David Ritchie

Research Project

Honors thesis abstract:

Inefficiencies in crop production due to drought negatively affect tens of millions of people across the globe every year. Much of this decrease in fruit production is caused by plant stress response pathways. The ultimate goal of this research is to prove that this stress response pathway can be optimized by the introduction of viral genes coding for silencing RNA’s. For both regulatory and environmental reasons, it would not be acceptable to release a plant virus with the ability to infect wild type plants in the natural ecosystem. Therefore, the virus has been deconstructed, meaning that a part that is necessary for reproduction and expression has been removed. In this case, the Tomato Mottle Virus (ToMoV) is being used as a model virus. The Replication Initiator Protein, or REP Gene, has been removed. The REP gene has then been inserted into the plant genome via a plant genetic transformation, allowing the plant to complement the deconstructed viral vector. To avoid gene silencing mechanisms caused by insertion of multiple gene copies, the plants being tested must be homozygous for the REP gene. The original T0 transformation can insert the gene onto multiple places within the plant chromosomes. To ensure a plant is homozygous, seeds must be taken from a hemizygous plant, which has the transgene inserted as a single gene copy. From there, Mendelian genetics for a diploid plant hypothesizes that 25% of the T1 offspring will be homozygous for the REP gene. The research performed in this thesis involves the genetic segregation testing using polymerase chain reaction (PCR) to identify the presence of the REP gene transformed into tomato plants under the control of a drought- inducible promoter. As of the completion of this thesis, several lines of homozygous plants are in the final stages of verification for testing of the ability to support the proliferation of REP-deficient viral vectors containing plant protective genes.