Page 202 - Plant Canada 2024 Proceeding

PLANT CANADA 2024

development. Thus, modulating the clock could lead to significant improvements in crop performance. In
this study, we employed two innovative gene editing methods- “Diploid editing” and “Haploid induction
(HI)-edit” to edit two clock genes; Clock-1 and Clock-2. The diploid method involves gene editing via
wheat transformation and produces transgenic edited lines, while HI-edit involves maize transformation
and the subsequent use of transgenic maize lines to pollinate wheat plants and thus, produces
transgene-free edited lines. We have successfully edited wheat plants using both methods and a
comparison between the two approaches shows that diploid edit has higher editing efficiency (42-44%)
than HI-edit (4.29%). While the HI-edit has lower efficiency, it has a shorter time to develop a fixed-edited
line. Evaluation of these mutant lines for both genes is ongoing. Thus, this study offers valuable insights
into wheat genome modification and serves as a significant resource for future wheat genome research.

*[O196] SPEED EDITING: HIGH THROUGHPUT GENE EDITING USING CRISPR/CAS9 SYSTEM IN
BRASSICA NAPUS. Rajbir Kaur , Mohamed Samir Youssef , Robert Duncan , and Harmeet Singh
1
1,2
1,2
1 1
Chawla . Department of Plant Science, University of Manitoba, 66Dafoe Road, Winnipeg, MB, Canada,
2
R3T2N2; These authors contributed equally to this work
Correspondence to: Harmeet.Chawla@umanitoba.ca

Brassica napus is an allotetraploid crop species with 2n=4x=38 developed through interspecific
hybridization between B. rapa and B. oleracea and following the events of chromosomal doubling and
genomic rearrangements. Even though canola/rapeseed ranks as the second most significant source of
vegetable oil globally, it still encounters gaps between achieved and potential yield due to various biotic
and abiotic factors. Addressing these challenges necessitates precise and innovative approaches to plant
breeding and genetics. Gene editing, particularly the CRISPR/Cas9 system, offers an innovative solution
to these limitations by enabling targeted modifications at specific genomic locations. By leveraging
CRISPR/Cas9 technology, it is possible to enhance the resilience of canola to abiotic and biotic stress.
Brassica napus has a complex polyploid genome that often requires editing of multiple homoeologous
genes, thereby complicating the process of gene editing for this species. For the concurrent editing of
multiple genes with CRISPR/Cas9, separate promoters must be employed to drive the expression of
Cas9 and each distinct single-guide RNA (sgRNA). This can result in overly large constructs, complicating
the vector's delivery into the host plant. To streamline this process, the current study adopts a more
efficient strategy where multiple sgRNAs are engineered into a single transcript, expressed under a single
promoter. Moreover, addressing the laborious and low-efficiency tissue culturing typically associated with
CRISPR/Cas9, this study proposes a direct spray-based transformation method that circumvents these
traditional bottlenecks. By enhancing the transformation and regeneration processes, this approach aims
to reduce soma clonal variation and accelerate the development of genetically edited B. napus.
Shattering tolerance, which is extensively documented in the scientific literature, will serve as the initial
trait to assess the efficacy of this approach. Using our high throughput CRISPR/Cas9 method, we
targeted three genes SHP, IND, and ALC, described to confer shattering tolerance in oilseed rape. We
have successfully generated Cas9-positive T1 plants and are in the process of phenotypic and genotypic
characterization of the CRISPR mutants.

[O197] FUNCTIONAL VALIDATION OF A CANDIDATE GENE CONTROLLING SOYBEAN ROOT
SYSTEM ARCHITECTURE BY CRISPR-CAS9 TECHNOLOGY. Benjamin Karikari , Waldiodio Seck ,
1,2
1,2
1,2
Davoud Torkamaneh , and François Belzile . Département de phytologie, Université Laval, Québec,
1,2 1
QC, Canada, G1V 0A6; and Institut de biologie intégrative et des systèmes (IBIS), Université Laval,
2
Québec, QC, Canada, G1V 0A6
Correspondence to: francois.belzile@fsaa.ulaval.ca

Soybean (Glycine max (L) Merr.) is a vital crop, providing plant-based protein and oil globally. To meet
increasing food security demands under unpredictable changes in the climate, enhancing soybean yield
and quality is crucial. Root systems are central to plant survival and productivity, enabling access and
uptake of nutrients and water. They are also key determinants of plants’ ability to withstand nutrient
depletion and extreme weather conditions including drought, heat and flooding. Considering this, we
employed a core set of 137 Canadian soybean lines with over 2 million high-quality single nucleotide
polymorphism (SNP) markers to perform a genome-wide association study to identify major loci and
candidate genes for root system architecture (RSA) traits. One locus explaining 21% of variation for the
total length of the roots (qTLR1) was located on chromosome 1. Gene models within/around qTLR1 were

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