Mason Holum

Global trends have increased the needs for improved crops, and plant gene editing is one of the strongest tools for meeting this need. Gene targeting is an especially useful technique for crop enhancement. However, gene targeting can be quite difficult to achieve in plants. Currently several methods are used to improve gene editing in plants such as the use of developmental regulators on gene editing constructs, usage of excisable donors, and the usage of virus replicons to increase gene editing material within the cell. Specifically, geminiviruses have been used to enhance gene targeting in dicotyledonous plants, but this method has thus far failed to work in monocotyledonous plants. This is possibly due to a fitness penalty incurred by plant cells that contain replicating geminivirus which lead to a failure to regenerate gene targeting events. Furthermore, utilization of these methods requires complex molecular cloning in order to produce vectors that can be transformed into Agrobacterium tumefaciens (Agrobacterium) for plant infection. I have designed several complex vectors aimed at measuring the relative effects of these tools, to test a molecular cloning system, and to test a replicon switch method to potentially make a functioning geminivirus gene targeting system in Oryza sativa (rice). Molecular cloning was done using an advanced golden gate toolkit, and final constructs were confirmed using whole plasmid sequencing. Rice callus were infected with Agrobacterium containing the newly designed constructs. Heat shock was used in order to induce the replicon switch. These constructs were successfully cloned, but the replicon switch constructs failed to demonstrate transgenesis. Information pertaining to the effect of certain construct designs and gene editing frequencies was obtained, but gene targeting was unsuccessful even in the replicon switch treatment groups. This represents a failure of this design, and further experimentation is required to determine if a gemini viral gene targeting system could work in monocotyledonous plants.