Field assessment of ultra-low asparagine, low acrylamide, gene edited wheat

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The presence of acrylamide in popular foods has become one of the most difficult problems facing the food industry and its supply chain. Acrylamide is a contaminant that forms from an amino acid called asparagine in its free (non-protein) form and sugars such as glucose, fructose and maltose. The reaction occurs during frying, baking, roasting, toasting and high-temperature processing. Acrylamide is classified as probably cancer-causing in humans and affects development and fertility at high doses. Products made from wheat and other cereals are major sources of dietary acrylamide.Acrylamide in food is covered by regulation (EU) 2017/2158 (2018), which set Benchmark Levels for the presence of acrylamide in food, described compulsory mitigation measures and required all food businesses to monitor the levels of acrylamide in their products. Methods developed to limit acrylamide formation during processing do not work for all products, may adversely affect product quality and are expensive to implement. Our approach has been to lower the acrylamide-forming potential of crop products: for wheat and other cereals that means reducing the concentration of free asparagine in the grain. The project arises from previous BBSRC-funded work in which we have demonstrated large varietal differences in free asparagine concentration in wheat grain and shown it to be highly responsive to sulphur deficiency and other nutritional and environmental stress factors, as well as disease. Asparagine is made by an enzyme called asparagine synthetase and wheat has five genes that encode this enzyme. One of these genes, TaASN2, has emerged as a genetic target because it is highly active specifically in the grain. In our current work, involving teams at Rothamsted Research, Univ. of Bristol and partners AHDB, KWS, Limagrain, RAGT, Saaten Union and Syngenta, we have used the genome editing technique, CRISPR-Cas9, to knock out TaASN2 genes. In addition, we have screened a wheat population in which mutations have been introduced by chemical mutagenesis (a much older technique that has been used in plant breeding since the 1950s) and identified lines with mutations in each of the six versions of TaASN2 in the wheat genome. The TaASN2 mutations in these lines are being 'stacked' by one of our partners and partial knockouts will be available by Year 2 of the project. The concentration of free asparagine in one of the CRISPR lines is reduced by approximately 90%. The aim of this project, which will continue the partnership with Univ. of Bristol, AHDB, Limagrain, RAGT, Saaten Union and Syngenta, is to undertake field trials of these ultra-low asparagine wheat lines with a view to the development of low acrylamide wheat varieties for the UK market. In year 1 we will bulk up seed for the trial. We will also investigate the germination rate of the CRISPR lines: they have shown poor germination so far but this can be reversed by treatment with low concentrations of asparagine. We will check the efficacy and practicality of the asparagine and other potential treatments. We will also develop genetic markers/tools to enable breeders to integrate the ultra-low asparagine trait into breeding lines. Lastly, we will prepare a risk assessment and submit an application for permission to hold the field trial. In year 2 we will conduct the field trial, analysing the low asparagine lines for emergence, physical characteristics, developmental differences, composition, yield and other agronomic characteristics. We will also conduct glasshouse experiments to ascertain whether the low asparagine lines respond to sulphur deficiency in the same way as normal wheat. The project would represent a landmark for crop gene editing, with the field trial, to our knowledge, being the first for gene edited wheat in the UK and Europe, while the involvement of AHDB and wheat breeders will provide a direct pathway to commercialisation if the plants perform well in the field trial.