Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36784
標題: 影響大豆農桿菌媒介基因轉殖因子之探討
Factors affecting Agrobacterium- mediated soybean transformation
作者: 蘇柏昀
Su, Po-Yun
關鍵字: Agrobacterium;大豆;soybean;transformation;農桿菌;基因轉殖
出版社: 農藝學系所
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摘要: 
利用基因轉殖技術以植物做為生物反應器,生產具有經濟價值的蛋白質或提高作物營養成分是一具有發展潛力的領域。大豆種子含有大量的蛋白質,約佔種子乾重的40 %,因此非常適合作為生產異源蛋白的生物反應器。但目前大豆轉殖仍是非常的困難,試驗的再現性差使得實際應用及研究均受限制,如能探討影響轉殖的重要因子,可作為日後發展穩定高效率基因轉殖平台的基礎,將有助於大豆基因體研究及新品種之育成。本研究選用大豆子葉節及胚尖為培植體,建立植株再生系統與不定芽誘導系統。在不定芽誘導試驗中,以不同濃度BA試驗,結果顯示無論在何種濃度BA條件下,子葉節皆無法形成不定芽;而胚尖於BA 0.2 mg/L條件下,有最佳的不定芽誘導效率(76 %)。因此以胚尖作為培植體,探討影響農桿菌感染效率的原因,並以報導基因GUS作為評估感染效率的依據。試驗使用CRWD、Jack、William82等三個品種大豆進行與農桿菌EHA105之親合性分析,結果顯示三個品種皆可被農桿菌感染;在不同傷害處理的比較發現,超音波震盪處理有最佳的感染效率,可有效的提升感染的面積與感染的培植體數。共培養的階段添加不同濃度之抗氧化劑L-cysteine (0-800 mg/L),可減少農桿菌轉殖過程中的褐化現象,濃度提高至200 mg/L時,有最佳的感染率(89.5 %),但過高濃度400及800 mg/L時,則會抑制培植體的生長。利用最佳的感染條件進行轉殖可得到再生擬轉殖植株,但由出瓶的擬轉殖株萃取其葉片基因組DNA進行PCR分析,無法擴增出目標基因與篩選基因片段,表示經由此再生與農桿菌感染系統無法獲得大豆轉基因植株,進一步探討何因素造成芽體於篩選培養基脫逸(escapes)的現象,觀察結果發現可能是由胚軸周圍所增生之癒合組織提供解毒作用,因而使非轉殖之芽體得以生長。本研究亦建立大豆雜交系統作為未來轉殖基因堆疊之依據。

Using plants as bioreactors to produce the high economic value proteins or improving nutrient content of crops through the transgenic technique is of great potential. Soybean seeds contain high protein content which accounts for about 40% of dry seed weight and can be served as a good bioreactor for the production of foreign proteins. The poor efficiency and difficult reproducibility of soybean transformation limits its applications. To overcome these problems, several critical factors including type of explants, hormone, antioxidant and treatments of the explants were analyzed to develop an efficient transformation. Cotyledonary nodes and embryonic tips were both used as the explants for establishment of plant regeneration and shoot induction system, respectively. The optimal concentration of benzyleamino-purine (BA) for adventitious shoot induction was determined and the results showed that no adventitious shoot was induced from cotyledonary nodes by BA. The use of embryonic tips provided the best induction frequency (76%) in 0.2 mg/L of BA. In this work, GUS and GFP genes were used as reporter to evaluate efficiency of transformation. Three cultivars, CRWD, William82 and Jack, were subjected to Agrobacterium-mediated transformation and the results displayed that all cultivars could be infected by Agrobacterium tumefaciens EHA105. “Sonication- assisted Agrobacterium-mediated Transformation” (SAAT) and carborundum treatment were also applied in the process of transformation. The results displayed that the transformation efficiency of SAAT was the best in all of the treatments because it could efficiently increase the infected area and amount of explants. Addition of L-cysteine (0- 800 mg/L) could reduce the browning of explant in the transformation and the best efficiency (89.5%) was found by addition of 200 mg/L L-cysteine, however the regeneration of explants was inhibited by the addition of high (400- 800 mg/L) L-cysteine. PCR analysis showed that no putative transgenic soybean plant was obtained after ex vitro acclimation. The result suggested that these untransformed and regenerated explants were escaped from the selection medium. It was speculated that the infected calli around the hypocotyl detoxified the selective antibiotics and caused the growth of uninfected shoots on the selective medium. Soybean hybridization system was also established for pyramiding the transgenes in the future.
URI: http://hdl.handle.net/11455/36784
其他識別: U0005-1708201109091300
Appears in Collections:農藝學系

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