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GrainGenes Reference Report: CRS-41-1697

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Reference	CRS-41-1697
Title	An improved whole-seed assay for screening wheat germplasm for polyphenol oxidase activity
Journal	Crop Science
Year	2001
Volume	41
Pages	1697-1705
Author	Anderson JV Morris CF
Abstract	Summary: Polyphenol oxidase (PPO) causes darkening and discoloration of wheat (Triticum aestivum L.) foods such as noodles. Consequently, a simple, nondestructive, quantitative assay for determining PPO on one to a few wheat seeds could identify superior germplasm and eliminate inferior breeding lines, thus greatly assisting the development of wheat cultivars with superior noodle color. We sought to (i) examine current PPO whole-seed assays and develop an improved assay that would facilitate rapid, efficient evaluation of wheat breeding lines and cultivars, be amenable to single seeds, and not adversely affect seed viability; (ii) use the assay to evaluate a large collection of wheat germplasm with the aim of identifying lines with very low PPO levels for crossing; and (iii) gain additional information on the location of PPO gene(s). Phenol, L-tyrosine, catechol, methyl catechol, 3,4 dihydroxyphenyalanine (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 3-(N-morpholino) propane sulfonic acid (MOPS) buffer, pH 6.5, with 3 to 5 seeds constantly rotated in a 2-mL microcentrifuge tube for 0.5 or 1 h at room temperature] was used to screen 1953 germplasm accessions grown in a common environment. Lines with low levels of PPO (i.e., 10% of the population) were identified; 24 of 66 lines displayed low PPO when 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
Keyword	[ Hide all but 1 of 49 ] 2d 50 accession acid assay breeding breeding lines caffeic acid cantonese noodle color chinese spring chromosome chromosome 2d collection color crop crossing durum wheat efficient environment enzyme flour germplasm germplasm accessions homoeologous group 2 information method methyl noodles oxidase ph polyphenol polyphenol oxidase polyphenol oxidase activities population ppo screen screening single seeds spring substitution substitution line triticum triticum aestivum tube viability wheat breeding wheat cultivar wheat germ lasm wheat seed

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