利用香蕉球狀芽團建立農桿菌轉殖系統之研究

Establishment of Agrobacterium-mediated transformation system by multiple shoot clumps of Musa. acuminata cv. ‘Pei Chiao’

蘇貴卿(Su-Kuei Ching)

指導教授 ： 何錦玟

大同大學/工程學院/生物工程學系所/碩士(2008年)

摘要

利用含有GUS報導基因及篩選標誌基因的pBI121質體，導入農桿菌EHA105品(株)系中，進行北蕉球狀芽團之農桿菌基因轉殖研究，測試北蕉轉殖及非轉殖球狀芽團在含不同抗生素kanamycin濃度(0、10、20、30、40 mg/L)培養基之反應。結果顯示轉殖及非轉殖培植體在含 kanamycin濃度高於30 mg/L的培養基中，大部份長出之芽體皆呈現白化現象且無法再生植株，僅少部份長出之芽體呈現綠色可再生植株；嚴重影響培植體再生與增殖，因此轉殖後培植體之再生及增殖時培養基不加入抗生素，直接以聚合酶連鎖反應(Polymerase Chain Reaction, PCR)分析檢測，做為擬轉殖株的篩選鑑定方法。從138個轉殖培植體經過農桿菌轉殖，經培養後所得到之再生植株中隨機取樣48株再生植株，抽取48株再生植株基因組DNA進行PCR分析檢測；結果顯示48株再生植株中有23株(47.9 ﹪)為擬轉殖株。將23株擬轉殖株進行GUS 報導基因組織化學染色，檢測其GUS 基因表現；結果顯示，僅部份擬轉殖株在根部有GUS基因表現，而抽取擬轉殖株總量 RNA以RT-PCR (Reverse Transcriptase PCR )分析檢測，有4株植物具有不同程度的GUS基因的RNA表現。未來可針對所得到之擬轉殖株利用南方轉漬法(Southern blotting)、北方轉漬法(Northern blotting)、西方轉漬法(Western blotting)等進行更進一步驗證分析。

關鍵字

球狀芽團 ； 農桿菌轉殖 ； 香蕉

並列摘要

An efficient protocol for the Agrobacterium tumefaciens-mediated transformation of banana (Musa acuminata. cv. ‘Pei Chiao’) is described. Cauliflower-like bud clumps (multiple bud clumps) were co-cultivated with A. tumefaciens EHA105 carrying a plasmid containing β-glucuronidase (GUS) reporter gene. In the absence of kanamycin, the bud clumps discs grew normally and produced shoots. In the presence of 30 mg/L kanamycin, the regeneration of the non-transform disc was inhibited. The growth and regeneration on both transformed and non-transformed discs were completely inhibited, and bleaching was observed under the concentration of 40 mg/L in the medium. In order to increase the regeneration of transformed plant, explants were cultured on growth medium without kanamycin after co-cultivation and putative plants were directly identified by Polymerase Chain Reaction (PCR) assay. Forty-eight out of 138 discs were found to have survived and produced shoots. Twenty-three plants of these were confirmed to contain the GUS transgene by PCR. The GUS expression in transgenic plants was only found in roots but not in leaves. Only four transgenic plants (PCR positive) found GUS expression at different RNA levels by RT-PCR (Reverse Transcriptase PCR) assay. The protocol is useful for the breeding of banana through genetic transformation.

並列關鍵字

multiple shoot clumps ； Agrobacterium-mediated transformation ； Musa. acuminata cv.

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