Ro, Dae-KyunRoth, Susan Alexandra2024-07-092024-07-092024-07-04Roth, S. A. (2024). Developing base-editing for the improvement of agronomic traits in field peas (Pisum sativum) using glyphosate resistance as a target (Master's thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.https://hdl.handle.net/1880/11915210.11575/PRISM/46748The world’s population is estimated to surpass 10 billion by 2059, and alongside the increasing challenges posed by climate change, current global food systems are not sustainable. With these challenges has come an increased push towards more plant-based diets as plant agriculture has lower greenhouse gas emissions (GHGs) and land use requirements than animal agriculture. Field peas (Pisum sativum), an important Canadian crop, lend themselves well to this shift due to their high protein content, which can be used to replace animal-derived protein. Expanding the gene-editing toolkit for field peas will allow for more agronomic traits to be targeted and increase the ease with which field pea crops can be grown. Canonical CRISPR/Cas systems have revolutionized the field of crop improvement; however, their application has been limited to imprecise knockout mutations through non-homologous end joining (NHEJ) of double-stranded breaks (DSBs). Cytosine base-editing (CBE) is a new CRISPR-based technology that increases the precision of gene editing to the single base pair resolution in plants and opens the doors to producing gain of function mutations. This technology has yet to be used in peas and will increase the number of agronomic traits that can be targeted. This study used CBE to target 5- enolypyruvulshikimate-3-phosphate (EPSP) synthase in yellow field pea hairy roots to induce resistance to the herbicide glyphosate (commercially known as RoundUp). The base-editing window that predicts where editing is most likely to occur with CBEs was found to be expanded when using the PmCDA1 cytidine deaminase. Novel mutations R105C and P106F were obtained and characterized to show a complete knockout of EPSP synthase function with an R105C mutation and a 100-fold increase in glyphosate resistance with a P106F mutation. A P106S mutation, which has previously been characterized in other species, was also obtained and presented an 18-fold increase in glyphosate resistance, consistent with previous works.enUniversity of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission.Base-editingGlyphosateHerbicide resistanceEPSPSAgricultureGeneticsBiochemistrymaster thesis