University of North Dakota

Induction of twin embryos by x-ray treatment of early proembryo stage embryos --William F. Sheridan An experiment was conducted with the goal of analyzing cellular autonomy of mutant expression of the dek1 mutation (for results regarding this question see the next report). Plants grown in pots were used as females. They were crossed and later administered a dose of about 800 Roentgens of soft x-rays over a seven minute period using the x-ray machine in Curtis Hall at the University of Missouri-Columbia. I thank Jerry Neuffer and Dan England for growing the experimental plants and for use of the equipment and I thank Evelyn Bendbow for assistance in treating the ears.

Ears were treated at 50 hours, 72 hours, and 104 hours after pollination in mid-July 1988. Ears were harvested, dried, and tagged. They were then shelled and the embryo side of each kernel was visually examined. The frequencies of kernels with twin embryos for each set of treatments among the ears analyzed to date are: 50 hours after pollination, 4 out of 170 (2.3%); 72 hours after pollination, 39 out of 752 (5.2%); and 104 hours after pollination, 4 out of 197 (2.0%).

Under the summer growing conditions in Missouri fertilization occurs probably by 18 hours after pollination. At 50 hours after pollination the zygote has divided to produce a few-celled proembryo, by 72 hours after pollination the proembryo is still very small, consisting probably of no more than 16 cells. Even if fertilization occurred at 12 hours after pollination and the zygote divided 12 hours later to form two cells, and there was a doubling of embryo cell number every 12 hours thereafter, at 72 hours after pollination, embryo cells would be dividing to form a 32 cell embryo, including the suspensor. An examination of the data on maize embryogenesis reported by L. F. Randolph (1936) reveals that "four days after pollination the embryos contained ordinarily 10 to 24 cells" and his Figure 5B showing a radial longitudinal section through a proembryo fixed 5 days after pollination reveals that there are only a few additional cells. Furthermore, Randolph noted that "except for the localization of growth, primarily in the upper portion, the proembryo is wholly undifferentiated up to and including the seventh day" after pollination.

X-ray treatment of early developing maize embryos was performed by R. S. Poethig, E. H. Coe, and M. M. Johri (1986). They performed a histological analysis on their greenhouse-grown materials. Their embryos were slightly ahead in their rate of development but at 48-53 hours after pollination the early proembryo consisted of four cells, and at four days (96 hours) after pollination, their midproembryo appears about equivalent in development and cell number to the 5 day proembryo figured by Randolph.

The results presented in this report show a frequency of twin embryos of 5.2% when x-ray treatment was administered 72 hours after pollination. In contrast, Poethig, Coe, and Johri observed, following treatment of developing embryos with 1000R of soft x-rays at two, three, four and five days after pollination the following frequency of twin shoots: two days, 0.5%; three days 0.3%; four days, 6.7%; and five days, 6.2%. A high twinning rate did not occur until treatment was given at four days after pollination and in most of these twins, twinning occurred at the first node or above.

The results described in this report are of particular interest because they indicate that the very early proembryo of maize is comprised of cells that can regulate when cell death is inflicted by x-ray treatment. The data on embryos treated 72 hours after pollination are particularly persuasive. Although a morphological analysis of the embryos was not undertaken these embryos were undoubtedly at the early proembryo stage. Yet analysis of kernels of five different ears irradiated at 72 hours after pollination revealed a twin embryo frequency ranging from 4.2% to 5.8%. Of additional interest is the observation that nearly all of the twin embryos examined were complete twins and were fused only in the suspensor region if they were fused at all.

The presence of complete twin embryos indicates that the cells of the early proembryo surviving the x-ray treatment were able to sufficiently regulate that they could proceed with complete embryo formation. The x-ray treatment may have killed a cell (or small group of cells) occupying an apical position in the proembryo. The death of this cell (or cells) may have had the effect of microsurgery so as to split the proembryo so that each of the two surviving portions proceeded to form an embryo. This idea is congruent with the fact that frequently one of the embryos was larger than its twin. It is also conceivable that the effect of x-ray induced cell death may have been to release the proembryo cells from apical dominance. Twinning may normally be absent because of the very early establishment of apical dominance in the proembryo. These studies will be continued with the addition of structural studies to seek evidence for the occurrence, timing, and location of cell death in treated embryos.

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