2013 Annual Report
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. Replacing 5325-21430-011-00D (June/2010).
This is the final report for this project, which has been replaced by 5325-21430-013-00D, "Improvement of Wheat Quality through Molecular Genetics". Research persists 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 2 cysteine amino acids in a glutenin protein that is linked with strong and elastic bread doughs. Solubility tests found that the glutenin variant that lacked 2 of its 5 cysteines was less efficiently incorporated into the gluten polymer, indicating that at least 1 of those 2 cysteines forms inter-molecular disulfide bonds. Small scale mixing experiments were initiated to further distinguish dough strength in these lines. Seed was also raised from true-breeding lines that contained a gamma gliadin with 9 cysteines instead of the 8 usually found in this protein. It can be incorporated into the gluten polymer via its extra cysteine, but not linked to more than one other protein. High levels of this gliadin variant are predicted to inhibit the formation of large gluten networks and thus to weaken doughs. Small scale mixing experiments are underway to test this prediction. Seed was also increased from true breeding lines of transgenic wheat that had increases in the “Dy10” glutenin subunit. Research with ARS collaborators in Lincoln, Nebraska, in the first year of the project had shown that the strong-dough characteristics of such lines could be transferred to hard red winter Midwest wheat varieties by genetic crosses. In FY13, collaborative work showed that flours derived from the winter wheat lines with high levels of Dy10 could be blended with flours from non-transformed winter wheats to improve their dough mixing tolerance. Some of the blends produced bread loaves with improved symmetry and crumb color scores compared to unblended wheats. So far, genetic crosses and genetic segregation have failed to separate the high-Dy10 transgenes from the herbicide resistance gene that was used to select these transformants. Thus, we have not yet produced transgenic wheats with high gluten strength that lack bacterially-derived marker genes. In future experiments, we plan to use site-specific recombination to remove marker genes. We continued to refine our wheat transformation protocol. In FY12, we employed DsRed, a new reporter gene that encodes a red fluorescent protein, for the first time in wheat to follow the fate of introduced DNA after transformation through selection of callus and regeneration of plants. This allowed us to identify inefficiencies in the way we use selection to identify transformants. Research with collaborators 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 being assessed. The knowledge obtained in this research is enabling biotechnologists and breeders to improve wheat end-use properties and disease resistance more efficiently.
Increases in glutenin content improve some dough mixing and bread loaf properties. The baking industry would benefit from the availability of wheat flours with high gluten strength for production of breads and buns. ARS scientists in Albany, California, and Lincoln, Nebraska, collaborated to show that transgenic wheat flours containing increased levels of a native high-molecular-weight glutenin subunit could be blended with non-transgenic control flours to improve the mixing stability of doughs prepared by a commercial baking method. Bread loaves prepared from the blended flours had better loaf symmetry and crumb color scores than non-blended controls. These results show that transgenic wheat flours with increases in one glutenin protein can be used via blending to improve dough mixing strength and tolerance in a commercially relevant bread-making process. Such flours could potentially substitute for imported wheat gluten in baked products requiring high gluten strength.
Blechl, A.E., Vensel, W.H. 2013. Variant high-molecular-weight glutenin subunits arising from biolistic transformation of wheat. Journal of Cereal Science. 57:496-503.
Graybosch, R.A., Seabourn, B.W., Chen, Y.R., Blechl, A.E. 2013. Effects of transgene-encoded high-molecular weight glutenin proteins in wheat flour blends and sponge and dough baking. Cereal Chemistry. 90(02): 164-168.
Robertson, G.H., Blechl, A.E., Hurkman II, W.J., Anderson, O.D., Cao, T., Tanaka, C.K., Gregorski, K.S., Orts, W.J. 2013. Physical characteristics of genetically-altered wheat related to technological protein separation. Cereal Chemistry. 90(1):1-12.