Standardization of the tyrosinase test for identification of wheat varieties Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/p2676z66b

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  • This study was undertaken to standardize and evaluate the tyrosinase test as a variety identification procedure for wheat. The potential value of the tyrosinase test lies in its ability to quantify the colored products, rather than relying on visual estimation as in the phenol test. The specific objectives of this study were to: (a) standardize the methodology of the tyrosinase test, (b) determine the effect of various non-genetic characteristics on the results of the test, (c) determine the ability of the tyrosinase test to distinguish Pacific Northwest wheat varieties, and (d) compare the relative merits of the tyrosinase and phenol tests as variety identification methods for wheat. The test parameters evaluated were soaking period, temperature, pH of tyrosine solution, concentration of tyrosine solution, and shaking effect on enzyme reaction. The following procedures are suggested for conducting the tyrosinase test on wheat seed: For the bulk-seed method soak 2 g of seeds in 10 ml of . 1% tyrosine solution in sodium citrate buffer .11 M, pH 8, for 2 hours at 35 C. For the single-seed method, soak the seed in 5 ml of . 1% tyrosine solution in sodium citrate buffer .11 M, pH 8, in a shaking water bath for 17 hours at 35 C. Then read the absorbance of the resulting solution at 470 nm against the substrate solution. Tyrosinase activity of WS-1 and Yamhill wheat seeds declined with increasing degrees of deterioration induced by artificial aging. Vitavax treatment caused a reduction in color development or enzyme activity PCNB (Pentachloronitrobenzene) depressed color development in WS-1 but not in Yamhill. HCB (Hexachlorobenzene) had no effect on either variety. Yamhill seeds produced in Pendleton developed a darker colored product or had more enzyme or higher enzyme activity than seeds produced in Moro, while Hyslop seeds from the two locations produced similar colored product or were comparable in enzyme activity. Tyrosinase activity of seeds of Hyslop variety was not affected by seed protein contents of 9, 11, and 13%, indicating that total seed protein does not affect pericarp enzyme (tyrosinase) quantity or activity. Larger seeds possessed greater tyrosinase activity than small seeds because of more pericarp tissue. Variation in enzyme activity also occurred in seeds of similar size within a variety. Comparisons with the phenol test showed that the bulk-seed tyrosinase test probably will differentiate more varieties than the phenol test and it is less affected by fungicide seed treatments. Both tests are affected by seed deterioration and production area. Both tests are relatively quick and can be completed in approximately the same amount of time. The phenol test is superior to individual-seed tyrosinase test for detecting varietal mixtures. Seeds within a variety differ in tyrosinase activity because of variations in seed size and other unknown factors. The bulk-seed test may be useful in differentiating between varieties belonging to the same phenol color group. Such a quantitative measurement may also be useful in describing varieties being entered under the Plant Variety Protection Act. The tyrosinase test may also provide more accurate results on fungicide treated seed.
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