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United States Department of Agriculture

Agricultural Research Service

Title: An Improved Whole-Seed Assay for Screening Wheat Germplasm for Polyphenol Oxidase Activity

Authors
item Anderson, James
item Morris, Craig

Submitted to: Crop Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 6, 2001
Publication Date: November 1, 2001
Citation: Anderson, J.V., Morris, C.F. 2001. An improved whole-seed assay for screening wheat germplasm for polyphenol oxidase activity. Crop Science.41:1697-1705.

Interpretive Summary: The countries of Eastern Asia are major wheat users with as much as 50% of the flour consumed in the form of noodles. A key quality trait factor of raw noodles (mainly white salted and yellow alkaline) in the Asian market is noodle color. The color of these noodles can vary dramatically depending on the source of wheat flour used to produce the noodles. Consequently, any means of predicting noodle color from wheat grain early in breeding programs is highly desirable. Since the enzyme polyphenol oxidase (PPO) has long been associated as the factor leading to the discoloration of raw noodles, many breeding programs have developed various assays for determining PPO activity in wheat grain. In this report, we describe the development of a simple, quantitative assay for determining the potential noodle color or discoloration characteristics of wheat germplasm which is also based on PPO activity of whole seeds. Our new assay is non-destructive to the seed and can be accomplished with as little as one seed. This is particularly advantageous for permitting propagation of plants from individual seeds or lines early in wheat breeding programs. We also show how this assay can be used to quickly screen thousands of varieties of wheat to identify breeding lines which can be used to develop wheat germplasm that will produce superior color in raw noodles. Additionally, we show how this new assay has multiple application as it can also be used to identify the chromosome location of PPO genes.

Technical Abstract: Polyphenol oxidase (PPO) causes darkening and discoloration of wheat foods. We sought to 1) examine current PPO whole-seed assays and develop an improved assay that would facilitate rapid, efficient evaluation of wheat breeding lines and cultivars, would be amenable to single seeds, and would not adversely affect seed viability; 2) use the assay to evaluate a large number of wheat germplasm with the aim of identifying lines with very low PPO levels for crossing; and 3) gain additional information as to the location of PPO gene(s). Phenol, L-tyrosine, catechol, methyl catechol, L-DOPA, and caffeic acid were evaluated as potential substrates. Kinetic studies indicated that L-DOPA and catechol at pH 6.5 produced the greatest enzyme activity. L-DOPA did not reduce seed viability, whereas catechol is reportedly toxic to seeds. A standard assay (1.5 mL of 10 mM L-DOPA in 50 mM MOPS buffer, pH 6.5, with 3 to 5 seeds constantly rotated in a 2-mL microcentrifuge tube for 0.5 or 1 hr at room temperature) was used to screen 1,953 germplasm grown under a common environment. Lines with low levels of PPO (i.e. 10% of the population) were identified; 66 were re-evaluated under a second environment. Lastly, chromosome 2D was identified as a location of PPO gene(s) based on 'Langdon' durum/'Chinese Spring' D-genome substitution lines, and homoeologous group 2 nullisomic/tetrasomic stocks of Chinese Spring. The L-DOPA standard assay described here provides a robust and efficient method of evaluating germplasm and cultivars for PPO.

Last Modified: 11/27/2014
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