Research Objective
To assemble the necessary molecular constructs for editing genes that confer herbicide tolerance; to evaluate the transformation and regeneration methods recently established for green lentil genotypes at NRC-Saskatoon using small red lentil germplasm.
Expanding on the limited options for herbicide tolerance in lentil is a priority for Saskatchewan lentil producers. The goal of this project was to establish methodologies and reagents needed for using gene editing to introduce new herbicide tolerance traits into small red lentils. The components of a framework for CRISPR-Cas9-based gene editing in red lentils was established including workflows used to:
1) Identify gene editing targets to introduce herbicide tolerance,
2) Design and rank candidate guide RNAs,
3) Prepare guide RNA and Cas9 for delivery into plants by packaging them in plasmids,
4) Guide RNA efficiency testing in isolated plant cells (protoplasts), and
5) Optimize transformation methodology used to deliver guide RNA and Cas9 constructs in elite lentil germplasm of the small red class.
We are now poised to combine these elements to create edited lines with mutations to genes known to govern herbicide tolerance in other species.