Construction of a plasmid containing the bar gene for use in selection of biolistically transformed orchids : an honors thesis (HONRS 499)
The long term goal in our lab is to develop a method to genetically engineer/transform orchid tissue. A gene gun has been constructed to transform orchid tissues which resist more conventional transformation techniques such as Agrobacterium tumefaciens mediated DNA uptake. The plasmid pG35barB, encoding the bar gene which confirms selection based on PPT resistance, has been successfully introduced into orchid tissues with this gena gun, and approximately 1 % of the tissues exhibited herbicide resistance. In order to mitigate viral symptoms in transformed orchids, PCR primers have been designed to amplify the Tobacco Mosaic Virus O-Strain coat protein gene from infected orchid tissues. The amplified fragment can then be ligated into pG35barB and used to biolistically transform orchid tissues. Transformant will be selected by resistance to phosphinothricin due to liar expression. These transformants will be challenged with the TMV-0 virus to determine if viral symptoms are reduced as a result of TMV-O coat protein gene expression.