An improved whole-seed assay for screening wheat germplasm for polyphenol oxidase activity

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 on4he 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 nullisomie/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.