Electronic Thesis and Dissertation Repository

Thesis Format



Master of Science




Szczyglowski, Krzysztof


Agriculture and Agri-Food Canada (AAFC-AAC)

2nd Supervisor

Grbic, Vojislava



The impact of intensive agriculture on the environment is immense. This is especially dire with regard to the natural nitrogen (N) cycle, where the human driven interference, primarily associated with industrial fertilization, has reached unsustainable levels. Unlike cereals, legumes, such as soybean, alfalfa and common bean, have the ability to use atmospheric nitrogen, which limits the need for industrial fertilization. A more wide-spread use of legumes could alleviate some of the negative impacts on the biogeochemical cycle while also providing a useful alternative to meat consumption, an important factor in sustainability. To reach this goal, further improvements of legume crops with regard to their nitrogen economy and yield are essential. In the model legume, Lotus japonicus, the HAR1 receptor kinase plays a central role in the plant nitrogen and phosphate nutrition by regulating beneficial symbioses and root system architecture. In this thesis study, I used the HAR1 locus as a paradigm for the development of a CRISPR/Cas-based toolkit, with the ultimate goal of generating a range of synthetic variation at agriculturally important traits. While genome modifications at the HAR1 locus are yet to be demonstrated, the toolbox required to perform these experiments was developed. It should facilitate rapid expansion on the repertoire of alleles available for accelerated breeding of new, high yielding legume varieties that are better attuned with the natural environment.

Summary for Lay Audience

There is an urgent need to improve crop yields while simultaneously reducing the use of nitrogen fertilizers. One way to address this problem is to turn to legumes, which can use atmospheric nitrogen. They achieve this by forming a symbiotic relationship with soil bacteria known as rhizobia. However, legumes are currently not able to produce as much grain as cereals do. Breeders can use natural variation to improve the yields of legumes, but the levels of natural variation that is available is relatively small. Using a modern gene editing technology, called CRISPR, it is possible to expand on the existing variation. This expansion allows crop breeders to identify beneficial variants much quicker than they could using traditional methods. Using CRISPR to expand on the variation in genes related to nitrogen uptake, for example, will help reduce the need for industrial nitrogen inputs. Improving legumes’ yields requires the knowledge of gene interactions which regulate nitrogen use. In this thesis study, gene editing tools were developed in order to expand on the natural variation that exist in the model legume, Lotus japonicus. A gene, called HAR1, which regulates the plant nitrogen economy was chosen as a paradigm. The gene editing toolbox, which can be applied to any agriculturally relevant plant function has been developed and testing these new tools is in progress. If successful, the knowledge gained by working in the model plant organism will be transferable to crop plants. It should facilitate accelerated breeding of new, high yielding legume varieties that are better attuned with the natural environment.