Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/21318
標題: 木瓜微體繁殖與營養器官基因轉殖
Micropropagation and Gene Transformation of Papaya (Carica papaya L.) Using in vitro Vegetative Organ
作者: 余聰安
Yu, Tsong-An
關鍵字: rooting
發根
plantlet survival
aeration
antibiotics
somatic embryogenesis
Agrobacterium-mediated transformation
multiple shoot
bud primordium
植株存活率
通氣
抗生素
體胚形成
農桿菌媒介轉殖
叢生莖
側芽原體
出版社: 植物學系
摘要: 木瓜為熱帶及亞熱帶的重要經濟果樹,由於木瓜輪點病毒(Papaya ringspot virus, PRSV)的危害,使得木瓜產業遭受嚴重的打擊。在傳統抗病育種尚無法解決目前的產業困境的情況下,利用植物基因工程將外來基因導入植物體內而達到抗病的目的是一條可行的途徑。本研究的目的是要建立一套有效率的木瓜組織培養技術,在低成本的原則下,生產具高品質且事先知性別的優良種苗,以因應目前產業栽培所需。另一方面,以營養器官(組培瓶內的莖段)為材料,希望能建立一套穩定、快速的基因轉殖法。 本試驗發現,低濃度IBA濃度進行根原體誘導後,再移入含1/2MS的培養液的蛭石,在瓶蓋通氣的條件,進行根的生長發育階段,可有效提高木瓜微體繁殖的發根率及日後的存活率。 在農桿菌的基因轉殖過程中,於培養基中添加抗生素是必要的處理過程,其目的是為了抑制農桿菌苗生長和篩選轉殖細胞。卡苯尼西林(carbenicillin)和西弗士林(cefotaxime)為兩種最常加入培養基中,用來抑制農桿菌生長的抗生素。由結果得知,與農桿菌共同培養兩天後,先以無菌水清洗培殖體後,有促進抑菌的作用。在含有較高濃度的卡苯尼西林培養基(250~500 mg/l),即可增加癒合組織的鮮重。同樣濃度的西弗士林,卻明顯降低癒合組織的鮮重。體胚形成方面,於125 mg/l卡苯尼西林和250 mg/l西弗士林濃度之培養基中,有較高的體胚形成率,但就單一癒合組織形成體胚而言,在含125 mg/l卡苯尼西林濃度的培養基中,有較多的體胚數目形成。體胚的發芽狀況方面,在含較低濃度卡苯尼西林培養基(125~250 mg/l)中,異常體胚的形成率有明顯下降的現象。 康黴素(kanamycin)和建那黴素(geneticin)是常被用來作篩選nptII轉基因株系的抗生素。本研究中發現,培養基只要添加康黴素,對轉基因體胚的形成會有很強抑制作用,從癒合組織形成的體胚數也很少。低於25 mg/l建那黴素濃度下,雖然癒合組織的形成率有稍微減少的現象,但從癒合組織形成體胚數目和性狀與對照組比較,並無明顯差別。體胚的發芽狀況方面,抗生素並不影響轉基因體胚的轉化成莖的機率。 本試驗以農桿菌為載體,組織培養瓶內的莖段節間附近的側芽原體為轉殖材料,除了可以突破未成熟胚的缺點外,也可以有效縮短轉殖時間(只需3-4個月)。從1259個莖段(日陞品種)中經過篩選後,得到27個轉基因株系(17個PRSV CP轉基因株系,10個GUS轉基因株系)。泰國品種木瓜也由201莖段得到4個PRSV CP轉基因株系。擬轉基因植株的外來基因表現,經由GUS活性分析、PCR、南方轉漬法、北方轉漬法或西方轉漬法,證實為轉基因植物。
Papaya ringspot virus (PRSV) is a major limiting factor for papaya production in tropical and subtropical areas through the world. Resistant commercial cultivars are still not available by conventional breeding, crop improvement to solve disease problems of papaya can be enhanced by plant biotechnology. This study attempted to establish a low cost and fast micropropagation protocol to produce the papaya plantlets with high quality of cultivar traits and desired sex type. In addition, an easy and highly stable gene transfer method by Agrobacterium-mediated transformation targeting on lateral bulbils of multiple shoots in vitro to produce transgenic papaya was developed. In our results found that root induction in low-concentration IBA agar medium followed by root development in vermiculite containing half strength MS medium under aerated conditions results in efficient rooting of in vitro papaya shoots. Carbenicillin and cefotaxime, two antibiotics commonly used for excluding Agrobacterium tumefaciens during plant transformation. A washing step with sterilized distilled water two days after co-culture enhanced the bacteria -suppressing effects of antibiotics. Proliferation of Agrobacterium was completely suppressed in the medium containing 125 mgl-1 carbenicillin or cefotaxime. Callus fresh weight increase was apparently enhanced in the media with higher concentrations of carbenicillin (250-500 mgl-1), but was extremely inhibited in media with the same concentrations of cefotaxime. Higher percentages of somatic embryos were found in the medium with 125 mgl-1 carbenicillin or 250 mgl-1 cefotaxime; however larger numbers of somatic embryos from the individual callus were obtained in the medium with 125 mgl-1 carbenicillin, than in the medium with 250 mgl-1 cefotaxime. Percentages of abnormal somatic embryos were lower in the medium with lower concentrations of carbenicillin (125-250 mgl-1). Kanamycin and geneticin is frequently used for selection of nptII-transformed plants. In this investigation, it was found that the development of somatic embryos-forming callus of nptII-transformed tissue was strongly inhibited and very fewer numbers of somatic embryos occurred among media containing all the kanamycin concentrations. Less adverse effects during regeneration process of nptII-transformed tissue was noticed in the medium containing lower concentrations of geneticin (< 25 mgl-1) and the numbers and traits of somatic-forming callus were similar as the control treatment. Conversion rate of npt II transformed somatic embryos to shoots of transformed tissue was not significantly different among all kanamycin or geneticin treatments. In our study, a method of Agrobacterium-mediated transformation of papaya using multiple shoots in vitro was developed to circumvent these obstacles and greatly shorten trnasformation times (only 3-4 months). Twenty-seven transgenic lines (17 PRSV CP lines and 10 GUS lines) were obtained from 1259 shoot explants of cv. Sunrise and 4 PRSV CP transgenic lines was obtained from 201 explants of cv. Thailand. The expression of the gus gene and the CP gene of PRSV in the putative transgenic lines was verified by GUS activity assay, polymerase chain reaction (PCR), Southern blotting, northern blotting and western blotting assays.
URI: http://hdl.handle.net/11455/21318
Appears in Collections:生命科學系所

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