J. Cell Sci. 12, 143-161 (i973)
143
Printed in Great Britain
THE EFFECT OF SPINDLE INHIBITORS
APPLIED BEFORE MEIOSIS ON MEIOTIC
CHROMOSOME PAIRING
G. A. DOVER AND R. RILEY
Cytogenetics Department, Plant Breeding Institute, Trumpington, Cambridge, England
SUMMARY
Injection of 0-5 % colchicine into immature tillers of genotypes of Triticum aestivum, T.
aestivum x Aegilops mutica and T. aestivum x Secale cereale hybrids induces asynapsis at first
meiotic metaphase irrespective of the homologous or homoeologous nature of the potential
pairing chromosomes. The induction of asynapsis occurs at a time during and immediately following the last premeiotic mitosis of pollen mother cells. No disruption of synapsis and chiasma
formation occurs in anthers having pollen mother cells originally at leptotene or immediately
prior to leptotene when cultured in White's medium plus colchicine. Tetraploid and octaploid
pollen mother cells resulting from the disruption of premeiotic spindles by colchicine show
pairing of chromosomes only in bivalents, in genotypes normally having a degree of multivalent
pairing configurations. The induction of multipolar mitotic spindles with o-oi % colchicine
results in the development of pollen mother cell mosaics with different numbers of chromosomes.
Such cells show high levels of chromosome pairing, including multivalents, in some genotypes
that normally have very little chromosome pairing. The injection of 05 % chloral hydrate during
the last premeiotic mitosis of the archesporium causes no disturbances of meiotic pairing. The
results are discussed with reference to the hypothesis that the control mechanism of meiotic
chromosome pairing involves centromeric microtubules of the spindle (not affected by chloral
hydrate) that are responsible for the positional adjustment, during the last mitotic anaphase,
of potential pairing partners.
INTRODUCTION
The long arm of chromosome 5B of hexaploid wheat is known to carry a locus Ph,
the activity of which prevents the pairing of homoeologous chromosomes of the 3
genetically similar geonomes (Riley & Chapman, 1958; Okamoto, 1957; Riley &
Kempanna, 1963). Varying the dosage of 5BL alters the patterns of meiotic pairing such
that homoeologous chromosome pairing takes place in the absence of 5BL whilst the
presence of 6 doses of 5BL reduces synapsis (Feldman, 1968). The genetic system
controlling meiotic pairing is of some complexity in that the pattern of pairing is a
result of a fine balance of forces determined by genes located on several chromosomes
of the complement (Feldman, 1966; Riley & Law, 1965). Furthermore, the effects of
genes in wheat can be suppressed, in certain instances, by genes carried on chromosomes of either of 2 diploid outbreeding relatives of wheat, Aegilops mutica and Aegilops
speltoides that carry a 4-allele 2-locus system of pairing control (Dover & Riley, 1972;
Vardi & Dover, 1972).
Two hypotheses have been advanced as to the cellular mechanisms by which the
pairing-control gene systems in Triticinae might operate. Feldman (1968) asserts that
144
G- A. Dover and R. Riley
the whole genetic variation in synapsis is explicable in terms of the relative co-orientation and spatial proximity of potential pairing partners. Measurements of the relative
proximity of homologous and homoeologous telocentric marker chromosomes in roottip cells of Triticum aestivum with 0-6 doses of 5BL suggested that the somatic association of chromosomes is under the control of 5BL (Feldman, Mello-Sampayo & Sears,
1966). In contrast to this, Darvey & Driscoll (1972) have found no evidence of somatic
association in T. aestivum in measurements involving homologous and non-homologous
nearness of nucleolar organizers and telocentrics. Riley (1968) has attempted to explain
the action of 5BL in terms of a proposal by Darlington (1940) tha t differences in
the number and positions of chiasmata may be caused by the differences in the time
available for chromosome pairing and chiasma formation. If pairing is a 2-stage process
as suggested by Faberge (1942) in which attraction of like chromosomes takes place in a
first stage, prior to synapsis in the second, then the 5BL dosage effects on pairing
could be the result of alterations in the duration of the attraction phase. 5BL-deficient
situations could sufficiently lengthen the phase to allow homologous and homoeologous
association. On this hypothesis increasing the dose of 5BL would lead to progressive
shortening of the phase, so that first homoeologues and finally homologues would not
have sufficient time to associate prior to synapsis.
Driscoll, Darvey & Barber (1967) showed that the application of colchicine to developing flowering spikes of T. aestivum induced asynapsis due to disturbance of processes
occurring during the premeiotic interphase. This is the first instance in plants showing
the control of pairing apparently to occur at this point in development. Similarly
Bayliss & Riley (1972) investigating the low temperature induction of asynapsis in a
temperature-sensitive genotype of T. aestivum and Buss & Henderson (1971) working
with high-temperature induction of interlocking bivalents in Locusta migratoria
have shown a premeiotic determination of meiotic pairing. The interlocking of bivalents, primarily of the longest chromosomes of the complement of Locusta migratoria,
has led to the suggestion that chromosome alignment during the last mitotic telophase
affects subsequent pairing behaviour (Buss & Henderson, 1971).
First indications of the cellular mechanisms responsible for the control of meiotic
events, other than chromosome pairing, have been shown in T. aestivum (Dover, 1972)
and Lilium species (Heslop-Harrison, 1971). In T. aestivum the overall polarity of
pollen mother cells, as reflected in the depositions of the spindle axis and the siting of
pollen apertures, was disrupted by colchicine applied some time during the premeiotic
interphase. The establishment of polarity during the premeiotic interphase is apparently a pre-requisite for subsequent meiotic development and appears to be under the
control of cell components, possibly microtubules, that are sensitive to colchicine.
The experiments described below were designed to test the idea that the premeiotic
determination of pairing is similarly dependent on cell components, that are sensitive
to colchicine; and to elucidate the nature of these premeiotic events.
Premeiotic spindles and chromosome pairing
Table i. Nature of chromosome pairing in genotypes of Triticum aestivum,
T. aestivum x Aegilops mutica and T. aestivum x Secale cereale hybrids
Genotype
Chromosome
no.
Nature of pairing
chromosomes
Euploid T. aestivum
T. aestivum nullisomic 5B
tetrasomic 5D
T. aestivum x diploid Ae. mutica
euploid
(2« = 14)
T. aestivum x tetraploid Ae. mutica
tetra 5B
(zn = 28)
T. aestivum x Ae. mutica
di-isosomic
(zn = 14)
L
zn = 42
zn = 42
Homologous
Homologous and homoeologous
2M =
Homoeologous (high-pairing
class)
Homologous and homoeologous
28
2n = 36
in = 28
SB
T. aestivum x Secale cereale
2M = 28
Homoeologous and intrachromosomal pairing of
isochromosomes
Homoeologous (mostly asynaptic)
MATERIALS AND METHODS
The following genotypes, with different degrees of meiotic chromosome pairing, were selected
for injection with colchicine: Triticum aestivum; T. aestivum nullisomic 5B tetrasomic 5D;
T. aestivum x diploid Ae. mutica; T. aestivum di-isosomic sBL x diploid Ae. mutica; T. aestivum
tetrasomic 5B x tetraploid Ae. mutica; T. aestivum x Secale cereale.
All genotypes of T. aestivum were of the variety Chinese Spring.
The genotypes shown in Table 1 had a range of pairing that varied from almost complete
absence of synapsis in ¥1 hybrids of T. aestivum x S. cereale to high levels of homoeologous
pairing in other Fx hybrid situations involving T. aestivum x Ae. mutica and in T. aestivum
deficient for chromosome 5B. Fx hybrids of T. aestivum x Ae. mutica segregate into 4 classes with
different levels of chromosome pairing (Dover & Riley 1972; Vardi & Dover, 1972) and plants
in the high pairing class were selected for treatment with colchicine (Fig. 2). The effects of colchicine on homologous, homoeologous and intra-chromosomal pairing could be compared in
the genotypes listed in Table 1.
Application of colchicine and chloral hydrate to intact anthers
Colchicine at 0-5 % and o-oi % or 0-5 % chloral hydrate was injected with a hypodermic syringe
through the leaf sheaths of a tiller into the space surrounding the developing spike. Tillers were
injected before the complete emergence of the flag leaf at a time when some archesporial cells
were undergoing their last premeiotic mitotic division. Knowledge of the rate of development
of anthers in T. aestivum (Bennett, Chapman & Riley, 1971), T. aestivum x Ae. mutica and T.
aestivum x S. cereale (G. A. Dover and M. D. Bennett, unpublished) was used to estimate the
stage of development reached by the anthers when first exposed to colchicine. Tillers were
sampled and fixed in 1:3 acetic alcohol at known time intervals after the time of injection and
first metaphase preparations were made by the Feulgen procedure and the stain was supplemented using propionic orcein. All plants were grown at 20 °C under continuous illumination.
Application of colchicine to excised anthers
Spikelets of wheat, rye and Ae. mutica contain 3 anthers per floret, which are approximately
synchronous in meiotic development. The 3 anthers were removed from a floret and 2 were
placed in modified White's medium (Ito & Stern, 1967) containing 0-25 % colchicine. The third
anther was fixed in 1:3 acetic-alcohol immediately after excision and the stage of meiosis
determined.
G. A. Dover and R. Riley
146
Time of application
of 0-5% coichicine
(2)
(1)
Mitosis
Penultimate
mitosis of P M C,
(3)
Last mitosis
of P.M.C.
(5)
(4)
Penultimate
mitosis in
tapetal cells
Leptotene in
PM.C. and
synchronous
division in
tapetal cells
First
metaphase
in P.M.C.
L
Developmental
stage of anther
(a) 2N-Bmucleate
(b) 2N-Unchanged
(c) 4-pore monad
(a) 2N + 4N
2N-Unchanged
(c) 4-pore monad
Consequences of
colchicine application
00 4N + 8N
*-— (b) 2N-Asynapsis
M Poreless monad
W 8N + 16N
(b) 4N-Bivalents
M
(a) 16N+32N
(b) 8N-Bivalents
(0
Fig. 1. Sequence of stages in the development of the archesporial cells and tapetal cells
from the penultimate premeiotic mitoses to metaphase I in T. aestivum. The time of
application is indicated at 5 separate points in the sequence with the consequences of
colchicine application noted for: (a) ploidy level of tapetal nuclei at metaphase I;
(b) ploidy level of P.M.C.s and degree of meiotic pairing; and (c) pollen type.
Symbols zN to 16 N indicate ploidy level.
The remaining 2 anthers were sampled and fixed after time intervals of 6 and 22 h and the
stage of meiosis reached during development in modified White's medium and colchicine was
determined.
RESULTS
The method used to relate the time of application of colchicine to meiotic irregularity is based on observations of the ploidy of the tapetal cells and the pollen mother
cells (Dover, 1972). The sequence of events occurring in the archesporial tissue and the
consequences of colchicine application at several points in development are shown in
Fig. 1. The induction of asynapsis occurred when colchicine affected premeiotic pollen
mother cells at a time between the last mitotic division and the penultimate division in
the tapetum. This was concomitant with the stage at which a poreless pollen condition
was induced by colchicine (Dover, 1972). No asynapsis occurred in sampled anthers
in which tapetal ploidy levels indicated that the colchicine affected the anther tissues
between the penultimate tapetal division and the last tapetal division. The last tapetal
division was synchronous in all cells, and occurred some time during meiotic leptotene.
In excised cultured anthers, 0-25 % colchicine did not prevent the intimate synapsis of
chromosomes once this has been determined prior to excision. Excised anthers containing pollen mother cells that had unpaired chromosomes in leptotene or immediately
Premeiotic spindles and chromosome pairing
147
Table 2. Mean chromosome pairing per cell in anthers from tillers injected
with 0-5 % colchicine and in untreated tillers of the same genotype
Genotype
T. aestivum
Control
Colchicine
Colchicine
T. aestivum, nullisomic 5Btetrasomic 5D
Control
Colchicine
T. aestivum x diploid Ae. mutica
di-isosomic 5B L (high pairing class)
Control
Colchicine
T. aestivum x tetraploid Ae. mutica
tetrasomic 5B
Control
Colchicine
Chromosome
number Univ.
Biv.
Triv.
0-07
20-93
IO-II
650
—
—
—
—
—
—
43-3°
2283
1816
0-38
o-88
40 -2O
I2-OO
—
—
l6-75
052
009
I2-I
24-1
II-7O
Quad. Chiasmata
42
42
42
29-0
42
42
18-00
28
28
91
5'6o
2-OO
I5-3
4-07
036
36
36
6-6
91
7"OO
i-8
o-8
20-5
o-66
0-08
0-78
17-69
io-oo
664
prior to leptotene, developed normally with synapsis and chiasma formation similar to
that observed in untreated tillers of the same genotypes.
Effect of 0-5 % colchicine on the pairing of homoeologous and homologous chromosomes
A 0-5 % solution of colchicine induced asynapsis in all genotypes listed in Table 1
(Figs. 3, 4), when applied at a time between the last mitotic division of the pollen mother
cells and the penultimate division of tapetal cells, irrespective of the genetic relationship of potential partner chromosomes (Table 2). In genotypes where both occurred,
homoeologous pairing was reduced first by colchicine and homologous pairing was
reduced later. For example, colchicine resulted in the complete absence of multivalents
in T. aestivum nullisomic for chromosome 5B whilst the frequency of bivalents was
little reduced. There is a reduction in multivalent frequency in early sampled tillers
of F± hybrids of T. aestivum di-isosomic 5BL x diploid Ae. mutica (high pairing class)
followed by a reduction in bivalent frequency in late sampled tillers. Similarly F x
hybrids of T. aestivum x tetraploid Ae. mutica, in which there were 2 genomes of
Ae. mutica with one genome of wheat, had a more pronounced reduction in pairing of
homoeologous than that of homologous chromosomes (Table 2).
Meiotic pairing of chromosomes after complete disruption of premeiotic spindles of the
archesporal cells
Anthers to which 0-5 % colchicine was applied prior to the last pollen mother cell
mitosis developed pollen mother cells with twice the normal number of chromosomes
(tetraploid P.M.C.s), due to failure of mitotic spindle formation. Pollen mother cells
with 4 times the normal number (octoploid P.M.C.s) developed after colchicine in-
148
G. A. Dover and R. Riley
Table 3. Mean chromosome pairing per cell, in untreated tillers of
genotypes subsequently injected with o-oi % colchicine
Genotype
Triticum aestivum
T. aestivum x Aegilops mutica
di-isosomic 5BL
T. aestivum x Secale cereale
in
Univ.
Biv.
Triv.
2-O
42
28
0-07
91
20-93
5-60
28
27-24
0-38
Quad. Chiasmata
433°
052
I2-I
O-38
duced spindle failure of both the penultimate and the last mitotic spindle. Tetraploid
pollen mother cells in T. aestivum (4W = \ix = 84) had chromosomes paired as ring
bivalents with occasional univalents. The close pairing of chromosomes as bivalents
took place despite the continued presence of colchicine which would have induced
asynapsis if applied at the premeiotic interphase and despite the tetrasomic condition of
each chromosome. This probably indicates that the paired chromosomes were derived
from sister chromatids that failed to separate during the last premeiotic mitosis and
remained in close juxtaposition throughout the intervening interphase to meiotic
prophase. Similarly pairing of chromosomes as bivalents was observed in tetraploid and
octoploid pollen mother cells of F± hybrids of T. aestivum x Ae. mutica (Figs. 5, 6).
This took place in genotypes that bore genes normally inducing high levels of pairing
of homoeologous chromosomes, and also with each chromosome in a tetrasomic condition in octoploid pollen mother cells, and in the continued presence of colchicine.
Tetraploid pollen mother cells (4W = 56) in hybrids of T. aestivum x Ae. mutica (high
pairing class) containing 2 isochromosomes showed pairing of all chromosomes as
bivalents with intrachromosomal pairing of the 2 isochromosomes (Fig. 6).
Pattern of pairing in pollen mother cells after induction of multipolar spindles with
o-oi % colchicine
The rationale behind the use of o-oi % (dilute) colchicine derived from the observations in pairing patterns in tetraploid and octoploid pollen mother cells just described. Octoploid pollen mother cells in Fx hybrids of T. aestivum x Ae. mutica, having
genes normally inducing high levels of homoeologous pairing, had only bivalents at
first metaphase. The disruption of spindle formation by 0-5 % colchicine had, presumably, prevented the wide separation of sister chromatids. It seemed possible that
components of the spindle or the products of chromosome-spindle interactions were
normally critical in determining the movement and relative positions of potential
pairing partners. If this is true then partial failure of a spindle due to colchicine sufficiently dilute to induce mitosis with multipolar spindles would cause the movement of
groups of chromosomes to the multiple poles; and subsequent patterns of chromosome
pairing might reflect this disturbance.
Colchicine at o-oi % was injected into tillers of euploid T. aestivum, into F1 hybrids
of T. aestivum di-isosomic 5BL x Ae. mutica (high pairing class) and into Fx hybrids
of T. aestivum x S. cereale. The mean chromosome pairing in untreated tillers is given
in Table 3. Cell mosaics were induced in pollen mother cells following premeiotic
Premeiotic spindles and chromosome pairing
149
Table 4. Chromosome pairing in pollen mother cell mosaics with a range of numbers
of chromosomes and in tetraphid (zn = 56) pollen mother cells in anthers of Triticum
aestivum di-isosomic 5B L x Aegilops mutica {zn = 28) treated with o-oi % colchicine
during the penultimate and last premeiotic mitoses
Chromosome
no. of cell
Univ.
Biv.
Triv.
> Triv.
Doubled pollen mother cells with 56 chromosomes only
—
—
8
23
28
—
—
—
26
—
—
3
2
—
—
27
1
—
23
7
—
2
—
27
56
56
56
56
56
56
56
56
56
—
2
Means
26
27
—
1
—
—
—
—
8
24
o-io
OIO
4-66
2566
All other cells with different numbers of chromosomes
Chiasmata
38
48
45
49
42
48
47
5°
43
45'5
48
2
—
4i
15
18
23
18
29
—
71
4
2
44
2
1
52
2
1
—
1
6
5
5
15
—
1
31
3
1
—
56
56
2
22
2
1
2
24
2
—
7
46
46
33
3
3
3
5
4
5
6
7
5
13
1
—
24
6
1
—
12
5°
—
—
86
10
1
—
12
1
2
17
31
8
7
1
1
21
1
1
9
—
—
86
8
40
14
18
103
28
39
29
28
8
1
1
19
17
14
43
11
13
2
—
28
13
5
—
—
10
13
2
—
29
15
17
3
3
3
3
6
5
—
—
11
—
1
3°
14
2
4
—
13
12
55*
74
8
14
2
2
—
10
27
2
1
62
11
19
—
3
46
38
25
20
9
3
7
7
28
12
3
9
5
3
8
25
28
35
21
20
56
35
7
4
23
14
2
1
10
—
—
15
1
—
9
1
1
10
—
—
10
1
—
1
—
42
27
* Undetermined chromosome number > 55-
150
G. A. Dover and R. Riley
treatment of T. aestivum di-isosomic 5BL x Ae. mutica (zn = 28). At first metaphase,
chromosome numbers ranged from 8 to 103 (Figs. 7-9). The level of pairing was high
in all pollen mother cells with chromosome numbers other than 56. Most cells with
56 chromosomes had bivalents only (Table 4). The full significance of these observations will be brought out in the Discussion.
Sampled anthers from treated tillers of T. aestivum euploid (zn = 42) had 2 types
of pollen mother cells. Those with 42 chromosomes had some asynapsis but with
occasional trivalents or quadrivalents (Fig. 10). The second type had 84 chromosomes
and bivalents and multivalents.
Injection of o-oi % colchicine into premeiotic tillers of F1 hybrids of T. aestivum x
S. cereale (zn = 28) caused the formation of pollen mother cells with chromosome
numbers varying from 22 to 28 (Figs. 11, 12). Many cells had high levels of pairing
although there was little or no pairing in untreated pollen mother cells. After treatment
with dilute colchicine during the last premeiotic mitosis, pollen mother cells with
trivalents and up to 5 bivalents were observed (Figs. 11, 12) although untreated cells
had a mean bivalent frequency of only 0-38 (Table 3).
The effect of chloral hydrate on meiotic chromosome pairing
Ris (1949) showed that chloral hydrate disrupts mitotic spindles in grasshopper
spermatocytes without disturbance of chromosome movement. Mole-Bajer (1969) has
followed the effects of chloral hydrate on different components of the mitotic spindle
in Haemanthus endosperm using the electron microscope. She found that both continuous and discontinuous microtubules of the spindle fibres are absent immediately
after treatment but that the discontinuous microtubules, originating at the centromeres,
and primarily responsible for chromosome movement, soon begin to reform. Cell
division was then able to proceed, although the poles were not well defined. Injection
of 0-5 % chloral hydrate into young premeiotic tillers of T. aestivum revealed no disturbance of meiotic pairing and 21 bivalents (zn = 42) were regularly observed in the
derived pollen mother cells (Fig. 13). No doubled pollen mother cells were seen in any
of the sampled anthers. Many aborted anthers and malformed archesporial tissues
resulted, indicating the presence of chloral hydrate in anther cells. Higher concentrations of chloral hydrate (1 and 2%), caused death of entire spikes.
DISCUSSION
Induced environmental changes during the premeiotic interphase and the premeiotic mitoses can cause corresponding changes in crossing-over and chiasma frequencies (Maguire, 1968; Grell, 1969; Lamb, 1971). High-temperature induction of
interlocking bivalents in Locusta migratoria (Buss & Henderson, 1971) takes effect
during the premeiotic interphase; and low-temperature induction of asynapsis occurs
in temperature-sensitive genotypes of T. aestivum nullisomic 5D (Bayliss & Riley,
1972), and in Fx hybrids of T. aestivum x Ae. mutica with B chromosomes (Vardi &
Dover, 1972) also during an early stage of the premeiotic interphase. It could be that
the determination of meiotic chromosome pairing, taking place during the premeiotic
Premeiotic spindles and chromosome pairing
151
interphase, arises from the premeiotic association of potential pairing partners. A
hint as to the nature of this control and the cellular mechanisms responsible came from
examination of the effects of colchicine on meiotic pairing (Barber, 1942; Levan, 1939;
Nebel & Ruttle, 1938; Driscoll et al. 1967; Driscoll & Darvey, 1970). The precise
polarity of pollen mother cells of T. aestivum, as reflected in the positions of meiotic
spindles and pollen grain apertures, is similarly determined at a time during the premeiotic interphase, a stage that is sensitive to colchicine (Dover, 1972) (Fig. 1). The
establishment of polarity during the premeiotic interphase may be related to the
organization of chromosomes necessary for pairing.
The results obtained from treatment with colchicine of intact and cultured excised
anthers, depicted in Fig. 1, show that the asynapsis by 0-5% colchicine is induced
between the last mitosis of the pollen mother cells and the penultimate mitosis of the
tapetum. This is the time when poreless pollen can be induced by colchicine (Dover,
1972). Furthermore, asynapsis occurs in all genotypes (Table 1) irrespective of whether
potential partners are homologous or homoeologous (Table 2). However, homoeologues possibly fail to pair before homologous chromosomes. This suggests either
differences in degree of the relative associations of homologous and homoeologous
chromosomes, or differences in the mechanisms responsible for their premeiotic
properties.
The absence of multivalents in tetraploid and octoploid pollen mother cells in
genotypes that were tetrasomic for all chromosomes, or that carried genes normally
inducing homoeologous pairing (Figs. 5, 6), suggests the residual association of the
derivatives of sister chromatids after the failure of spindle formation following 0-5 %
colchicine treatment. Chromosomes from sister 'chromatids' apparently form ring
bivalents, after replication at premeiotic S. The movement of chromosomes during the
last mitotic anaphase appears to be an essential prerequisite for chromosome pairing in
multivalents in genotypes having multiple copies of the same chromosome and also in
genotypes normally having pairing of homoeologous chromosomes.
Injection of dilute colchicine (o-oi %) induces multipolar spindles during the last
mitosis and pollen mother cell mosaics with varying numbers of chromosomes are
observed at first meiotic metaphase. The high levels of pairing in cells developing
after the partial disturbance of the last mitotic spindle (Figs. 7-12; Table 4) strongly
suggest that the random movement of chromosomes taking place under such conditions predetermines subsequent abnormal pairing patterns during meiosis. This is
strikingly seen in pollen mother cells with 22 to 28 chromosomes in hybrids of Triticum
aestivum x S. cereale (zn = 28) in which the formation of trivalents and bivalents had
been induced by the action of dilute colchicine on the last premeiotic spindle (Figs. 11,
12). Untreated tillers of the same hybrids had a mean chiasma frequency of less than
0-5 per cell.
The non-interference of chloral hydrate in meiotic pairing when applied at all
stages from the last mitosis to meiotic prophase can be interpreted as the result of its
discriminatory effects on different components of the spindle (Mole-Bajer, 1969).
Chloral hydrate, whilst preventing the polymerization of protein subunits comprising
the continuous microtubules, does not grossly disturb the re-initiation of centromeric
152
G. A. Dover and R. Riley
microtubules soon after application. The continued activity of centromeric microtubules, involved with chromosome movement, could possibly be the cause of undisturbed regular pairing in treated tillers of T. aestivum. Such an interpretation would
need to be confirmed by electron-microscope studies of the differential response of
spindle components to chloral hydrate and other spindle disruptors in T. aestivum.
The observations, presented above, of the effects of different concentrations of
colchicine and chloral hydrate on meiotic pairing, lead to the conclusion that the
mechanism of pairing control is involved with chromosome movement at the last
premeiotic anaphase.
The evidence for continuous somatic association of homologous chromosomes in
T. aestivum is contradictory (Feldman et al. 1966; Darvey & Driscoll, 1972). However,
the last mitotic anaphase could be unique and critical in controlling the relative positions of related chromatids prior to pairing. Aneuploid genotypes of T. aestivum
having different doses of the pairing control locus (Ph) on chromosome 5BL differ in
their degree of homologous and homoeologous pairing. These pairing differences
observed in T. aestivum with different doses of 5BL could be the result of differences in
the cellular mechanisms reponsible for chromosome association prior to synapsis. If,
as the above results indicate, the control of pairing is through a type of chromosomespindle interaction that determines the relative positions of chromosomes during the
last mitotic anaphase then the alternative patterns of pairing with different doses of
5BL could be the result of differences in spindle properties as a result of the activities
of 5B1'. Avivi, Feldman & Bushuk (1969, 1970a, b) in an extensive series of experiments on the spindle system of root-tip cells of T. aestivum found that the affinity of
the spindles for nucleoside triphosphates and colchicine alters with increasing doses of
5BL. They postulated that this occurs through alteration of the structure of the
spindle subunits, determined by the activity of 5BL. If a similar 5BL-dependent alteration of spindle subunits occurred in the last premeiotic mitosis then the variation in the
degree of meiotic pairing controlled by 5BL would find a plausible explanation. The
explanation would be in terms of the premeiotic control mechanism of pairing involving components of the spindle during the last mitotic anaphase.
Supporting evidence for this conclusion is found in genotypes of T. aestivum in
which abnormal pairing has been induced by the introduction of the genes controlling pairing on a chromosome of Ae. mutica. Plants of T. aestivum with an additional
alien chromosome of Ae. mutica that bears the high-pairing loci have pairing of homoeologous chromosomes and also exhibit a high degree of pollen mother cell mosaics and
abnormal multipore pollen (G. A. Dover, in preparation). Both the occurrence of cell
mosaics and multipore pollen are the result of the establishment of multiple polar
determinants in species hybrids in the Triticinae (Dover, 1972). Genotypes of T.
aestivum carrying addition chromosomes of Ae. mutica that do not affect the regular
bivalent pairing of the wheat background exhibit no pollen mother cell mosaics and
have normal single pore pollen. Similarly the B chromosomes of Ae. mutica are able
to induce asynapsis at low temperatures in hybrids of T. aestivum x Ae. mutica during
the premeiotic interphase and also frequently prevent the formation of the last mitotic
spindle (Vardi & Dover, 1972). Both these genotypes show a striking correlation
Premeiotic spindles and chromosome pairing
153
between malfunctioning of the last mitotic spindles and abnormal meiotic pairing
situations.
If the proposed mechanisms for the premeiotic control of meiotic chromosome
pairing, based on positional adjustment at the last mitotic anaphase, were to be substantiated in other genera using other spindle inhibitors, then part of the problem of
homologous chromosome recognition would have been removed.
REFERENCES
AVIVI, L., FELDMAN, M. & BUSHUK, W. (1969). The mechanism of somatic association in com-
mon wheat, Triticum aestivum (L.). I. Suppression of somatic association by colchicine.
Genetics, Princeton 62, 745-752.
AVIVI, L., FELDMAN, M. & BUSHUK, W. (1970a). The mechanism of somatic association in
common wheat, Triticum aestivum (L.). II. Differential affinity for colchicine of spindle
microtubules of plants having different doses of the somatic-association suppressor. Genetics,
Princeton 65, 585-592.
AVIVI, L., FELDMAN, M. & BUSHUK, W. (19706). The mechanism of somatic association in
common wheat, Triticum aestivum (L.). I I I . Differential affinity for nucleotides of spindle
microtubules of plants having different doses of the somatic-association suppressor. Genetics,
Princeton 66, 449-461.
BARBER, H. N . (1942). The experimental control of chromosome pairing in Fritillaria. J. Genet.
43, 359-374BAYLISS, M. W. & RILEY, R. (1972). Evidence of premeiotic control of chromosome pairing in
Triticum aestivum. Genet. Res. (in the Press).
BENNETT, M. D., CHAPMAN, V. & RILEY, R. (1971). The duration of meiosis in pollen mother
cells of wheat, rye and Triticale. Proc. R. Soc. B 178, 259-275.
Buss, M. & HENDERSON, S. A. (1971). Induced bivalent interlocking and the course of meiotic
chromosome synapsis. Nature, Lond. 234, 243-246.
DARLINGTON, C. D. (1940). The prime variables of meiosis. Biol. Rev. 15, 307-321.
DARVEY, N. L. & DRISCOLL, C. J. (1972). Evidence against somatic association in hexaploid
wheat. Chromosoma 36, 140-149.
DOVER, G. A. (1972). The organization and polarity of pollen mother cells of Triticum aestivum.
J. Cell Sci. 11, 699-711.
DOVER, G. A. & RILEY, R. (1972). Variation at two loci affecting homoeologous meiotic chromosome pairing in Triticum aestivum x Aegilops mutica hybrids. Nature, New Biol. 235, 61-62.
DRISCOLL, C. J. & DARVEY, N. L. (1970). Chromosome pairing: effect of colchicine on an isochromosome. Science, N.Y. 169, 290—291.
DRISCOLL, C. J., DARVEY, N. L. & BARBER, H. N. (1967). Effect of colchicine on meiosis of
hexaploid wheat. Nature, Lond. 216, 687-688.
FABERGE, A. C. (1942). Homologous chromosome pairing: the physical problem. J. Genet. 43,
121-144.
FELDMAN, M. (1966). The effect of chromosomes 5B, 5D and 5A on chromosome pairing in
Triticum aestivum. Proc. natn. Acad. Sci. U.S.A. 55, 1447-1453.
FELDMAN, M. (1968). Regulation of somatic association in meiotic pairing in common wheat.
Proc. 3rd int. Wheat Genet. Symp. (ed. K. W. Finlay & K. W. Shepherd), pp. 169-178.
Canberra: Australian Academy of Science.
FELDMAN, M., MELLO-SAMPAYO, T . & SEARS, E. R. (1966). Somatic association in Triticum
aestivum. Proc. natn. Acad. Sci. U.S.A. 56, 1192-1199.
GRELL, R. (1969). Meiotic and somatic pairing. In Genetic Organisation, vol. I (ed. E. W. Caspari
& A. W. Rowin), pp. 361-492. New York: Academic Press.
HESLOP-HARRISON, J. (1971). Wall pattern formation in angiosperm microsporogenesis. In
Control Mechanisms of Growth and Differentiation, Symp. Soc. exp. Biol. 25, pp. 277-300.
Cambridge: University Press.
ITO, M. & STERN, H. (1967). Studies of meiosis in vitro I. In vitro culture of meiotic cells.
Devi Biol. 16, 36-53.
154
G. A. Dover and R. Riley
LAMB, B. C. (1971). Some details and effects of the premeiotic control of recombination frequencies in Neurospora crassa. Genet. Res. 18, 255-264.
LEVAN, A. (1939). The effect of colchicine on meiosis in Allium. Hereditas 25, 9-26.
MAGUIRE, M. (1968). Evidence of the stage of heat induced cross-over effect in maize. Genetics,
Princeton 60, 353-362.
MOLE-BAJER, J. (1969). Fine structural studies of apolar mitosis. Chromosoma 26, 427-448.
NEBEL, B. R. & RUTTLE, M. L. (1938). The cytological and genetical significance of colchicine.
J. Hered. 29, 3-9.
OKAMOTO, M. (1957). Asynaptic effect of chromosome V. Wheat Inf. Serv. Kyoto Univ. 5, 6.
RILEY, R. (1968). The basic and applied genetics of chromosome pairing. Proc. 3rd int. Wheat
Genet. Symp. (ed. K . W . Finlay & K.W. Shepherd), pp. 185-195. Canberra: Australian
Academy of Science.
RILEY, R. & CHAPMAN, V. (1958). Genetic control of the cytological diploid behaviour of hexaploid wheat. Nature, Lond. 182, 713-715.
RILEY, R. & KEMPANNA, C. (1963). The homoeologous nature of the non-homologous meiotic
pairing in Triticum aestivum deficient for chromosome V (5B). Heredity 18, 287-306.
RILEY, R. & LAW, C. N . (1965). Genetic variation in chromosome pairing. Adv. Genet. 13, 57114.
Ris, H. (1949). The anaphase movement of chromosomes in the spermatocytes of the grasshopper. Biol. Bull. mar. biol. Lab., Woods Hole 96, 90-106.
VARDI, A. & DOVER, G. A. (1972). The effect of B chromosomes on meiotic and premeiotic
spindles and chromosome pairing in Triticum/Aegilops hybrids. Chromosoma (in the Press).
(Received 24 May 1972)
Fig. 2. Meiotic chromosome pairing in untreated T. aestivum di-iso 5BL x diploid
Ae. mutica (high pairing class): 9 univalents, 3 bivalents, 3 trivalents and 1 quadrivalent. Isochromosome sB L arrowed, x 1000 approx.
Fig. 3. First metaphase in P.M.C. of T. aestivum x Ae. mutica (high-pairing class)
after injection of 0-5 % colchicine during the premeiotic interphase: 24 univalents and
2 bivalents. x 1000 approx.
Fig. 4. First metaphase in P.M.C. of T. aestivum after injection of 0-5 % colchicine
during the premeiotic interphase: 28 univalents and 7 bivalents. x 1000 approx.
Premeiotic spindles and chromosome pairing
4
I
4
156
G. A. Dover and R. Riley
Fig. 5. First metaphase in P.M.C. with double the number of chromosomes (zn = 56)
of T. aestivumx Ae. mutica (zn = 28) (high-pairing class) after injection of O'5 %
colchicine at the last premeiotic mitosis: 18 univalents and 19 bivalents. x 1000
approx.
Fig. 6. First metaphase in P.M.C. with double the number of chromosomes (271 = 56)
of T. aestivum di-isosomic 5BL x Ae. mutica (high-pairing class) after injection with
o-oi % colchicine at the last premeiotic mitosis: 27 bivalents and 2 isochromosome
rings (arrowed), x 1000 approx.
Fig. 7. P.M.C.s with varying numbers of chromosomes (see Table 4) of T. aestivum
di-isosomic sBL x Ae. mutica (high-pairing class) after injection with o-oi % colchicine
at the last premeiotic mitosis, x 400 approx.
Premeiotic spindles and chromosome pairing
•V
\
.1.57
158
G.A. Dover and R. Riley
Fig. 8. First metaphase in P.M.C. with a low chromosome number {zn = 21) from
anthers treated with o-oi % colchicine (see legend Fig. 7). 8 univalents, 3 bivalents,
1 trivalent and 1 quadrivalent, x 1500 approx.
Fig. 9. First metaphase in P.M.C. with a high chromosome number (zn = 71) from
anthers treated with o-oi % colchicine (see legend Fig. 7). High-pairing situation
with univalents, bivalents and multivalents. x 1000 approx.
Fig. 10. First metaphase in P.M.C. of T. aestivum after injection of 001 % cochicine
during the last premeiotic mitosis: 6 univalents, 16 bivalents and 1 quadrivalent,
x 1000 approx.
Premeiotic spindles and chromosome pairing
159
8
i
10
160
G. A. Dover and R. Riley
Fig. 11. First metaphase in P.M.C. of T. aestivum x S. cereale (zn = 28) after injection
of o-oi % colchicine during the last premeiotic mitosis: 17 univalents, 1 bivalent and
1 trivalent (zn = 22). x 1000 approx.
Fig. 12. First metaphase in P.M.C. of T. aestivum x S. cereale (2w = 28) after injection of o - oi % colchicine during the last premeiotic mitosis: 14 univalents and 5 bivalents (2M = 24). x 1000 approx.
Fig. 13. First metaphase in P.M.C. of T. aestivum after injection of 0-5 % chloral
hydrate during the premeiotic interphase. 21 bivalents. x 1000 approx.
Premeiotic spindles and chromosome pairing
11
12
13
161