1a.Objectives (from AD-416):
Determine the genetic basis of wheat end-use properties, specifically the structural attributes of high-molecular-weight glutenins that determine dough strength, the effects of over-expression of gliadins with extra cysteine residues on polymer formation, the types of low-molecular-weight glutenins and gliadins that form the largest and strongest gluten polymers, and the molecular and physiological basis for the increase in grain protein content associated with the presence of a gene introgressed from wild durum wheat. Develop transgenic wheats with high gluten strength whose only non-wheat DNA is a short non-protein-encoding sequence for site-specific recombination.
1b.Approach (from AD-416):
Use molecular biology to make coding regions for expression of variant gluten proteins in wheat. Use genetic transformation to introduce genes encoding variant and natural gluten proteins into wheat. Characterize transgene inheritance using genetics and transgene expression levels using molecular biology, biochemistry, and cereal chemistry. Determine dough mixing properties and gluten polymer characteristics in flours with transgene-encoded gluten proteins. Collaborate to test transgenes for their effects on wheat grain protein content and agronomic traits in field trials.
FY12 was the second year of a three-year bridging project migrating from National Program 302 to 301. Research continued with the goal of understanding the roles played by wheat seed proteins in the functionality of wheat flours. Seed was increased from true-breeding transgenic wheat lines that contained genes designed to test the gluten polymer-building roles of two cysteine amino acids in a glutenin protein that is associated with strong and elastic bread doughs. Solubility tests found that the glutenin variant that lacked two of its five cysteines was less efficiently incorporated into the gluten polymer, indicating that at least one of those two cysteines forms intermolecular disulfide bonds. We initiated small scale mixing experiments to further characterize dough strength in these lines. True-breeding lines were derived from transgenic plants with higher levels of the Dy10 glutenin subunit. Those lines lack bacterial DNA and antibiotic resistance genes. However, attempts to identify sublines that had lost the herbicide resistance gene used to select the transformants were unsuccessful. Wheat transformation experiments were initiated with a new reporter gene, DsRed, which encodes a red fluorescent protein. The reporter allows us to follow the fate of introduced DNA from bombardment through selection of callus and regeneration of plants. Collaborative research produced transgenic wheat plants containing 6 different constructions designed to understand the Yr 36 stripe rust resistance gene or to improve wheat’s resistance to the stripe and stem rust fungi. The disease resistance of these plants is currently being evaluated. The knowledge obtained in this research is enabling biotechnologists and breeders to improve wheat end-use properties and disease resistance more efficiently.