II.20 Lack of chromosome breakage by azide in embryonic shoots and microspores of barley.
R. A. Nilan and 0. W. Pearson. Department of Agronomy and Soils, Washington State University, Pullman, Washington 99163, USA.
Azide is a very potent mutagen in barley in terms of induction of chlorophyll-deficient and a variety of morphological and physiological mutations. It is also a highly efficient mutagen, since it appears to induce literally no chromosome aberrations in barley (Sideris, 1968; Sideris et al., 1973; Nilan et al., 1973; Sander and Nilan, 1974) and Vicia (Kihlman, 1959).
In previous studies, chromosome aberrations in barley were scored during the first anaphase of embryonic shoots from seeds treated with azide. To further analyze the action of azide on chromosomes, aberrations were scored at first metaphase of cells in embryonic shoots and first metaphase and first anaphase of microspore mitoses. In scoring the aberrations in embryonic shoots, the effect of azide on the mitotic cycle (Pearson, 1973) was taken into account.
For embryonic shoot analyses, barley seeds were pre-soaked at room temperature in oxygen for 8 or 12 hours and then treated with 10-3 M azide solution at pH 3 for two hours at room temperature. These treatments have been the most effective in inducing mutations in barley (Sander and Nilan, 1974). Embryonic shoots were removed 20-24 hours after azide treatment, treated with 0.05% solution of colchicine, and fixed in Carnoy's (3:4:1). Squashes were stained with aceto orcein.
No significant frequency of chromosome aberrations was induced by azide in embryonic shoots. Among 800 metaphase cells treated with azide at pH 3, 6 fragments and no dicentrics were observed. In 200 cells that were treated at pH 3 but with no azide 4 fragments and no dicentrics were observed, while in 200 cells not exposed to buffer or azide, 2 fragments and no dicentrics were scored.
In scoring for aberrations during microspore mitosis all treatments were made on the anthers of the two-row barley variety Akka. All sodium azide solutions were buffered at pH 3 and all treatments were 1-1/2 hours in duration.
Anthers were removed and treated at stages between 9 and 12 hours prior to the first mitosis. Since the three anthers of a floret are at about the same stage, one was checked to determine the stage of the other two. One of the two remaining anthers was placed in the sodium azide solution and the other in the buffer as a control. At the end of the treatment, the anthers were washed for 5 minutes with distilled water, and plated on Linsmaier and Skoog's medium (Linsmaier and Skoog, 1965) without optional constituents and 1 p.p.m. each of IAA and BAP. They were then placed in an incubation chamber set at 25 C. The microspores were examined cytologically for aberrations from 15 to 33 hours after treatment, depending on the stage and severity of the treatment.
No aberrations were observed in any of the treated pollen. A total of 771 first pollen mitosis metaphase and anaphase cells were scored--261 treated with 10-4 M azide and 510 treated with 5 x 10-5 M azide. These concentrations were lower than those used on the embryo, since microspores are somewhat more sensitive to sodium azide than are cells of the embryo. A 1-1/2 hour treatment at 10-3 M killed nearly 100% of the pollen. The lower concentrations severely affected the pollen--10-4 M killing about 50% of the pollen and delaying the remainder between 6 and 12 hours, and 5 x 10-5 M killing about 12% and delaying the remainder about 3 hours.
During a recent visit to Washington State University, Dr. B. K. Vig analyzed the effects of azide on chromosomes in human leukocytes. The frequencies of chromatid and chromosome fragments and exchanges did not differ significantly from control frequencies. These results plus those obtained with barley will be published in detail.
References:
1. Kihlman, B. A., 1959. The effect of respiratory inhibitors and chelating agents on the frequencies of chromosomal aberrations produced by X-rays in Vicia. J. Biophys. Biochem. Cytol. 5: 479-490.
2. Nilan, R. A., E. G. Sideris, A. Kleinhofs, C. Sander, and C. F. Konzak, 1973. Azide--A potent mutagen. Mutation Res. 17: 142-144.
3. Pearson, Orin W., 1973. The effect of sodium azide on the cell cycle of the embryonic barley shoot. M. S. Thesis, Program in Genetics, Washington State University. 57 pp.
4. Sideris, E. G., 1968. Physiological and cytogenetical aspects of the effect of sodium azide on irradiated and non-irradiated barley seeds. Ph.D. Thesis, Program in Genetics, Washington State University. 88 pp.
5. Sideris, E. G., R. A. Nilan, and T. P. Bogyo, 1973. Differential effect of sodium azide on the frequency of radiation-induced chromosome aberrations vs. the frequencies of radiation-induced chlorophyll mutations in Hordeum vulgare. Radiation Botany 13: 315-322.
6. Sander, C. and R. A. Nilan, 1974. Increasing the mutagenic efficiency of sodium azide in barley. Barley Genetics Newsletter 4: 63-65.