasing concentrations (from 10 to 50 or 80 mg•L^(-1)) of geneticin (G418), or in a constant high concentration (25 or 50 mg•L^(-1)) of G418 during the selection period for shoot regeneration. The results showed that the rates of forming adventitious shoot clumps in putatively transformed plants were higher when cultured in a medium containing sequentially increasing concentrations of G418 than when cultured in the constant high-concentration of G418 (11.7%-22% vs. 2.9%-4.6%). PCR analysis and GUS histochemical staining confirmed that the transgenes had been successfully inserted into the plant genome and expressed. In this study, 11 independent transgenic lines were obtained. The efficiency of transformation did not differ significantly between the two treatments, but the regeneration time in the treatment of constant high concentration G418 was shortened to 6 months. Therefore, we suggest that callus explants after co-cultivation should begin selection by cultivating in a callus induction medium with 10 mg•L^(-1) G418 for 21 days, then being transferred to a shoot regeneration medium with 25 mg•L^(-1) G418 for 90 days, and finally being cultivated in a root regeneration medium without G418 for 30-45 days.