CHIA TET FATTINSTITUTE OF MOLECULAR & CELL BIOLOGY2020-06-172020-06-171992CHIA TET FATT (1992). GENETIC ENGINEERING OF CROSS-PROTECTION AGAINST CYMBIDIUM MOSAIC VIRUS AND THE TRANSFORMATION OF ORCHID. ScholarBank@NUS Repository.https://scholarbank.nus.edu.sg/handle/10635/170162Virus infection in orchids is a serious problem in the orchid industry. There is no cure once a plant is infected and traditional breeding of orchids resistant to viruses has been unsuccessful. Cymbidium mosaic virus (CyMV) and odontoglossum ringspot virus (ORSV) are the two most prevalent viruses infecting orchids. A sensitive method for detection of the two viruses was developed using tissue prints and viral cDNAs as hybridization probes. A survey comprising 2,622 plants randomly collected from various local farms was conducted. Among the five most important cultivated orchid genera in Singapore, namely: Aranda, Dendrobium, Makara, Oncidium and Vanda, 69.1% of the matured plants were found to be infected by CyMV, 23.2% doubly infected with CyMV and ORSV and only 7.7% were uninfected. Therefore, plants from the local farms are heavily infected by the two viruses, of which CyMV is the predominant culprit. Infected orchid plants have greatly reduced productivity and a shorter economic life-span. The coat protein (CP) gene of CyMV from a local isolate was cloned, sequenced and characterized. This gene was placed under the control of the cauliflower mosaic virus (CaMV) 35S promoter and the chimeric gene was then transferred into Nicotiana benthamiana, as transformation of orchid had not yet been established. Transgenic tobacco plants were used as a model system to investigate whether over-expression of the CyMV CP would confer protection against virus infection. It was found that transgenic plants over-expressing the CP were protected against CyMV infection. The protection was specific and was not dependent on the health of the plant. A reproducible system for the generation of transgenic orchid plants have been developed. Cells from orchid calli were transformed with the firefly luciferase gene using microprojectile bombardment. Transformed cell were selected by screening the bombarded celli for light emittance with a video imaging machine (VIM) after the addition of luciferin. Transgenic orchid plants regenerated from these selected calli appeared normal and were morphologically indistinguishable from the untransformed plants. Southern and Northern analyses of these plants revealed the integration of 35S-luciferase (chimeric) gene into the orchid genome and the expression of transcript of the correct size. The successful establishment of this transformation system opens the door for the introduction of novel genetic traits (eg. viral resistance) into orchidsThesis