Ethidium bromide ineffectiveness and production of all male-sterile progeny by combining tillering and genetic male sterility

Research initiated in 1977 was to study the mutagenic induction of cytoplasmic fertility restoration of genetic male sterility in maize. Mutagenesis was attempted using ethidium bromide, an acridine dye used extensively to induce cytoplasmic mutations in yeast and reported to induce cytoplasmic male sterility in plants. To facilitate selection and recovery of any altered cytoplasm, genetic male steriles were incorporated into high tillering lines. In this way high tillering plants that also were male sterile could be examined for differences in sterility between the main tassel and the tiller tassel(s). Any sterility/fertility differences between the tassels could suggest a cytoplasmic change restoring fertility to the genetic male sterile. Recovery of the altered cytoplasm would be possible in these high-tillering lines because the entire tiller is represented by approximately two cells in the mature kernel; cells for the ear and tassel primordia have not been differentiated for the tiller. Therefore, a cytoplasmic change induced in one of these cells should be expressed in at least a sector throughout the tiller. Any cytoplasmic change noted in the tiller tassel also would be expected to be present in the ear of that tiller. This can be compared with the tassel and ear of the main stalk where the tassel is represented by two to eight cells in the mature kernel, and the ear is represented by a different two to four cells. A cytoplasmic change induced in a cell destined to become part of the main stalk tassel will not necessarily occur in a cell destined to become part of the main stalk ear.

Lines established for mutagenesis included nine different genetic male-sterile loci in two inbred backgrounds (A632 and Oh43) used individually in crosses with three high-tillering lines. Genetic male-sterile loci included ms1, ms2, ms5 ms8, ms9, ms10, ms14, ms17, and po. The high tillering lines included 'Butter and Sugar' sweetcorn, WAIC0, and a multihybrid for plant color (from Dr. E. H. Coe, Univ. of Missouri). Sufficient quantities of segregating lines were obtained by backcrossing the ms/high-tillering F1 with the appropriate male-sterile plant. Approximately 27,000 kernels from these lines were subjected to various ethidium bromide treatments (Table 1). Although long ethidium bromide treatments would completely inhibit kernel germination, there was no decrease in plant height and no increase in frequencies of seedling mutants, chlorophyll mutants, kernel mutants, etc. in any of the treatments.

Table 1. Ethidium bromide (EB) treatment variables. *

EB Conc. (M): 0.005, 0.05, 0.1

Treatment Length (h): 12, 18, 24, 36, 40, 42, 48, 58

Temp. ( C): 4, 24, 35

Solvent: H20, 5% DMSO, pH 3 phosphate buffer

*31 different treatments conducted from these variables.

Several instances of extruded anthers were noted on tiller tassels of otherwise male-sterile plants in one ms/high tillering combination. In most cases, pollen was not observed from these extruded anthers. There were three instances, however, where tiller tassels from plants in two consecutive cultures had extruded anthers that were shedding pollen. These plants were BC1 progeny from an A632 ms14 x Coe's multihybrid cross. Self pollinations were made on the tillers with extruded anthers, and an ear was formed from each of the three tillers with kernel numbers ranging from approximately 50 to 300 kernels per ear. One cross with a fertile sib (heterozygous for ms14) was made onto the main stalk ear of one of the three plants.

We planted kernels from each of the three self pollinations in our Hawaiian nursery but were limited to kernels from two of the three selfs in St. Paul. An average of 13 kernels per self germinated in Hawaii and produced all male-sterile plants. Observations from St. Paul corroborated that all progeny were male-sterile (Table 2). A total of 337 kernels from two of the three self pollinations germinated and produced male-sterile plants. Of these plants, 20% had extruded anthers either on the main stalk tassel or tiller tassel or both. Progeny from the sib cross to Ms14 ms14 resulted in a 1:1 segregation of male fertiles to male steriles. This segregation ratio was expected if the plant was sterile from being homozygous recessive for the ms14 locus. The 1:1 ratio also suggested that a cytoplasmic male sterile had not been induced by ethidium bromide as all progeny would have been expected to be male sterile if such a mutation had occurred.

Table 2. S1 progeny from self pollinations of tillers with extruded anthers on otherwise male-sterile plants.


% male steriles (no.)

% plants with anther extrusion (no.)










The occurrence of all male-sterile progeny from a self pollination made on the tiller of a genetic male-sterile plant seems to be another example of how a gene can be expressed differently in the maize main stalk compared with the tiller. We do not believe that ethidium bromide had a significant role in the extruded anther observations as other types of mutations were not increased. Anther extrusion may be more the result of the tiller physiology and represent an interaction of ms14, A632, and Coe's tillering multihybrid.

M. C. Albertsen and R. L. Phillips

Please Note: Notes submitted to the Maize Genetics Cooperation Newsletter may be cited only with consent of the authors.

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