橫山梨組培苗生長之研究

為了建立橫山梨(Pyrus pyrifolia Nakai cv. Hengshan)健康種苗完整的生產系統，本研究調查橫山梨莖頂組織培養植株生長，與一般實生苗繁殖後嫁接之苗木進行生長之比較，探討組培苗取代現行嫁接苗作為生產主要苗木之可行性。經由兩種苗木生長情形之比較發現，盆栽及田間試驗之組培苗的新梢長與橫徑、葉片數、葉片鮮重、葉面積等均比嫁接苗較長、較大且較多。組培苗之葉片數、鮮重可達嫁接苗的三倍以上，主枝橫徑及長度亦達嫁接苗之三倍以上。田間組培苗木之根圈半徑達1m，嫁接苗僅35cm。綜合本試驗之結果可知組培苗之幼年期生長較嫁接苗佳。The growth of two-year-old tissue culture propagated and grafted pear (Pyrus pyrifolia Nakai cv. Hensha) plants was investigated. The tissue culture propagated plants grew much faster than the grafted ones. The total fresh weights of leaf, shoot and root of tissue culture propagated plant all were more than threefold of those of grafted plant. The leaves of tissue culture propagated plant had higher chlorophyll content and last longer on the tree than those of grafted plant

Development of the techniques for eliminating the Phalaenopsis viruses found in Taiwan

蝴蝶蘭為台灣最重要的出口蘭花，本研究之目的為建立台灣常見蝴蝶蘭病毒的去病毒技術，策略以組培誘導新生擬原球體結合藥劑處理去病毒，在擬原球體增殖培養基中添加病毒抑制劑 ribavirin以重新再生無病毒苗株，測試不同濃度及條件，去病毒處理苗株將以ELISA、RT-PCR定期檢測病毒，持續篩選無病毒苗株及追蹤其生長及開花情形，本計畫之完成期能解決臺灣蝴蝶蘭生產所面臨之病毒感染問題並幫助建立可實際運用於產業界之健康種苗生產系統。Phalaenopsis spp. are one of the most important exporting orchids in Taiwan. The main objectives of this proposal are to develop viruses elimination techniques of Phalaenopsis orchids. The strategy was combine tissue culture and chemotherapy by treating PLBs with virustatic agent ‘ribavirin' in PLBs propagation medium to regenerate virus-free plantlets. Different ribavirin concentrations and conditions were tested. ELISA and RT-PCR were used for confirm the efficiency of viruses elimination and observation of virus-free plantlets phenotype and flowering. Through these efforts, we hope the techniques developed in this project would provide a very efficient and useful system for establishing healthy Phalaenopsis orchids seeding production system and resolve the virus infection problem in Phalaenopsis orchids production

Development and Application of Biotechonology on Plant Protection

一、 轉殖文心蘭抗蘭花病毒之抗性試驗：文心蘭為台灣僅次於蝴蝶蘭之重要經濟生產蘭花，但嚴重被惠蘭嵌紋病毒(Cymbidium mosaic virus, CymMV)及齒舌蘭輪點病毒(Odontoglossum ringspot virus, ORSV)所感染。本計畫之長期目標為轉殖病毒鞘蛋白基因，以培育抗病毒的轉殖文心蘭品系，以根本解決病毒感染文心蘭所造成之經濟損失。在之前的計畫中，我們已建立文心蘭組織培養再生系統以及轉殖和篩選之條件，並將鞘蛋白基因轉殖至文心蘭。因此本期計畫將分析轉殖文心蘭轉殖基因之表現及測試其抗病表現與穩定性，以期獲得具抗性之轉殖文心蘭植株。 二、 番茄青枯病皁期防禦基因之研究與應用：植物病害一直是全球性的嚴重問題。為有效地將長期廣效抗病之性質轉入農作物，探討並瞭解植物早期抗病防禦機制之整體相關資訊及重要參與基因極為重要。番茄青枯病(又稱為細菌性萎凋病)為一極為複雜之重要土壤傳播性病害，基於抗病育種之困難性與抗病策略需求迫切性等考量，我們選擇此病害為本計畫之研究目標，研究可能參與番茄抗青枯病的早期關鍵性基因。本年度計畫將針對一或兩個特定基因，深入了解這些基因在抗病及感病番茄植株遭受青枯病菌感染後的表現情況，同時也以植物基因轉殖策略進一步研究目標基因在抗病反應中所扮演的角色，並冀可培育出可能的新番茄品系。同時，我們也計畫分離並研究所選目標基因之啟動子。我們的長期目標是要將所獲得的重要訊息與資源，應用於番茄及其他相關作物的抗病育種工作上，並且期望對臺灣其他生物及農業之研究與發展將有所助益。 三、 β-1,3-葡萄聚醣水解酶與幾丁質水解酶在番茄抗真菌性病害上的應用：番茄為台灣重要蔬果，惟常受包括真菌在內的各種病源菌的侵害，本計畫將來自細菌的β-1,3-glucanase與來自愛玉子的chitinase基因轉殖入台中亞蔬四號的親本、Micor Tom及CL5915品系，寄望藉由β-1,3-glucanase與chitinase的額外表達，提高轉殖株對真菌病害的抵抗能力，強化番茄作物對真菌性病害的防禦機制，達到減少作物損失的目的。最終以提高番茄栽種的經濟效益，與降低農藥的使用量為目的。所得的經驗可作為將β-1,3-glucanase與chitinase基因轉殖入其它重要農作物的參考。強化番茄作物對真菌性病害的防禦機制，達到減少作物損失的目的。 四、 辣椒防禦相關基因之選殖：本研究擬利用基因選殖的方式選殖辣椒之PRs、NPR1與NIF等防禦相關基因，並利用定序比對、北方雜合分析法、南方雜合分析法確認所選殖序列為辣椒之防禦相關基因，以便將來利用遺傳工程技術開發出可持續表現不同的防禦相關基因且對真菌病害(特別是針對萎凋病、炭疽病或晚疫病)具有抗性之轉基因茄科作物。此外，並以「北方雜合分析法」、「RT-PCR」，或「real-time PCR」等方法，分析對炭疽病具不同抗、感性之辣椒品種於接種前後各防禦相關基因之表現情形，藉此研究抗病或感病反應發生時可能之分子途徑，以便未來利用這些分析結果，找出影響茄科作物抗上述病害之相關因子。 五、 軟腐病菌細菌素基因選殖在軟腐病防治法上之應用：Genomic DNA Libraries的做成與新規低分子量細菌素基因序列的在確認：我們首先從對台灣存在軟腐菌株具有最大抗生能力的3F-3菌株插入Tn5使其插入低分子量細菌素基因中而喪失低分子量細菌素基因的表現能力後，再以TAIL-PCR方法解析其周圍基因序列；在確認為軟腐菌的細菌素基因後，我們將腐菌菌體中分離出Genomic DNA，並且以限制酵素處理後將其載入質體DNA製成Genomic DNA Libraries，然後我們再利用我們已知的序列製成各種不同的probe，再利用這些probe進行南方菌落雜合法將含有我們已知序列片段的菌落分離出來，並且將其載體所含有的DNA片段的基因序列解析，以做最後的序列確認；並且利用這些DNA片段做為進行細菌素蛋白表現實驗時所需的DNA片段。細菌素基因序列的在確認我們將在本年度內確認軟腐病菌所生產低分子量細菌素基因的全長基因序列，以期在下一年度計畫中能與carocin S1基因一童被殖入其他宿主系統中(如蘭花等)表現確認，並且能被大量生產表現，以期能於生物防治上應用。 六、 由楊桃斑點病菌Pseudomonas syringae pv. averrhoi選殖位於CEL區域的effector基因並探討其應用於茄科及葫蘆科植物抗病之可行性：Pseudomonas syringae這類病原菌在寄主植物上常引起斑點或壞疽的病害，造成重大經濟損失，而其分子致病機制，主要受控於所謂的病原島嶼(Hrp Pai)上所轉譯的蛋白質。Hrp Pai主要分為3個區域，中心區域(core一、 轉殖文心蘭抗蘭花病毒之抗性試驗: Oncidium is the second most economically important orchid compared to Phalaenopsis in Taiwan. Infection of Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) usually results in the great loss of yield and quality of flowers. The long-term goal of this project is directed to develop the transgenic Oncidium orchid expressing coat protein (CP) gene, to confer the resistance to virus infection. In the previous project, we have established the regeneration system of Oncidium orchid and condition for transformation and selection of the transgenic plants were established. In this project, we will characterize the transgene expression at the molecular level and test the resistance of transgenic Oncidium orchid after challenging with CymMV or ORSV. The stable and resistant transgenic lines will be selected and cultured. 二、 番茄青枯病皁期防禦基因之研究與應用: Plant disease is a major factor that limits crop yield, storage, production and quality worldwide. To effectively integrate durable resistance to crop diseases, comprehensive information on genes involved in early plant defense response would be highly desirable and necessary. We choose tomato bacterial wilt (BW) as the target disease to be studied in this proposal as it is one of the most complex and serious crop diseases worldwide, and breeding for resistance for this disease is certainly important and urgent. We therefore set out to study responsive genes involved in a disease-defense or pathogenesis pathway(s) of this disease. In current proposal, we attempt to study the roles of one or two specific BW-responsive genes in the resistance to BW by taking molecular and transgenic approaches. In addition, we intend to isolate and study the gene promoters. Through this study, we hope to identify a group of agronomically important and useful genes (and promoters) that may benefit our future work on tomato or relevant crop improvement. 三、 β-1,3-葡萄聚醣水解酶與幾丁質水解酶在番茄抗真菌性病害上的應用: In this proposal, we attempt to introduce the β-1,3-glucanase gene of Paenibacillus and the chitinase gene of jelly fig into the tomato chromosome by means of Agrobacterium tumefaciens infection. We expect that the tolerance of the transgenic tomato to pathogenic fungi, such as Alternaria solani, Phytophthora infestans, and Fusarium oxysporum, will be enhanced thanks to the stronger hydrolytic activity of the transgenic tomato toward the invasive fungal cell wall. The results of this study may reduce the production loss of tomato, and the experience learned from this study is valuable for the following works of introducing β-1,3-glucanase and chitinase genes into other important produces in Taiwan. 四、 辣椒防禦相關基因之選殖: The goal of this research project is to isolate defense-related genes (PRs, NPR1 or NIF) from chillies pepper. The idenfication of the obtained defense-related genes is based on Southern hybrization, northern hybrization and DNA sequence analysis. The genes will be used for generation of resistant transgenic Solanaceae crops overexpressing the above-mentioned defense-related genes. These transgenic plants will be tested for resistance to some fungal pathogens: C. acutatum, Fusarium oxysporum, or Phytophthora parasitica. The differential gene expression in control and inoculated resistant and susceptible chillies pepper leaves will be analyzed to help elucidating the factors affecting disease resistance. Total RNA will be isolated and subjected to northern hybridization using DNA probes of the obtained defense-related genes. The gene expression profile may also be analyzed by RT-PCR or real-time PCR techniques if necessary. 五、 軟腐病菌細菌素基因選殖在軟腐病防治法上之應用: We insert the tran

Functional Study of the Nuclear Shuttle Protein of Tomato Leaf Curl New Delhi Begomovirus and Its Relationship to Mechanical Transmissibility and Host Range

本研究室在2007 年於田間表現嵌紋、葉片捲曲變形的罹病東方甜瓜上，分離到一個新德里番茄捲葉病毒分離株，並將其命名為新德里番茄捲葉病毒東方甜瓜分離株(Tomato leaf curl New Delhi virus -oriental melon isolate, ToLCNDV-OM)。ToLCNDV-OM是一個可以被機械接種傳播至多種瓜類寄主的新德里番茄捲葉病毒分離株，但是在機械接種的實驗中卻發現其無法感染番茄。序列分析結果顯示，ToLCNDV-OM NSP(nuclear shuttle protein) 基因的N 端序列，與同樣被報導指出可以被機械接種傳播回原寄主的馬鈴薯分離株 (ToLCNDV-Pot; NCBI accession number: AY158080) 較為相似，而與無法機械接種回原寄主的番茄分離株 (ToLCNDV-Svr; NCBI accession number:U15017)、胡瓜分離株 (ToLCNDV-Cuc; NCBI accession number: AB330080) 有明顯差異。過去的研究指出NSP 的功能與協助病毒基因體穿透核膜運送、協助病毒基因體在細胞內定位與運輸、病徵表現、病源性及病毒的系統性感染相關。依據本研究室先前的研究結果，我們提出了NSP 基因可能與病毒的寄主範圍及可機械接種傳播特性有關的假設性看法，為了進一步了解其間的關聯性，我們研擬了這次的研究計畫。首先，我們會進行ToLCNDV-OM NSP 基因N 端修飾，使其擁有番茄或胡瓜病毒分離株N 端多出來的序列，並分析重組病毒的病源性、感染後病徵發展，及其寄主範圍。另一方面，我們將與亞洲蔬菜中心合作，取得不同作物來源、且以機械接種及粉蝨接種方式分析過寄主範圍的ToLCNDV 分離株，分析這些病毒分離株的NSP 基因序列，並選殖其中表現出不同感染特性的分離株之NSP 基因，用以取代ToLCNDV-OM 分離株DNA-B 上的NSP 基因產生重組病毒株，同樣地進一步分析這些重組病毒株的病源性、感染後病徵發展，以及其寄主範圍。我們預期研究的結果將能闡明ToLCNDV 的NSP基因在病毒寄主範圍及機械接種特性上所扮演的角色。In 2007, a virus culture was isolated from a diseased oriental melon plant showingsymptoms of mosaic, leaf curl and puckering by our Lab and was designated as Tomato leafcurl New Delhi virus oriental melon isolate (ToLCNDV-OM). ToLCNDV-OM is amechanically transmissible isolate of ToLCNDV which can infect cucurbitaceous plants butcannot infect tomato plant with mechanical transmission. Sequence analysis showed that theN terminal sequence of the nuclear shuttle protein (NSP) of ToLCNDV-OM is similar inlength to the NSP of a mechanically transmissible potato isolate (ToLCNDV-Pot; NCBIaccession number: AY158080) of ToLCNDV but is different from those of a tomato(ToLCNDV-Svr; NCBI accession number: U15017) and a cucumber (ToLCNDV-Cuc; NCBIaccession number: AB330080) isolate. Previous, various studies have discussed on thefunction of NSP, viz. transport viral genomes across the nuclear membrane, intracellulartranslocation of viral genomes, cell-to-cell movement, symptom development, pathogenicity,and virus systemic infection. In our study, we hypothesize that the NSP may also relate to thehost range and the mechanical transmissibility of ToLCNDV. To investigate the relationshipbetween the NSP and the host range or the mechanical transmissibility, we design severalexperiments in this project. First, the N terminus NSP of ToLCNDV-OM will be modified asthose of ToLCNDV-Svr or ToLCNDV-Cuc, and the recombinant viruses will be analyzed forpathogenicity, symptom development and host range with mechanical or whiteflytransmission. On the other hand, we will cooperate with AVRDC for getting differentisolates of ToLCNDV and the host range of these ToLCNDV isolates will be confirmed withthe mechanical and whitefly transmission. The NSP genes from ToLCNDV isolates thatshowed differences on the host range and mechanical transmissibility will be cloned forgenerating recombinant viruses. These recombinant viruses will also be analyzed forpathogenicity, symptom development and host range. We expect that the results willelucidate the role of the ToLCNDV NSP gene on host range and mechanical transmissibility