Multi-faceted Approach for Engineering Novel Herbicide Resistance in Canola

Canola, Brassica napus, is a plant species of vital importance to both the Canadian economy and cultural history. For canola to remain the powerhouse commodity crop that it is, effective herbicide resistance technologies will need to be advanced in order to combat the growing threat of herbicide resistant weeds. In order to control weed populations with effective herbicides without causing harm to the planted crop, growers often utilize herbicide resistant cultivars. While resistant cultivars of canola already exist that are tolerant to highly effective herbicides such as Roundup and Liberty, overuse of few herbicides will only lead to greater likelihood of resistant weed populations developing. Cellulose Biosynthesis Inhibitors (CBIs) are known pre-emergent herbicides with no known field resistance but have currently had very limited application in commodity farming. These herbicides directly interfere with a plant’s ability to produce cellulose, necessary for their cell walls as a vital structural component. Current literature agrees that CBIs likely interfere directly with CELLULOSE SYNTHASE (CESA), an enzyme known to play a vital role in cellulose biosynthesis. If canola cultivars were to be developed with resistance, either GMO or non-GMO, to CBI herbicides such as isoxaben or flupoxam, it could represent a major economic boon for canola growers. In the present work, I utilize Ethyl methanesulfonate (EMS) mutagenized B. napus seed to screen for novel alleles conferring isoxaben resistance. By Whole Genome Sequencing (WGS) candidate resistant seedlings, I was able to find three potentially causal mutations in BnCESA3 that may independently confer isoxaben resistance. Transgenic lines of B. napus were also developed containing a synthetic version of BnCESA1 containing three previously characterized flupoxam-resistant alleles on a single transgene. Pot screening of these transgenic lines revealed moderate level of tolerance to flupoxam. Lastly, CRISPR/Cas9-deaminase lines of B. napus were also developed to base edit BnCESA1 and BnCESA3 to generate novel alleles that could confer tolerance to CBI herbicides