嘉德麗雅蘭再生及轉殖系統建立

Abstract

本論文係以嘉德麗雅蘭為材料，建立組織培養無性繁殖系統，並利用農桿菌媒介法建立完整之轉殖系統，以供後續轉殖抗病與延緩老化基因之用。 再生系統部分，以實生苗原球體和葉片作為培植體，分別處理不同種類之鹽類（KC、1/2 MS）和不同植物生長調節劑（NAA、2,4-D、BA、TDZ）。研究結果顯示，以實生苗原球體為培植體置於含 0.5 mg/l NAA 和 2 mg/l BA 之 KC 培養基中，以及用葉片作為培植體置於含 3mg/l TDZ 之 1/2 MS 培養基中，可使其產生類原球體，完成再生。 為了建立轉殖系統，使用葉片和類原球體作為培植體，測試農桿菌菌種、菌液濃度、感染時間、介面活性劑添加物和超音波震盪輔助法對轉殖效率之影響，結果顯示嘉德麗雅蘭類原球體較佳的轉殖流程如下：將培養48小時之農桿菌 (A281菌系) 菌液（OD600=0.8）稀釋至OD600=0.1，加入Acetosyringone使總濃度達300μM，加入培植體於黑暗條件下震盪感染三十分鐘，取出培植體，置於不含抗生素之固態培養基，黑暗條件下共培養三天，再進行殺菌，並換至含有抗生素之培養基中進行篩選。嘉德麗雅蘭葉片較佳之轉殖流程如下：將培植體於液態再生培養基中黑暗條件下震盪預培養一天，將培養48小時之農桿菌 (A281菌系) 菌液 （OD600=0.8） 稀釋至OD600= 0.1-0.5，添加 Acetosyringone 使總濃度達 300 μM 後，並加入培植體於黑暗條件下震盪感染一小時，取出培植體，置於不含抗生素之液態培養基，黑暗條件下共培養三天，再進行殺菌，並換至含有抗生素之固態培養基中進行篩選。 以 GUS活性組織化學染色結果計算轉殖率，以類原球體為培植體轉殖率為26.6%，葉片之轉殖率約 20%。聚合酶連鎖反應結果亦顯示，擬轉殖株基因組中可偵測到GUS基因之表達，顯示外源基因已成功轉殖進入基因組中。此外，農桿菌媒介法轉殖 pGcET-CyORi 於嘉德麗雅蘭類原球體，經 GUS 組織活性染色分析，初步推測已轉殖成功。 關鍵字:嘉德麗雅蘭、超音波震盪法、農桿菌媒介法、類原球體轉殖、葉片轉殖

In this study, plant regeneration and Agrobacterium-mediated transformation systems were established in Cattleya. Regeneration system has been optimized using protocrom and young leaf as explants, testing different basal mediums (KC or 1/2 MS) and plant growth regulators (NAA, 2, 4-D, BA, TDZ). The result showed that when using the protocorm as explant, addition of 0.5 mg/l NAA and 2 mg/l BA in KC medium is found to be effective to induce PLBs. For explants using young leaf, addition of 3 mg/l TDZ in 1/2 MS medium is the best one. For transformation, Agrobacterium-mediated transformation method was optimized by testing different factors such as Agrobacterium strains and their concentration, infection time, and presence or absence of surfactants. Furthermore, the effect of sonication to enhance the transformation rate was studied. The result showed that, for Agrobacterium-mediated transformation of PLBs, A281 was the best Agrobacterium strain for transformation. Agrobacterium suspension was cultured for 48 hr till the concentration reached to OD600= 0.8, and diluted to OD600= 0.1 by regeneration medium. PLBs were infected with A. tumefaciens, which had been activated with 300 μM acetosyringone, for 30 min. After infection, PLBs were co-cultivated for 3 days on the regeneration medium. Following this, the PLBs were transferred to selection medium containing Hygromycin, Cefotaxime and Timentin to inhibit the growth of Agrobacterium and screened for the transformants. Pre-cultured is needed when using the young leaf as explant. Young leaves were cultured on liquid regeneration medium for 1 day under dark and shaking condition. The procedure of Agrobacerium-mediated transformation of young leaves is similar to Agrobacerium-mediated transformation of PLBs, with a minor modification in the infection duration. Leaf need much longer infection duration (extended to 1 h) than PLB, for Agrobacerium to infect the young leaves. A transformation efficiency of 26.6% and 20% was obtained when histochemical GUS staining was used to assess the putative transgenic PLBs and leaves respectively. Besides GUS staining, PCR analysis also confirmed the foreign DNA was integrated in the Cattleya genome. Moreover, PLBs were infected with A. tumefaciens harboring pGcET-CyORi. After 2 months of selection, the putative transgenic PLBs also showed the GUS activity, indicating the presence of putative transformants. Key words: Cattleya, SAAT, Agrobacterium-mediated transformation, PLB transformation, leaf transformation