Development of metribuzin tolerant chickpea: Proof of concept validation of CRISPR based gene editing tools in chickpea
- University of Saskatchewan
Dr. Bunyamin Tar'an
Lead Investigator(s) Institution
University of Saskatchewan - CDC
To develop tools to facilitate high throughput genome screening of key traits in chickpea..
To develop the gene editing capability in this study, we used five gRNAs (guide ribonucleic acid) designed to target exon 1 of the phytoene desaturase (PDS) gene in chickpea cultivar CDC Frontier. From the result of the in vitro cleavage assay, we decided to transform (guide ribonucleic acid) gRNA #2, 4, and 5 to chickpea protoplast. We investigated a number of factors that may affect the delivery of CRISPR/Cas9 reagents into chickpea protoplasts and obtained maximum viability and yield using a combination of 1.5% cellulase and 0.75% macerozyme 4 hours digestion of leaf tissues collected from 10 day old seedlings. Using multiple Cas9 and sgRNA (single guide ribonucleic acid) constructs and ribonucleoprotein (RNP) complex, we present the evidence for the seamless introduction of targeted modifications in the chickpea PDS gene. This study demonstrates the value and feasibility of combining protoplasts technology and CRISPR/Cas9-based gene editing for trait discovery and improvement in chickpea and potentially in other pulse crops. This is the first report of an effective CRISPR/Cas9 modification system in chickpea. This editing platform will be very helpful for the future development of herbicide tolerance, disease resistance and enhanced nutritional quality in chickpea.
Duration/Timeline of Project (Year to Year)
2017 - 2019