Development of viral RNA isolation methods from CyMV and TMV-infected orchids and analysis of DNA isolation protocols for subsequent use in DNA fingerprinting of orchids : an honors thesis (HONRS 499)
With the presence of the Wheeler Orchid collection at Ball State, we have focused this research on developing methods to isolate nucleic acids from orchids for future studies involving mitigation of virus symptoms and DNA fingerprinting.In the first step, we isolated RNA from plants infected with the RNA viruses, CyMV and TMV-0. We hoped to distinguish the viral RNA from normal plant RNA. In order to isolate and genetically manipulate it, we would then reintroduce it into the plant to cause mitigation of the viral symptoms. However, there were problems with the isolation of RNA due to the presence of RNases and other degradative enzymes. Although typical procedures were used and precautions were taken to minimize enzymatic activity, most of the RNA isolated was either degraded or degraded within a few weeks of its isolation. Samples degrading more slowly than the others were obtained from using a 4M guanidium thiocyanate procedure. We isolated 100ug of RNA from 6.7 grams of healthy, mature orchid tissue. From mature, virused tissue, 200ug was isolated from 4.5 grams of a plant exhibiting an intermediate form of CyMV and 100ug from 5 grams of a severely virused plant.With the absence of strict taxonomic guidelines in defining species groups, it is important to develop alternate methods of naming and defining interspecies relationships. In a second group of experiments we attempted to develop a reliable method for DNA isolation yielding high quality DNA from small amounts of plant tissue. We investigated many different procedures utilizing different combinations of cell lysis, CsCl/EtBr density gradients, and phenol/chloroform extractions. Since orchids have numerous phenolic compounds in addition to tough and fibrous tissue and a high concentration of polysaccharides, many procedures failed to deal with all of these factors. The DNA isolated was either degraded or simply could notbe isolated. However, using a 4X CTAB procedure, we obtained between 50 ug and 100 ug per orchid leaf (approximately 4 to 6 grams per leaf). Electrophoresis of these samples showed that the use of early, meristematic tissue resulted in cleaner bands with less degradation. Mature tissue, though, provided as much DNA as early tissue from approximately similar amounts of starting tissues. Digestion of the DNA isolated by restriction endonuclease Eco RI is underway to determine if this relatively simple method will yield sufficient quantities of relatively clean nucleic acids to permit future DNA fingerprinting experiments.