Abstract:
A major goal of our laboratory is to confer resistance specifically to the Odontoglossum Ringspot virus (ORSV; sometimes referred to as Tobacco Mosaic Virus Strain 0 (TMV-O)) in orchids, which may also provide cross-protection to other pathogens. The experimental design for the entire project is presented here along with the results of several preliminary experiments. Our approach involves RT-PCR amplification of the viral coat protein gene and digestion of the cDNA into oligonucleotides. These fragments will be cloned into a selectable vector (which confers herbicide resistance) in both sense and antisense orientations, coated with tungsten beads, and shot into orchid callus tissue using a makeshift biolistic gun. The callus will be selected for transformants by herbicide resistance, and analyzed to determine which oligonucleotide was received and the effect each oligonucleotide has on pathogen resistance. The viral coat protein gene was successfully amplified using RT-PCR with specific primers. This cDNA was cloned into the TA Cloning kit vector pCR 2.1, and was amplifiable by PCR using the same virus-specific primers. The oligos have been prepared using a DNase I digestion, verified by gel electrophoresis, and currently are ready to be ligated into the plasmid vector pG35barB. Callus tissue is currently being cultured, and once matures, will be used in transformation experiments. The remaining steps in this project will be completed for my masters project.
