National Grassland Research Institute
I planned the experiment to prove the hypothesis in 1989, and an interesting result was observed. The method is the supplementary cross using the purple embryo marker R-scm2 gene.
I crossed Stock 6 (C-I/C-I, y/y, white kernel, white scutellum) to Oh43 (c1/c1, Y/Y, v1/v1, yellow kernel, white scutellum) thinly. Twenty-four hours later I crossed X18G (A1/A1, A2/A2, C1/C1, C2/C2, R-scm2/R-scm2, Y/Y) to the same ears. The R-scm2 gene makes the scutellum and aleurone deep purple. I obtained 609 seeds. One-hundred-ninety-eight seeds were yellow kernels with white scutella which means they are Oh43 x Stock 6 hybrids. Four-hundred-nine seeds were purple kernels with purple scutella which means they are Oh43 x X18G hybrids. Two seeds were purple kernels with white scutella. Seedlings from the two seeds did not exhibit virescence, and the fertility was normal. Genotypes of the two plants were both (C-I/c1, Y/y) so they are clearly Oh43 x Stock 6 hybrids. This means the egg cells were fertilized with Stock 6 and polar nuclei were fertilized with X18G. Such a phenomenon is known as heterofertilization.
I suppose it was the case that the pollen tube of Stock 6 released only one sperm cell and it fertilized the egg cell, and the polar nucleus failed to be fertilized. Twenty-four hours later the polar nucleus was fertilized with X18G sperm cell. If so this phenomenon supports the hypothesis described by several investigators.
However, in this case there is another
possibility, that the pollen of Stock 6 germinated slowly and reached the
embryo sac at the same time as the pollen of X18G, and they heterofertilized
the polar nucleus and the egg cell. I think such a case must be very rare.
But further experimentation is needed.
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