ABSTRACT 1: Abrasive treatment and subsequent electroporation of the basidiomycete Pleurotus ostreatus in a hypertonic buffer was investigated as a potential method of transforming of intact hyphæ. Mycelia, which are not capable of being transformed via electroporation due to interference by the cell wall, were abraded in an attempt to mechanically degrade the cell wall prior to electroporation. An electroporation field strength of 12,500 V/cm for 500 μs to 1.25 ms was found to be optimal based upon mortality effects. A 32 μm carborundum abrasive was initially evaluated but was quickly found to be inappropriately large so a corundum abrasive with an average size of 300 nm was focused on. Vortexing as well as low and medium power ultrasonic agitation with the corundum abrasive were investigated for their potential to cause mechanical degradation of the cell walls. Vortexing and low power sonication were found to be ineffective at causing adequate degradation while medium power sonication was found to be both ineffective and super lethal. While the possibility of mechanical abrasion facilitating transformation via electroporation remains, it is unlikely that conventional methods of agitation will be effective. ABSTRACT 2: The dose response of the basidiomycetes Pleurotus ostreatus, Agrocybe aegerita and Cordyceps millitaris to the antibiotic hygromycin B was tested on two common and one in-house solid growth media. The three species were grown on Potato dextrose agar, malt extract agar and an agar containing maltose, yeast-extract, peptone and glucose with concentrations of hygromycin B from zero to 100 μg/ml for 11 to 14 days. Micrometer measurements were used to determine the growth rate of each species on each media. Significant differences in hygromycin B tolerance for each species between the three media types were evident (p-value < .0001 by ANOVA for all). Neither the media type nor the growth rate on hygromycin B free plates were useful predictors of effective hygromycin B doses so optimization should be performed on every strain and media type used for selection.
| Identifer | oai:union.ndltd.org:vcu.edu/oai:scholarscompass.vcu.edu:etd-1172 |
| Date | 01 January 2008 |
| Creators | Cordesman, Alexander |
| Publisher | VCU Scholars Compass |
| Source Sets | Virginia Commonwealth University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Theses and Dissertations |
| Rights | © The Author |
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