A Database for Triticeae and Avena
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).
Table 1. Chromosome elimination in bi-mitotic cells
in roots of a diploid interspecific hybrid of Hordeum.
Figures 1 to 4. Bi-nucleate cells exhibiting chromosome
elimination: 1) Bimetaphase cell with one metaphase exhibiting one non-congressed
chromosome. 2) Bi-telophase cell with one mitosis exhibiting a lagging
chromosome. 3) Bi-telophase with both mitoses exhibiting lagging chromosomes
(a micro-nucleus is present in the far right of the cell). 4) A recently
divided bi-nucleate cell exhibiting lagging chromosomes from both mitoses.
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.
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
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.
BGN 12 toc
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