For the last two summers I have been testing the effects of gamma irradiation on the Mutator system. Shedding central spikes were collected early in the morning just as pollen was beginning to shed. Only plants which were not shedding pollen the previous day were selected. The shedding central spikes were placed in glass test tubes stoppered with a cork. The tassel of each plant sampled was bagged and selfed and outcrossed to produce the controls. The central spikes were exposed to gamma irradiation from a cobalt 60 source sufficiently long to provide dosages of 600, 1,200 and 1,400 rads. The irradiated central spikes were removed from the test tubes (in the fieldhouse) and placed in an earshoot bag. A second earshoot bag was used to cap the first. These samples were placed in a protected sunny spot until pollen was shedding well, and were pollinated on standard silks that had been cut back the day before.
In 1981, a treatment of 600 rads resulted in very little observable effect (Table 1). But there are two slight trends that can be detected: 1) The irradiation slightly reduced the number of ears obtained. For the Mu test, 200 seeds from both controls and irradiated crosses were planted. For the standards, 150 seeds of each were sown. Neither the reduction for the Mu or the standard crosses is statistically significant. 2) For the Mu crosses, there is reduction in the mutation frequency in the irradiated material. This reduction was not statistically significant.
In 1982, plants were irradiated with 1,200 and 1,400 rads. In most of these crosses we obtained good seed set (normal looking ears). Thus I thought we still were not getting much of a biological effect. Therefore, only two Mu plants were tested at 1,200 rads and one standard at 1,400 rads (Table 2).
Again, as with the lower doses, there is a reduction in the number of ears produced. For the Mu crosses 200 seeds were sown, and for the standard crosses 100 control and 200 seeds from irradiated pollen were planted. The reduction in number of ears recovered is statistically significant at the 1% level in both sets of crosses. In the Mu crosses, the reduction in mutant frequency in the outcrosses from irradiated pollen is significant at the 1% level.
Some gamma-ray-induced recessive seedling mutants could be expected in the progeny from irradiated pollen of standard plants, but none were observed. However, a sample of only 63 plants is too few for a good test.
As far as Mutator activity is concerned, gamma irradiation markedly reduces the frequency of mutants obtained in the outcross progeny. Why is there this reduction? I believe it might be telling us something about the timing of Mutator activity. It has been shown that Mutator acts late in development (Robertson, Genetics 94:969-978,1980; MGNL 58:11-12, 1984). Small sectors of allelic mutants have established this fact. However, many Mu-induced mutants are singular events. Some of these may be postmeiotic (gametophytic or zygotic). It may be that gamma irradiation inactivates the Mutator system in pollen so that no or a greatly reduced number of mutations are taking place between the time of pollen shedding and the first division of zygotic DNA. Thus, the mutations we observe in the irradiated Mu crosses could be predominantly those that occurred before pollen maturation. The synergistic response between Mu activity and U.V. (MGNL 56:2-4, 1982; 58:19-20, 1984) also involves pollen irradiation and hence is active in the same developmental stages as the gamma irradiation. Thus both types of irradiation act in the same time frame. It is improbable that Mu transpositions occur in the sperm since no DNA replication is occurring. However, the direct effects of irradiation impinge upon the sperm. It is likely that the irradiation damage is repaired in the zygote at the time of the first DNA replication. This is also the first opportunity for Mu1 transpositions to occur. If, at this time, Mu1 transposes to an unreplicated portion of a chromosome, a plant could result that is heterozygous for a new Mu-induced mutant. The results from U.V. and gamma irradiation suggest that such mutations do occur, and that changes induced in the male genome by both types of irradiation have a marked but different effect on the frequency of such mutants.
Perhaps gamma irradiation is completely inactivating Mu and hence eliminating the zygotic transposition. Tests will be made next year to determine if the gamma irradiation effect is a transitory or permanent change. If the latter, gamma irradiation may provide a method for eliminating Mu transposition and thus facilitate the location of genic DNA by Mu mutagenesis.
This last summer we performed similar experiments with gamma dosages of 1600 and 1800 rads. In these crosses, a marked reduction in seed set was observed and hence greater biological effects can be expected.
Table 1. The effects of 600 rads gamma irradiation of Mu and standard pollen.
Table 2. Effects of 1,200 rads gamma irradiation an Mu pollen and 1,400 rads
Donald S. Robertson
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