Comparing orchid transformation using agrobacterium tumefaciens and particle bombardment

The Wheeler Orchid Collection is home to some of the most endangered species of orchids in the world. This fantastic reservoir of endangered species has been enhanced and broadened by its function as a plant rescue station for the U.S. customs service. Unfortunately, this responsibility increases the risk of bringing orchids, which harbor contageous diseases, into the greenhouse where sap transmitted diseases such as the Tobacco Mosaic Virus (TMV), can run rampant. Although manipulation of orchid characteristics is typically done by classical plant breeding techniques, genetic engineering is emerging as a useful technique for the introduction of desirable traits into the orchid genome. Through the use of genetic engineering techniques it may be possible to mitigate the symptoms associated with this destructive virus. Virus resistance may be achieved through the expression of either the sense or antisense viral coat protein gene in orchid tissues if an efficient means of orchid transformation is developed. In this research two transformation protocols were examined for their ability to efficiently transform orchid tissue. The first transformation protocol explored utilized the native ability of Aq bacterium tumefaciens to incorporate DNA into host plants to achieve transformation. The second mechanism explored was particle bombardment transformation.Many strains of A. tumefaciens were employed using direct exposure of Cattleya_ orchid protocorm and callus tissue. Particle bombardment using DNA coated 0.5 um diameter tungsten particles and high pressure helium tank acceleration was employed. The particle bombardment procedure employed the pG35barB plasmid which confers herbicide resistance to the herbicide basta when integrated and expressed in plant tissues.GUS fluorescence assays and PCR analysis indicate that T-DNA is present in orchid tissues, while Southern blot analysis was unable to display that integration had occurred. Particle bombardment yielded herbicide resistant orchid tissues which have yet to be analyzed by Southern blot analysis to confirm integration due to limited tissue quantities.Department of BiologyThesis (M.S.