II. 27. Chromosome elimination in bi-nucleate cells of a (2X) H. vulgare X (2X) H. bulbosum hybrid.
W. G. Wheatley and K. J. Kasha, Crop Science Department, University of Guelph, Guelph, Ontario, Canada, N1G 2W1. "R"
Following interspecific hybridization between (2X) Hordeum vulgare and (2X) H. bulbosum, the bulbosum chromosomes are selectively eliminated during the mitotic divisions of the developing embryo. After embryo culture two types of plants are generally recovered: a relatively high frequency of haploid vulgare plants which, after colchicine doubling of the chromosomes, yield pure lines and a low frequency of diploid interspecific hybrid plants (Kasha and Kao, 1970). The hybrid plants exhibit spontaneous chromosome elimination in meristematic regions such as the root-tips (Noda and Kasha, 1981) and for this reason are useful in the study of the factors which influence chromosome elimination in Hordeum.
Several hypotheses have been suggested to explain the mechanism of chromosome elimination. These hypotheses include asynchrony of cell cycle times between the two genomes in the hybrid nucleus and asynchrony of factors regulating chromosome movement during mitosis such as spindle malfunction or centromere mistiming (Kasha, 1974). The complexity of the events involved in mitosis makes it difficult to distinguish between many of the hypotheses. In particular, it is difficult to separate the effect of cytoplasmic factors from those which originate in the hybrid nucleus.
In an attempt to distinguish between the effect of nuclear and cytoplasmic factors we have recently studied chromosome elimination in binucleate cells, induced by treatment with caffeine, in root meristems of a diploid interspecific hybrid of Hordeum. Roots were grown hydroponically in White's media (without hormones) treated for 6 h with 500 ppm 5-aminouracil to partially synchronize the cells at the end of DNA synthesis, released from the 5-aminouracil blockade for 10 h and, as the synchronized cells passed through mitosis, treated for 10 h with 0.1% caffeine. Caffeine acts to inhibit cytokinesis resulting in the formation of bi-nucleate cells. About 10 to 12 hours after the end of the caffeine treatment bi-nucleate cells were observed in mitosis. Roots were fixed in acetic acid: alcohol (1:3) and later slides prepared using Feulgen staining of the nuclei and Fast Green to counterstain the cytoplasm. Bi-nucleate cells were viewed under phase microscopy and distinguished by the absence of a cross wall between the nuclei. Bi-nucleate cells in mitosis, i.e. bi-mitotic cells, were scored by observing whether neither mitosis, one mitosis or both mitoses within the same cell exhibited chromosome elimination. The cytological characteristics which were used in this classification were: noncongressed chromosomes present at metaphase, lagging chromosomes during anaphase and telophase and the presence of micronuclei in the products of recently divided bi-nucleate cells.
In bi-mitotic cells of the hybrid root-tips chromosome elimination was observed most frequently in only one of the two mitoses (Table 1 and Figs. 1 and 2). The frequency of bi-mitotic cells which exhibited chromosome elimination in only one mitosis was 0.161 whereas both mitoses (Figs. 3 and 4) exhibited chromosome elimination at a frequency of 0.024. The occurrence of chromosome elimination in one mitosis of a bi-nucleate cell appears to be independent of the response of the other mitosis in the same cell, i.e. the square of the frequency in which only one mitosis exhibits chromosome elimination (0.161 X 0.161 = 0.026) is similar to that observed where both mitoses exhibit chromosome elimination (0.024).
A number of deductions about the factors influencing chromosome elimination in roots of the interspecific hybrid can be drawn from our observations. 1) Since the two nuclei of a bi-nucleate cell which share a common cytoplasm appear to undergo chromosome elimination independently of each other, it would appear that the factors resulting in chromosome elimination originate in the nucleus rather than the cytoplasm. Furthermore, the two nuclei of bi-nucleate cells appear to pass through mitosis synchronously and thus, chromosome elimination is not influenced by cytoplasmic factors which may regulate the passage of the chromosomes through the various phases of mitosis. 2) Since the nuclei of bi-nucleate cells share a common cytoplasm, the independent chromosome elimination observed by the two mitoses suggests that cell cycle is not a major influence on chromosome elimination.
It would appear that chromosome elimination in the bi-nucleate cells of the diploid interspecific hybrid we have studied is likely due to inherent differences between the two nuclei as suggested by the work of Armstrong and Davidson (1980). Bi-nucleate cells induced by caffeine treatment may be useful in future studies concerning the mechanism of chromosome elimination. For example, observations on the frequency with which both mitoses in a bi-mitotic cell respond similarly to a chemical treatment could be used as a criterion for judging the specificity of action and time of action of that chemical on chromosome elimination.
References:
Armstrong, S. W. and D. Davidson. 1980. Cell cycle duration and time of DNA synthesis in binucleate cells induced in V. faba meristems by caffeine or isobutyl-methylxanthine. Protoplasma 102:281-293.
Kasha, K. J. 1974. Haploids from somatic cells. In: "Haploids in higher plants: Advances and Potentials" (K. J. Kasha, ed.) pp 67-87. Univ. of Guelph.
Kasha, K. J. and K. N. Kao. 1970. High frequency haploid production in barley (Hordeum vulgare L.). Nature 225:874-876.
Noda, K. and K. J. Kasha. 1981. Chromosome elimination in different meristematic regions of hybrids between Hordeum vulgare L. and H. bulbosum L. Jap. J. Genet. 56:193-204.
