Thirty-five EMS (ethyl methane sulfonate) induced defective kernel mutants were selected for study of their endosperm protein differences. These mutants were crossed by a large embryo stock Aho (Alexander high oil) and backcrossed for several generations. Progenies had fairly uniform phenotypic expression. Fifteen normal kernels from a good F2 segregating ear were planted. Among these kernels 2/3 are heterozygous ( +/dek) and 1/3 are homozygous (+/+) for the mutant gene. Plants were selfed or double pollinated by selfing half the silks and outcrossing the rest by the arm-locating B-A translocation. Phenotypic difference between normal and mutant kernels can be identified 16 to 20 days after pollination. Good segregating ears were harvested 18 days post-pollination and were stored at -20 C. From each segregating ear, 10 normal and 10 mutant kernels were selected, and the pericarps and embryos were removed. The weight of 10 normal or 10 mutant endosperms was measured, 5 ml sample buffer was added and tissues were ground. Extractions were made by centrifugation and soluble proteins in the supernatant were denatured 5 minutes at 80 C and stored in a freezer. Samples were loaded in equal amount and proteins were separated by SDS-PAGE method. The results are: three mutants did not have protein differences, five mutants showed single or few protein differences and the rest (27 mutants) had many differences with low storage proteins (zeins) as listed in Table 1.
Ming T. Chang and M. Gerald Neuffer
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