SDS-PAGE of seed proteins for identification of varieties and species of ryegrass (Lolium spp.) Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/mc87pv208

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  • The number of varieties of ryegrass (Lolium spp.) has increased greatly in recent years. An accurate and rapid laboratory technique to identify these varieties would benefit the consumer as well as protect an organization's Plant Variety Protection rights. There is also a need for a technique to complement the seedling fluorescence test to differentiate annual (L. multiflorum Lam.) from perennial (L. perenne L.) ryegrass. Electrophoresis has been successful in species and variety identification of ryegrass, but often differences are based only on band intensity or require analysis of 100 plants or more. The purpose of this study was to develop electrophoretic procedures which would identify varieties of perennial ryegrass, differentiate between annual and perennial ryegrass species and detect seed mixtures of these two species. Proteins were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SOS-PAGE). Extractions were made on ground seed from bulk samples and from single seeds. In the characterization study of perennial ryegrass, 28 varieties were tested. Many of the protein bands were common for all of the varieties. Individual varieties were characterized by presence or absence of specific bands and by band intensity ratios calculated from densitometer scans. Most of the varieties were differentiated by unique banding patterns. The varieties 'Pennant' and 'Premier', however, were not successfully differentiated from each other nor was 'Omega' found to be different from the variety 'Birdie.' In a study to determine the feasibility of detecting mixtures of species, SDS-PAGE was conducted on 17 annual, 3 intermediate, and 28 perennial ryegrass varieties. The annual and intermediate varieties possessed protein bands at R[subscript f] .71 and .73 that were not found in any of the perennial varieties. Bands were present in the perennial varieties at R[subscript f] .80 and .88 that were absent or very faintly stained in the annual and intermediate varieties. The intermediate species could not be differentiated from the annual species. Attempts were made to use SDS-PAGE to detect contamination of perennial ryegrass seed lots with small percentages of annual ryegrass seed. Annual and perennial ryegrass seeds were mixed together in different proportions to make concentrations of 0, 1, 3, 5, 10, 25, 50, 75, and 100% annual seed. Visible detection of the annual bands was possible in the mixtures of 25% or more annual seed. Densitometer scans could detect the presence of these annual bands in mixtures of 10%, but not in lower concentrations. Protein extracts of individual seeds were electrophoresed to determine whether species mixtures can be detected on an individual seed basis. When individual seeds were used, the resulting banding patterns were different than those produced from bulk seed extracts from the same variety. Furthermore, no two seeds within a variety showed the same banding patterns. However, the characteristic annual bands at R[subscript f] .71 and .73 were still evident in most single seeds from annuals. Likewise, the characteristic perennial bands at R[subscript f] .80 and .88 were normally present in individual seeds of the perennials. Banding patterns of SDS-PAGE of seed proteins were not affected by year and location grown, class of certification or viability or vigor of the seed. This one procedure can be used to differentiate varieties as well as species of ryegrass, making this SDS-PAGE system adaptable to seed testing needs.
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