Forrest.Wold-McGimsey@colostate.edu, Caitlynd.d@hotmail.com, Rocio.Alarcon-Reverte@rothamsted.ac.uk, Karl@tesorogenetics.com, Andrew.Katz@colostate.edu, John.Stromberger@colostate.edu, Esten.Mason@colostate.edu, Stephen.Pearce@rothamsted.ac.uk

Acrylamide is a processing contaminant formed during high-temperature cooking processes of starchy foodstuffs, including bread products, and is a potent neurotoxin and probable carcinogen. There is now a growing movement among regulatory agencies to limit the permissible amount of acrylamide in food products sold to the public.

The amino acid asparagine provides the carbon backbone for acrylamide formation in baked products. Because free asparagine concentration in wheat grains is highly correlated with acrylamide levels in bread products, the development of bread wheat lines with low free asparagine content in their grains is a favorable method of producing end-use wheat products with low acrylamide levels.

A common wheat line carrying a chemically induced null allele in one of the three ASPARAGINE SYNTHETASE 2 (ASN2) homoeologues exhibited reductions in free asparagine content in their grains between 9 and 34% compared to wild-type sister lines. We hypothesize that loss of function in all three ASN2 homoeologues will confer more extreme reductions in free asparagine content.

To do this, a CRISPR/Cas9 construct with one single guide RNA was used to introduce knockout edits in all three ASN2 genes in five elite bread wheat varieties. In the greenhouse, edited wheat plants exhibit no reduction in germination rate or overall vigor. We will measure free asparagine content in the edited wheat grains using mass spectrometry coupled with ultra-performance liquid chromatography.

We expect these results from at least one edited variety to be available by June of 2024 and field trials for edited lines will be conducted within the next two years. It is our hope that this method can be used in complement with traditional breeding efforts to produce healthier, higher quality bread products with no loss to the favorable qualities exhibited in existing elite lines.