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In vitro production of virus-free Phalaenopsis orchids through chemotherapy of ribavirin and multiplex RT-PCR detection of Capsicum chlorosis virus and Phalaenopsis chlorotic spot virus
|關鍵字:||Phalaenopsis orchid-infecting viruses|
|摘要:||蘭花是台灣重要之花卉作物之一，其繁殖多仰賴分生組織培養技術，因病毒易經由組織培養傳播而感染蘭花，故病毒病害成為蘭花生產過程中最重要的限制因子。本研究主要為建立蝴蝶蘭擬原球體(protocorm-like bodies, PLBs)增殖階段添加病毒抑制劑ribavirin之去病毒技術(virus elimination technique)，以及建立蝴蝶蘭新興病毒之檢測技術。將感染病毒之蝴蝶蘭病株以花梗誘導定芽及增殖，再以小芽葉片誘導產生擬原球體，測試單獨於液態培養基中添加 ribavirin 及之後持續於固態培養基進行 ribavirin 處理之去病毒效果。試驗結果發現 ribavirin 濃度愈高，去病毒率亦愈高，但擬原球體存活率卻越低且易出現變異；此外，擬原球體所感染之病毒種類與蝴蝶蘭品系皆會影響去病毒效率，ELISA 檢測結果顯示擬原球體於液態培養基處理 ribavirin 後持續再於固態培養基進行處理可以提升去病毒率。感染 Odontoglossum ringspot virus (ORSV) 之 Phal. Taisuco Grace 持續以 ribavirin 處理可以獲得 72.73~81.82% ELISA 檢測為陰性之擬原球體；感染 Cymbidium mosaic virus (CymMV) 之 Phal. aphrodite subsp. formosana 持續處理 ribavirin 可以獲得 69.23~80.00% ELISA 檢測為陰性之擬原球體，而感染 Cucumber mosaic virus (CMV) 之 Phal. aphrodite subsp. formosana 則可獲得 45.45~85.71% ELISA 檢測為陰性之擬原球體。以反轉錄聚合&;#37238;連鎖反應 (Reverse transcription-polymerase chain reaction; RT-PCR) 檢測單獨於液態培養基處理 ribavirin 後增殖之小苗，感染 ORSV 之 Phal. Taisuco Grace 於 50 ppm ribavirin 處理後可獲得 13.33% 無病毒苗株，25 ppm ribavirin 之處理可獲得 9.09% 無病毒苗株；感染 CymMV 之 Phal. aphrodite subsp. formosana 經 50 ppm ribavirin 處理後可獲得100%無病毒苗株，25ppm ribavirin之處理可獲得83.33%無病毒苗株，ELISA檢測約有5.91~16.67%之植株呈現偽陰性。感染ORSV之Phal. Taisuco Grace 經 ribavirin 處理後，擬原球體(50 ppm: 35.00%, 25 ppm: 33.33%) 以 ELISA 檢測為陰性之比例高於小苗(50 ppm: 25.00%, 25 ppm: 15.00%)，顯示病毒感染擬原球體後有潛伏期，在擬原球體分化為小苗過程中，病毒慢慢繁殖累積並且逐漸系統性的分佈到小苗全身組織中；而受CymMV感染之擬原球體以ELISA檢測為陰性之比例較感染ORSV者高，顯示CymMV在擬原球體階段之累積量低，之後隨著新生組織之增殖，CymMV似乎較ORSV不利於在擬原球體細胞間移行，所以獲得無病毒之擬原球體比例較高(61.90,53.33%)；不添加藥劑之對照組也可獲得無病毒苗株，顯示亦有少量新生組織(20~30%)可自然躲避病毒感染。本研究所建立之蝴蝶蘭擬原球體以ribavirin處理之去病毒策略可以成功去除CymMV、ORSV及CMV，此策略未來可應用於去除其他蝴蝶蘭病毒，如Capsicum chlorosis virus (CaCV-Ph)及Palaenopsis chlorotic spot virus (PhCSV)。除了研發蝴蝶蘭去病毒技術，另外亦建立可同時檢測蝴蝶蘭新興病毒CaCV-Ph與PhCSV之多重反轉錄聚合&;#37238;連鎖反應(multiplex RT-PCR)檢測系統，所使用之CaCV-Ph(FJJ2008-17/FJJ2008-18)及PhCSV(FJJ2008-31/FJJ2008-32)引子不論在單對引子或是多對引子RT-PCR中，皆可穩定增幅出預期條帶且具高度專一性，未來此技術可以運用在同時檢測多種蘭花病毒，提供快速且全面性之病毒篩檢，以維護我國蝴蝶蘭健康種苗生產系統之品質，並結合去病毒技術以建立更完善之蝴蝶蘭健康種苗體系。|
Orchid is an important economic ornamental in Taiwan and often infected by viruses via the propagation of tissue cultures. Therefore, virus diseases are the major limiting factor of orchid production. The objectives of this study are to develop 1) a technique to eliminate viruses from the Phalaenopsis protocorm-like bodies (PLBs) using ribavirin treatments and 2) a detection method for new Phalaenopsis-infecting viruses using reverse transcription-polymerase chain reaction (RT-PCR). PLBs, which were induced from the leaves of the virus-infected explants that regenerated from the buds of Phalaenopsis stalks, were treated first with ribavirin only in the liquid medium and then kept on the solid medium supplemented with or without ribavirin. The viruse elimination rate from PLBs was positively correlated to the concentrations of ribavirin. However, the PLBs treated with higher concentration of ribavirin resulted in lower survival ratio with unusual variations. The elimination rates varied based on the Phalaenopsis-infecting viruses and the Phalaenopsis cultivars. The ELISA results indicated that higher elimination rates were obtained when the virus-infected PLBs were cultured in the liquid medium with ribavirin and then maintained on solid medium supplemented with ribaririn. About 72.73~81.82% of PLBs of Phal. Taisuco Grace infected by Odontoglossum ringspot virus (ORSV) were virus-free after ribavirin treatment both in liquid and solid medium based on ELISA results. After the same treatments and ELISA tests, about 69.23~80.00% or 45.45~85.71% of the PLBs of Phal. aphrodite subsp. formosana with Cymbidium mosaic virus (CymMV) or Cucumber mosaic virus (CMV) were virus-free, respectively. Based on the reverse transcription-polymerase chain reaction (RT-PCR) results, 13.33% and 9.09% of the plantlets regenerating from ORSV-infected PLBs were ORSV free after being treated with ribavirin in the liquid medium only at 50 and 25 ppm, respectively, while 100% (treated at 50 ppm) and 83.33% (treated at 25 ppm) of those from CymMV-infected PLBs were CymMV free. After being confirmed with RT-PCR, it was clear that there were 5.91~16.67% of the plantlets that were falsely identified as negative in ELISA tests. In the study of ORSV elimination from Phal. Taisuco Grace, the ELISA negative ratio from the treated PLBs (35.00% when treated at 50 ppm and 33.33% at 25 ppm) was higher than that of plantlets (25.00% at 50 ppm and 15.00% at 25 ppm). This may indicate that the titer of ORSV in plantlets were higher than that in PLBs, because ORSV had a latent period after infection in the PLBs and multiplied and accumulated slowly in the regenerated plantlets during the regeneration process. The ELISA negative ratio in the PLBs from CymMV-infected Phal. aphrodite subsp. formosana was higher than that in ORSV-infected PLBs. It seemed to suggest that the titer of CymMV in the PLBs was lower. A possible explanation for having more CymMV-free PLBs than ORSV-free PLBs was that CymMV moved not as readily in the PLBs as ORSV. There were 20-30% virus-free plantlets regenerated from the virus-infected PLBs that received no ribavirin treatment indicating the escape of virus infection during regeneration. The ribavirin treatment strategies used in this study are useful for the elimination of CymMV, ORSV and CMV, and can be tried for the elimination of Capsicum chlorosis virus (CaCV-Ph) and Palaenopsis chlorotic spot virus (PhCSV) in the future. In addition to the study of the strategies for virus elimination, the multiplex RT-PCR of CaCV-Ph and PhCSV was developed for virus detection. The amplification was stable and specific when using the primers of CaCV-Ph (FJJ2008-17/FJJ2008-18) and PhCSV (FJJ2008-31/FJJ2008-32) in the simplex and multiplex RT-PCR. The detection method developed in this study could be useful for the detection of multiple orchid viruses. The combination of the virus elimination system and the multiplex RT-PCR detection protocol will definitely help improve the quality of the orchid production especially in the industry of producing healthy orchid seedlings in Taiwan.
|Appears in Collections:||植物病理學系|
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