| Summary: | 碩士 === 國立中興大學 === 植物病理學系所 === 98 === 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.
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