Construction and evaluation of transgenic

封面 誌謝 中文摘要 英文摘要 第一章 前人研究 一、木瓜輪點病害之發生 二、木瓜輪點病毒之特性 三、馬令鈴薯Y群病毒貸研究近況 四、病毒病害之防治 五、鞘蛋白轉基因植物之研究近況 六、木瓜之組織培養及PRSV鞘蛋白轉基因之研究 七、參考文獻 第二章、木瓜輪點病毒台灣分離株系鞘蛋白轉基因煙草之構築及其抗病評估 Abstract Introduction Materials and Methods Plant materials Construction ofPRSV cp gene In vitro expression ofthe cp gene Plant transformation DNA extraction andpolymerase chain reaction Western blotting analysis Inheritance analysis ofthe RI progeny ofthe PRSV cp transgenic plants Evaluation of the resistance of the transgenic plants Results Construction ofthe PRSV cp gene In vitro expression ofthe cp gene Expression ofthe PRSV cp gene in transgenic tobacco lines Inheritance and segregation ofthe cp gene in the RI progeny of the transgenic lines Evaluation of transgenic plant lines inoculated with TEV, PepMoV, and PVY Discussion References Figures and Tables 第三章、以金鋼砂在擬胚組織上造成傷口之最高效率農桿菌木瓜轉型作用 Abstract Introduction Materials and Methods Plant materials Construction ofPRSV cp gene Plant transformation and regeneration DNA extraction andpolymerase chain reaction Western blotting analysis Results Development of somatic embryos on the kanamycin medium Establishment of transgenic lines PCR detection Western blotting Discussion References Figures and Table Abstract Introduction Materials and Methods Plant Materials Construction ofPRSVcp gene Plant transformation and regeneration DNA extraction andpolymerase chain reaction Western blotting analysis Inoculation of transgenic papaya with PRSV Detection of virus replication in transgenic papaya by ELISA Detection of virus replication in transgenic papaya by bioassay Results Development of somatic embryos on the kanamycin medium Establishment of transgenic lines PCR detection Western blotting Inoculation of transgenic papaya with Taiwan strain Inoculation of transgenic papaya with Hawaii strain Inoculation of transgenic papaya with Thailand strain Discussion References Figues and Table

The Current Status of Geminiviruses Occurred on Passion Fruit in Taiwan

在台灣感染百香果的病毒計有胡瓜嵌紋病毒 (Cucumber mosaic virus, CMV)、東亞百香果病毒(East Asian passiflora virus, EAPV)、百香果斑駁病毒(Passion fruit mottle virus, PaMV)、百香果漣葉病毒(Passion fruit crinkle virus, PCV) 4 種。2011 年3 月在埔里地區採集到黃綠不均且葉片變形百香果樣本，利用ELISA 檢測未受到上述4 種病毒感染，利用滾輪法(rolling circle amplification, RCA) 卻測到疑似雙生病毒感染。RCA 產物以BamHI 處理，得到約3.0 Kb 之產物，選殖至載體，選殖株以EcoRI 酵素處理確認時出現2 類不同切位DNA 片段。定序分析後分別有2745 與2732 個核苷酸，與GenBank 登錄之序列比對結果，2745 個核苷酸者(Accession No. KC161185) 與一品紅捲葉病毒(Euphorbia leaf curl virus, EuLCV) DNA-A 有98.5%的相同度，2732 個核苷酸者(Accession No. KC161184) 與番木瓜捲葉廣東病毒(Papaya leaf curl virus, PaLCuGDV) DNA-A 有91.7%的相同度。根據2 病毒序列分析結果設計PCR 引子對，利用電泳分析調查2 種病毒發生情形。此2 病毒應已普遍發生於百香果台農1 號多時，於宜蘭、花蓮、苗栗及南投地區等百香果栽培區均有發現，因為台農1 號百香果需經由嫁接苗繁殖，因此該2 種病毒隨著嫁接苗普遍發生。病毒感染後造成百香果葉片斑駁、葉面不平整，各百香果園觀察到的病徵嚴重程度不一，但進入春夏季之後氣溫回升，葉片變形情況趨於輕微至觀察不到病徵，百香果生長回復正常，而造成忽略。 The main variety of passion fruit (Passiflora edulis x Passiflora edulis f. flavicarpa) cultivated in Taiwan is ‘Tainung No. 1 ’which is a hybrid and propagated only by grafting. In the spring of 2011, plants with severe systemic mosaic and malformation on leaves were found in some orchards located in Puli, Nantou in the middle of Taiwan. However, after growing up to three months, most of these diseased plants became symptomless when the weather became warmer. In March of 2011, 2 leaf samples exhibiting mosaic and 3 samples showing malformation were collectec and tested by DAS-ELISA; none were positively reacted with antibodies against Cucumber mosaic virus (CMV), East Asian passiflora virus (EAPV), Passion fruit mottle virus (PaMV) or Passion fruit crinkle virus (PCV) that have been occurred in Taiwan. The rolling circle amplification (RCA) with hexamer primers was adopted to analyze the potential begomoviruses which were prevalent on the other crops in Taiwan. The RCA amplified products were digested with BamHI and separated on 1.2% agarose by gel electrophoresis. A fragment, about 3 kb, was purified from each gel and cloned into the respective site of pBluescript SK(-) individually. Clones were screened by EcoRI digestion and two types of restriction fragement length patterns were found among them. One type clone containing 2745nucleotides (Accession No. KC161185) with 98.5% identity to Euphorbia leaf curl virus (EuLCV) and the other type clone containing 2732 nucleotide (Accession No. KC161184) with 91.7 identity to Papaya leaf curl Guangdong virus (PaLCuGDV) were revealed by nucleotide comparisons of their DNA-A in the GenBank. Accordingly, we confirmed the existence of passiflora isolates of EuLCV and PaLCuGDV. In a brief survey, allpassion fruit leaf samples were detected EuLCV and/or PaLCuGDV infectionTo our knowledge, this is the first report of begomoviruses associated passion fruit in Taiwan and in the Asia

(62(4):360-371) Identification and Occurrence of Pepper mottle virus Isolated from Peppers in Taiwan

甜椒（Capsicum annuum）葉片出現嵌紋及扭曲變形疑似病毒感染的病徵，經直接法ELISA檢測，這些樣本可與辣椒葉脈斑駁病毒（Chilli veinal mottle viru；ChiVMV）的抗體反應，且汁液接種可感染Nicotiana benthamiana，但N. benthamiana中的病毒卻不與ChiVMV的抗體反應，也不感染紅藜、奎藜。經汁液系列稀釋後接種，可分離得一病毒純系，將其接種保存於N. benthamiana上，經寄主範圍試驗，結果只感染茄科植物，回接甜椒即在葉片出現嵌紋及扭曲變形病徵。抽取接種的N. benthamiana葉片之總量RNA，利用potyvirus的簡併式引子（HRP5/dT）進行反轉錄聚合酵素連鎖反應，可增幅得到1.3 kb的DNA片段。進行選殖及定序分析並與GenBank之核苷酸序列比對，與番椒斑駁病毒（Pepper mottle virus；PepMoV）VB分離株的鞘蛋白基因（Access No. AB126033）相同度達100%，因此鑑定本病毒是一株PepMoV。純化上述接種繁殖於N. benthamiana 的病毒分離株（PepMoV-TW1）製備多元抗血清，利用其抗體以ELISA進行病毒偵測，自2007至2011年採自台灣各地969個甜椒樣本中有20%可測到PepMoV，且多數與ChiVMV或番椒葉脈斑駁病毒（Pepper veinal mottle virus；PVMV）複合感染。因PepMoV與馬鈴薯病毒Y（Potato virus Y；PVY）、PVMV及ChiVMV等病毒有血清關係且彼此產生交互反應，使以往ELISA的結果之判別及病毒種類鑑定受到干擾，且甜椒中病毒複合感染的情況極為普遍，因此推測PepMoV可能之前就已存在於台灣。Bell pepper (Capsicum annuum L.) plants exhibiting systemic leaf mosaic and malformation were collected. By DAS-ELISA, these samples were positively reacted with antibody against Chilli veinal mottle virus (ChiVMV). After mechanical inoculation with sap extracted from these samples, unexpected mosaic symptoms were developed on upper leaves of Nicotiana benthamiana. The virus infecting N. benthamiana was non-reacted with antibody to ChiVMV when tested by DAS-ELISA. And the virus was non-infectious to Chenopodium amaranticolor and Chenopodium quinoa tested by sap-inoculation. Pure virus isolates were obtained from infected plants of N. benthamiana by mechanical inoculation with serially diluted saps of infected plants. Not only pepper plants were developed the mosaic and leaf distortion symptoms, but also some species of Solanaceae plants were found to be infected by this virus in the host range test. The total RNA extracted from leaves of inoculated N. benthamiana was subjected to RT-PCR. The potyvirus degenerate primers (Hrp5/Pot1) were used to amplify a RT-PCR product of 1.3 kb DNA fragment, which was then cloned and sequenced. Results of nucleotide sequencing revealed that the CP gene of the isolate was 100% identical to Pepper mottle virus (PepMoV)-VB (Access No. AB126033) and was concluded as an isolate of PepMoV. Polyclonal antiserum against PepMoV Taiwan isolate (PepMoV-TW1) was prepared by immunizing rabbit with purified virus that has been propagated in N. benthamiana. Use of its antibody in ELISA for virus detection, a total of 969 sweet pepper samples were collected from different areas of Taiwan and tested in 2007-2011. About 20% of all the survey samples were identified as PepMoV infection and most of them were complexly infected with ChiVMV, or Pepper veinal mottle virus (PVMV). It is speculated that PepMoV might already exist in Taiwan earlier, but it was ignored because the closely serological relationships among PepMoV, Potato virus Y (PVY), PVMV and ChiVMV could form cross-reactions with each other and that interfered with the judgment of ELISA. Besides, the virus complex infections were very common in plants of sweet pepper

Diagnostic Methods for Plant Virus Diseases

植物病毒曾造成作物的重大損失，與其他植物病原不同，病毒病害無法以農藥加以防治，目前以抗病育種、健康種苗和防治媒介昆蟲為重要病害管理方法。觀察病徵加以分類判斷是何種病害是病害診斷的第一步，但大部分的病毒病害不易從病徵判斷感染的病毒種類，需要進行檢測，才能診斷係何種病毒感染造成病害。生物檢定法 (bioassay) 是最早應用作為植物病毒診斷與鑑定之技術為，接著有利用電子顯微鏡 (electron microscopy) 觀察感病植物體內的病毒顆粒為直接之鑑定。50 年代末期科學家開始利用抗血清檢定法 (serological methods) 進行病毒病的鑑定與診斷，至今仍為病毒診斷與鑑定的主要技術。血清檢定法的應用包含免疫擴散法 ( immunodiffusion )、免疫漬染法 (immunoblotting)、組織壓片法 (tissue printing)、螢光免疫法 (immunofluorescence)、酵素連結免疫法(ELISA)、西方漬 染法 (Western blotting) 及免疫試紙 (immunostrip) 等。除了利用抗體所進行之診斷方法外，近年也盛行核酸鑑定法 (nucleic acids methods)，例如核酸發生雜合法 (hybribization)、聚合酵素聯鎖反應 (polymerase chain reaction , PCR)、即時-聚合酵素聯鎖反應 (real time-PCR) 、生物晶片 (biochip) 與恆溫環狀擴增反應(loop-mediated isothermal amplification, LAMP)等。 Plant viruses cause major losses to several agricultural and horticultural crops around the world. Unlike bacterium and fungus pathogens, there are no chemical methods available to control viruses and, consequently, the current measures rely on resistant breeding, healthy seedlings and control insect vectors to manage the viral diseases. Diagnostic methods for detection and identification of viruses are not reliable based only by symptomatology since many factors may affect the appearance of visible symptoms so that it is necessary to apply some more sensitive techniques to diagnosis or identify viral diseases. The first technology used to diagnose and identify plant viruses is bioassay, followed by electron microscopy. In the late 50's, scientists began to use serological methods for the identification and diagnosis of viral disease, still were the major technology for virus detection. In addition to the diagnostic methods performed by antibodies, nucleic acids methods have also been prevalent in recent years. It is expected that more detection methods will be developed in the future so that virus diagnosis and identification is no longer a complicated task. These advances will help to ensure that plant quarantine, disease-resistant breeding and disease management are implemented accurately and essential for sustainable agricultural development

(64(2):135-144)Development of Primers for Detection of Whiteflies Carrying Leaf Curl Disease:

以銀葉粉蝨 (Bemisia argentifolii Bellows & Perring) 為媒介所傳播的南瓜捲葉病毒 (Squash leaf curl Philippines virus, SLCPHV)，係近年來在台灣中南部瓜類上常見的病毒病害，造成農民嚴重的損失。除了此病毒外，銀葉粉蝨尚可傳播其他的豆類金黃嵌紋病毒屬 (Begomovirus) 的病毒。因此，必須開發比原有簡併式CP1up/dw 更具專一性的引子對，以提高在田間進行南瓜捲葉病毒病害監測及銀葉粉蝨體內帶毒率偵測的準確度。本試驗利用滾環式擴增法擴增SLCPHV 及3 種田間常見的豆類金黃嵌紋病毒屬病毒番茄捲葉新德里病毒 (Tomato leaf curl New Delhi virus, ToLCNDV)、藿香薊葉脈黃化病毒 (Ageratum yellow vein virus) 及木瓜捲葉廣東病毒 (Papaya leaf curl Guangdong virus)，以Bam HI 進行酶切，經選殖、定序、比對後，設計出可針對SLCPHV 的專一性引子對SLCVup/dw，並在田間常見的8 種瓜類作物與銀葉粉蝨蟲體上進行測試。結果顯示，使用專一性引子對SLCVup/dw 只會偵測到感染瓜類的SLCPHV 或ToLCNDV，以其檢測結果估算田間帶毒粉蝨之族群密度逐漸增加，與洋香瓜罹病率逐漸升高之趨勢相符。相較於簡併式引子對，顯示此專一性引子對具有更好的靈敏度及準確度，未來可應用於田間病害偵測及帶毒銀葉粉蝨之監測。 A viral disease caused by Squash leaf curl Philippines virus (SLCPHV), a begomoviurs transmitted by Bemisia argentifolii Bellows & Perring, is one of the most popular and serious diseases in muskmelon cultivation during the last decade in the central and southern Taiwan. In addition to SLCPHV, there are many begomoviruses transmitted by whitefly in Taiwan. Polymerrase chain reaction (PCR) assay with degenerated primers has been widely used for general detection of begomoviruses. The degenerated primer pairs CP1up/dw were designed based on the capsid protein gene of recorded begomoviruses in Taiwan and could amplify the viral DNA fragment of ca. 500 bp. The other degenerated primer pairs SLCVup/dw were designed to specifically target capsid protein gene of SLCPHV and Tomato leaf curl New Dali virus, i.e., two causing agents of squash leaf curl disease in Taiwan, and could amplify the viral DNA fragment of ca. 250 bp. In field survey of whitefly infection rate, results indicated that detection with CP1up/dw resulted in higher positive ratio than SLCVup/dw. Viruliferous whieflies could be detected with primer pairs CP1up/dw rather than with SLCVup/dw. Results showed that whitefly carried other begomovirus instead of SLCPHV. Comparison with CP1up/dw, the primer pairs SLCVup/dw possessed higher sensitivity and accuracy and therefore, they were more suitable for inspection of squash leaf curl disease and infection rate of B. argentifolii

Evaluation of cryotherapy for its industrial application in virus elimination of several important vegetative propagation crops in Taiwan

超低溫冷凍技術主要應用於種原保存，去病毒為附加效果。目前去病毒技術主流仍為生長點組織培養，但在不易得到無病毒生長點培植體且已建立超低溫冷凍保存系統的作物，可評估使用超低溫冷凍去病毒技術（冷療處理）之可行性。比較生長點組織培養與超低溫冷凍技術對去除病毒的效果，過去的研究案例顯示，以生長點培養的再生率較高，惟去病毒率則以超低溫冷凍處理較高，不同品種間之再生率與去病毒率差異極大。本報告嘗試以經濟效益評估蝴蝶蘭、文心蘭、百香果、紅龍果、馬鈴薯、甘藷、草莓、柑橘及綠竹等9種作物應用超低溫冷凍技術去除病毒的可行性。其中草莓及柑桔已有應用超低溫冷凍技術之報告，在有新病毒發生且無法以生長點組織培養去病毒之情況下，建議可嘗試超低溫冷凍去病毒技術之開發應用；蘭花種類繁多，目前農政單位並無提供健康種苗，業者通常以確認無病毒之植株進行分生苗繁殖，農業試驗所（農試所）亦利用擬原球體 （Protocorm-Like Body, PLB）加上薄層培養技術建立蝴蝶蘭去病毒技術；馬鈴薯及甘藷生長點培養去病毒效率較超低溫冷凍技術高；百香果、紅龍果及綠竹則尚未建立超低溫冷凍相關技術。總結評估推論目前蝴蝶蘭、文心蘭、百香果、紅龍果、馬鈴薯、甘藷及綠竹等7種作物在臺灣較無發展超低溫冷凍技術去除病毒之急迫性與必要性。 Cryopreservation is a technique commonly used to preserve plant tissues in liquid nitrogen. This method is one of the most important tools to preserve germplasms. When shoot tips are used as explants, cryopreservation may have extra functions in virus elimination. This phenomenon is known as cryotherapy. Studies have shown that using shoot-tip or meristem culture could improve the efficacy of regeneration but fails to completely eliminate viruses occasionally. Cryotherapy could greatly improve the efficiency of virus elimination in some crops. Plant cells in the top layers of apical dome and primordia of two youngest leaves are able to survive after cryotherapy. However, the regeneration rate and virus elimination vary considerably even among different varieties of the same crop. This report evaluates the economic feasibility of cryotherapy applying to eliminate viruses in nine vegetatively propagated crops including Phalaenopsis, Oncidium, passion fruit, pitaya, potato, sweet potato, strawberry, citrus and bamboo in Taiwan. Cryopreservation has been reported in strawberry and citrus. To deal with newly invasive viruses which can't be eliminated by meristem culture, cryotherapy would be a better alternative. Unlike strawberry and citrus, cryotherapy may not be economically practical in potato and sweet potato because viruses in these two crops can be effectively removed by meristem culture. Cryopreservation has never been developed for passion fruit, pitaya and bamboo and it remains unclear whether or not the method will be effective to eliminate viruses in these three crops. There is an urgent need to eliminate viruses using cryotherapy in orchids. When orchid plants are infected by viruses, meristem culture coupling with chemotherapy and/or thermotherapy is often used to generate virus-free plants. In addition, researchers in the Taiwan Agricultural Research Institute have recently developed a new technique using thin sections from protocorm-like body (PLB) and ribavirin treatment to eliminate viruses in Phalaenopsis and Oncidium. Through our assessments, we conclude that there is no urgent need to develop cryotherapy to eliminate viruses in Phalaenopsis, Oncidium, potato, sweet potato, passion fruit, pitaya and bamboo in Taiwan

Ti二位元載體媒介的木瓜轉型作用

本瓜(Carica papaya L.)是臺灣最重要的熱帶果園之一, 其產量, 品質都深受本瓜輪 點病毒(papaya ring spot virus)病害影響, 交互保護為防治此病害較有效的方法, 以病毒鞘蛋白基因產並入植物染色體, 使植物產生鞘蛋白面有防止病毒侵入感染的現 象稱為遣傳工程的交互保護, 此一現象已在多種病毒獲得證實, 外來基因曾利用具有 腫瘤基因的SEV 載體系統導入木瓜細胞, 本實驗則利用不具腫瘤基因的二位元載體系 統嘗試將抗kanamycin 基因及GUS(β-glucuronidase) 基因導入木瓜細胞, 另外亦構 築一轉型 PRV CP 基因的載體: 帶有抗kanamycin 及GUS 基因的pBI 121 在E. coli 複殖後經 triparental mating 轉移到 Agrobacterium tumefaciens。本瓜葉柄移殖體和 A. tumefaciens LBA 4404 /pBI 121共同培養兩天後, 移到含kanamycin 的愈合組織生長培養基篩選轉型細胞, 利用 GUS之DNA 為探針, 可偵測到並入轉型細胞染色體之GUS 基因及轉錄出之mRNA以 GUS 之抗血清可偵測到轉型翦台組織中之GUS 蛋白, 另外, 以螢光基因分析法可偵測 到GUS 的酵素活性。 構築轉型PRV CP基因的載體時, 必須突變產生一個含有起始碼(initiation codon)的 監識 切位, 首先將GUS 基因的 SmaI/SacI片段轉殖到pGEM質體含有pGEM之E. coli 受到協助噬菌體(helper phege) R408 感染時, pGEM以單股型態受到噬菌體鞘蛋白包 被, 穿過細胞質游離在培養液中, 單股DNA 抽出後作為生體外突變

Resistance Evaluation of the Transgenic Papaya Lines Expressing the Coat Protein Gene of Papaya Ringspot Virus

為了利用植物遺傳工程方式將木瓜輪點病毒(PRV)鞘蛋白(coat protein, CP)基因轉移至木瓜以產生抗病性狀，木瓜轉型作用之進行係以金剛砂製造傷口，經由Agrobacterium tumcfaciens媒介，將GUS或PRV 5’前導序列表現的PRV 鞘蛋白基因導入木瓜幼胚分化組織中，並再生成轉基因植株。經由PCR 及西方漬染法分析，確定CP 基因存在轉型木瓜株系中，並可表現產生PRV的鞘蛋白，計獲得包含GUS 前導序列的10個株系及PRV 5’前導序列的3 個株系。先以組培養技術繁殖其R0植株，經由發根、馴化，使其在溫室生長兩個月後，進行挑戰接種，以測定其抗病性狀。實驗中以來自臺灣、夏威夷及泰國三個不同來源的PRV YK、HA 及TH 系統供試“接種後在溫室中觀察其結果，不表現病徵之植株再以酵素連結免疫呈色法（E LlsA ）及生物分析偵測是否有病毒增殖。抗病篩選得到四個抗同源PRV YK的株系：GCP 16-0、GCP 17-0、GCP 17- 1及GCP 17-3，在此四株系中，GCP 17-1及GCP 17-3對異源的PRV HA 及TH 系統仍有高度抗性，GCP 16-0則對異源病毒只有中度抗性，而GCP 17-0的抗病能力為系統專一性的抗病現象，只對同源病毒系統有抗病力。目前此四抗病毒株系仍繼續於隔離溫室中培養，其生長情形與一般木瓜一致。但此四株系均為雌株，以日昇之花粉授粉，果實生長情形與外觀形狀也都正常。目前GCP 17-1之R1 種子已發芽，並於溫室中栽培，再次進行抗病檢定中。由於GCP 17-1及GCP 17-3對不同來源的三個病毒系統都有極高的抗病力，其未來可望不受地區限制而能廣泛應用以防治木瓜輪點病毒。 The coat protein (CP) of a local mosaic strain of papaya ringspot vrius (PRV YK) was previously constructed in the Ti-vector for generation of transgenic papaya resistant to PRV infection. The CP gene with a GUS or the PRV leader sequence was transferred to embryogenic tissues derived from immature embryos of papaya via Agrobacterium -mediated transformation assisted by carborundumwounding treatment. The presence and the expression of the transgene in the putative transgenic lines regenerated were confirmed by PCR detection and Southern blotting. R0 plants of ten CP-transgenic lines with the GUS leader and three lines with the virus leader were established by micropropagatio n The plants were challenged with three different strains of PRV originated from Taiwan (YK), Hawaii (HA), and Thailand (TH). The infection of PRV was determined by symptom observation, ELISA and bioassay. Four lines of GCP 16-0, 17-0, 17-1, and 17-3 were found to be resistant to the homologous YK strain. Among them, GCP 17-1 and 17- 3 were highly resistant to the heterologous HA and TH strains, GCP 16-0 were moderately resistant to the heterologous strain HA and GCP 17-0 was specifically resistant only to the homologous YK strain. All four lines were female and their horticultural properties were similar to the untransformed papaya. The R1 plants of GCP 17-1 crossed with Sunrise papaya have been obtained and their resistance to PRV infection are currently evaluated under greenhouse conditions. The broad resistance in GCP 17-1 and 17-3 indicated that they have a great potential to be applied in different areas for control of the notorious PRV