Annals of Botany 78: 7381, 1996

Evidence for Heterochromatin Involvement in Chromosome Breakage in Maize

Callus Culture*
A. FLUMINHAN Jr, M. L. R. DE AGUIAR-PERECIN and J. A. DOS SANTOS
Departamento de GeneTtica, Escola Superior de Agricultura Luiz de QueiroTz, Uniersidade de Sago Paulo,
13400-900, Piracicaba, SP, Brazil
Received: 6 July 1995 Accepted: 22 January 1996
Mitotic instability was investigated in maize embryogenic callus cultures induced from tropical genetic stocks. Bridges
resulting from delayed separation of chromatids and typical bridges were observed in Feulgen preparations. The
analysis of C-banded anaphases showed that delayed chromatids were held together at heterochromatic knob sites
(primary event), and the presence of typical bridges with and without bands corresponding to knobs. These events
suggest the occurrence of breakage-fusion-bridge (BFB) cycles initiated by chromosome arms broken during the
primary event. Additional evidence for such a mechanism was the presence of gross aberrations involving
chromosome 7, detected in several C-banded metaphases of some cultures. It is hypothesized that such aberrations
are duplication deciencies produced by BFB cycles and chromosome healing that would have occurred after some
cell divisions. # 1996 Annals of Botany Company
Key words: Zea mays L., maize, tissue culture, chromosome breakage, heterochromatin, C-banding.
INTRODUCTION
Several studies have shown that changes in chromosome
number and structure can occur in plants regenerated from
tissue culture and that chromosome instability can be
induced by media components, culture age, explant tissue
and even by plant genotype (for a review see Lee and
Phillips, 1988; Phillips, Somers and Hibberd, 1988; Peschke
and Phillips, 1992). Cytological analysis of regenerated
plants has shown that chromosome breakage and its
consequences (deciencies, duplications, translocations and
inversions) are events quite frequently observed in plant
tissue culture, and that breakpoints are often associated
with late-replicating chromosome regions. This was rst
observed by Sacristan (1971) in Crepis capillaris, where
82% of rearrangements induced in itro involved chromo-
some breaks at the heterochromatic middle region of the
long arm of the Sat-chromosome. McCoy, Phillips and
Rines (1982) found a high frequency of chromosome
breakage near the centromere of oat chromosomes, a region
shown to be late-replicating and heterochromatic by
Johnson, Phillips and Rines (1987). Break-points involved
in translocations and deletions were also found in hetero-
chromatic regions in regenerated wheatrye hybrids
(Lapitan, Sears and Gill, 1984). Meiotic studies of re-
generated maize have shown that most breakpoints are
located between the centromere and the heterochromatic
knobs or the nucleolus organizer (Lee and Phillips, 1987;
* The data are part of MS theses by A. Fluminhan and J. A. dos
Santos.
For correspondence.
Present address: Institute of Genetic Ecology, Tohoku Uni-
versity, 980-77 Sendai, Japan.
Benzion and Phillips, 1988). A hypothesis proposed to
explain the role of heterochromatin in inducing chromosome
breakage is that normally late-replicating heterochromatic
regions may replicate even later in the culture environment,
leading to the formation of chromosome bridges, due to
delayed separation of sister chromatids at heterochromatic
regions (Lee and Phillips, 1987, 1988). In addition, quanti-
tative changes in repetitive DNA sequences have been
observed in regenerated plants of several species, such as
amplication of repeated sequences observed in tissue
culture-regenerated plants derived from wheatrye hybrids
(Lapitan, Sears and Gill, 1988) and one line of rye (Karp et
al., 1992).
Chromosome variation in regenerable maize callus cul-
tures have been investigated in few studies and only changes
in chromosome number have been reported. Edallo et al.
(1981) found 8 and 15% of non-diploid cells in cultures
derived from two inbreds. McCoy and Phillips (1982)
reported that 5% of the dividing cells in calli of reciprocal
hybrids had other than diploid numbers. Balzan (1978)
investigated two nonregenerable cultures derived from
mesocotyl tissue and found in one of these cultures, a high
frequency of cells with abnormal chromosome numbers
(79% tetraploid cells) and also dicentrics, anaphase bridges
and fragments. Wang, Phillips and Mi (1986) observed a
low mitotic index (about 4%) in a maize cell suspension
culture and based on this result, Phillips et al. (1988) stated
that due to the low mitotic index, relatively little is known
about chromosome variation in maize cell cultures them-
selves.
In this study, the occurrence of mitotic instability was
investigated in maize embryogenic callus cultures, induced
from genetic stocks derived from a tropical variety. Bridges
03057364\96\070073j09 $18.00\0 # 1996 Annals of Botany Company
74 Fluminhan et al.Chromosome Breakage in Maize Callus Culture
and chromosome fragments were detected in anaphase and
telophase cells in Feulgen preparations. With the application
of the C-banding technique, it was possible to detect the
presence of knobs (C-bands) in chromosome arms involved
in breakage. The analysis of C-banded anaphases and
metaphases provided evidences of the occurrence of
breakage-fusion-bridge cycles involving broken chromatids.
MATERIALS AND METHODS
The genetic stocks used were sister inbred lines and hybrid
combinations, obtained from an S
#
progeny derived from a
brazilian int variety (Jac Duro, Sementes Agroceres,
Brazil). They were homozygous for the presence of some
heterochromatic knobs, as shown in Table 1 (references in
Aguiar-Perecin and Decico, 1988).
T 1. Chromosome knob positions of inbred lines
Knobs*
Designation
of inbreds K6L
#
K6L
$
K7S K7L K8L
"
K8L
#
K9S
1315 (S
(
) jj jj jj jj jj jj jj
13213 (S
)
) jj jj jj jj jj jj jj
132331 (S
*
) jj jj jj jj jj jj jj
13342\7 (S
(
) jj jj jj jj jj jj 00
* K, knobs; numbers refer to chromosomes possessing knobs; S,
short arm; L, long arm (Knobs at 6L
#
, 6L
$
and 8L
"
, 8L
#
observed at
pachytene, appear as single bands on mitotic chromosomes).
jj (presence), 00 (absence) of knobs.
T 2. Frequency of mitotic abnormalities in callus cultures
Anaphases Telophases
Total
Stocks Culture* Weeks Total
DSC
(%)
1 Bridge
(%)
2 Bridges
(%)
Others
(%) Total
1 Bridge
(%)
2 Bridges
(%)
abnormalities
(%)
1315\1i13213\1
1-1 17 77 0n0 3n89 1n30 0n0 54 0n0 0n0 5n19
1-2 21 76 1n31 3n95 1n31 0n0 85 0n0 0n0 6n57
1-2 23 163 0n61 5n52 1n23 0n0 74 0n0 0n0 7n36
1-2 28 195 1n52 2n52 1n01 1n52 216 0n46 0n46 7n49
1-2 30 37 0n0 5n41 0n0 0n0 54 1n85 0n0 7n26
3-8 34 21 0n0 0n0 4n76 0n0 20 0n0 0n0 4n76
3-8 38 149 1n34 2n68 0n67 1n34 107 2n80 0n93 9n76
3-57 45 152 1n97 0n66 1n97 2n63 111 0n0 0n0 7n23
9-17 24 49 4n08 4n08 0n0 2n04 10 0n0 0n0 10n20
9-16 28 21 4n76 0n0 4n76 0n0 8 0n0 0n0 9n52
9-4 32 250 1n20 2n80 0n80 1n20 226 0n44 0n0 6n44
9-15 34 192 1n56 2n60 0n52 1n56 237 0n42 0n0 6n66
10-4 10 22 0n0 0n0 4n55 0n0 10 0n0 0n0 4n55
1315\5i1315\4
5-5 19 57 0n0 1n75 3n51 0n0 19 0n0 0n0 5n26
5-4 23 314 0n32 1n27 0n64 0n64 262 0n76 0n0 3n63
5-4 32 257 0n78 1n56 1n56 0n0 239 0n42 0n0 4n32
13342\7
13-2 23 48 0n0 2n08 0n0 0n0 18 0n0 0n0 2n08
132331\1
14-1 22 94 0n0 2n38 1n06 2n38 18 0n0 0n0 5n82
* First number designates the donor plant of each of the seven ears used.
Anaphases with fragments or three bridges.
F
#
plants were used.
DSC, Delayed separating chromatids.
Mitotic abnormalities were investigatedinFeulgenstained
preparations from 13 callus cultures, each derived from an
individual embryo, from seven donor ears (Table 2).
Tissue cultures were initiated from immature embryos
(1n02n0 mm long), placed on Petri dishes containing agar
solidied MS medium (inorganic components of
Murashighe and Skoog, 1962; 99 mg l

" inositol, 39n4 mg l

"
cystein and vitamins according to Prioli and Silva, 1989)
supplemented with 2 mg l

" 2,4-dichlorophenoxy-acetic acid

(2,4-D), 20 g l

" sucrose, 20 mg l

" casein hydrolisate and

8n0 g l

" agar with pH adjusted to 5n8. Embryogenic callus

cultures (Type II) initiated through this procedure were
maintained in this medium at 26 mC under dark conditions
and subcultured every 1520 d.
For mitotic analysis, samples of proembryoids (globular
stage) were taken from callus cultures with dierent ages,
5 d after transfer to fresh medium and were xed in 3: 1
alcohol : acetic acid overnight and stored in 70% alcohol at
4 mC. Feulgen staining was carried out as described
previously (Aguiar-Perecin and Vosa, 1985). The tissues
were macerated in 5% pectinase for 510 min at 37 mC and
squashed in 1% acetic carmine.
For the investigation of the involvement of knobs in the
occurrence of bridges and chromosome breakage, C-banded
anaphases were examined. Preparations with very clear
band patterns at anaphase bridges proved to be very
dicult to obtain, due to the highly stretched state of
chromatin in some cells. So, only few preparations of good
quality of cultures 1-2, 3-8, 5-4, 9-4 and 9-15 were used in
this analysis (Table 3). C-banded metaphases were examined
Fluminhan et al.Chromosome Breakage in Maize Callus Culture 75
T 3. C-banded anaphases analysed
1 Bridge 2 Bridges
Culture Weeks Total DSC j 0 j 0
1-2 36, 37, 38 43 2 7* 2 0 2
3-8 40 5 0 0 1 0 0
5-4 38 5 0 1 0 0 0
9-4 36 13 1 1 1 1 1
9-15 45 12 1 1 1 0 0
j, Bridge with band.
0, Bridge without band.
* Two anaphases with one fragment with band.
Anaphase with one fragment without band.
Anaphase with one fragment without band.
in some cultures for the investigation of the consequences of
the mitotic abnormalities detected. Cultures 1-2, 3-57, 9-4
and 14-1 were studied.
A B
C D E
F. 1. Feulgen stained anaphase cells with a laggard chromosome showing delayed separating chromatids (A), two typical bridges (B), one bridge
with stretched regions (possible break points) at dierent positions (C, D) and a fragment (E). Bars l10 m.
The C-banding technique employed followed the pro-
cedure described by Aguiar-Perecin (1985) with some
modications. For metaphase preparation, the cells were
pretreated with 0n03% 8-hydroxyquinoline for 2n5 h. The
materials were stored in the xative at 4 mC until the
preparation of squashes . The tissues were macerated in 5%
pectinase for 35 min at 37 mC, washed in distilled water and
softened in 45%acetic acid for 1015 min, before squashing.
The treatment in satured solution of barium hydroxide was
made at 37 mC for 25 min, then the material was washed in
deionized water and incubated in 2X SSC at 60 mC for 1 h.
The preparations were stained in 1% solution of R66
Giemsa (BDH) for 13 min, rinsed in distilled water, air-
dried and mounted in Canada balsam.
RESULTS
The analysis of anaphases in Feulgen preparations showed
two types of events which we identied as bridges: (a)
76 Fluminhan et al.Chromosome Breakage in Maize Callus Culture
F. 2. C-banded anaphase cells showing bridges with and without bands (knobs). A, B, Early anaphase showing one chromosome with delayed
separating chromatids held together at a C-banded region. It is particularly clear that this region corresponds to K7L in (B) ; note the homologous
chromosome showing normal chromatid separation (small arrows). C, Anaphase with two typical bridges with bands. D, Anaphase with one
typical bridge without band. Bars l10 m.
laggard chromosomes with delayed separating chromatids
held together at or near their ends (Fig. 1A) ; (b) typical
bridges (Fig. 1B). Bridges under stress with very stretched
chromatin regions and breakpoints at dierent positions
were observed (Fig. 1C and D). Fragments were also found
in some anaphase and telophase cells studied (Fig. 1E).
The frequency of these events was evaluated in the 13
cultures studied (Table 2). Dierences in sample size, to
some extent, are due to dierences in mitotic index between
the samples examined. The frequency of anaphase cells with
delayed separating chromatids ranged from 4n76 to 0n32%.
Cells with one typical bridge ranged from 5n52 to 0n66%,
with two typical bridges, from 4n76 to 0n52%. Anaphases
with three bridges or fragments were observed only in some
cultures. Of the telophase cells examined, 2n8 to 0n42% had
one bridge and 0n93 to 0n46% had two bridges.
The analysis of C-banded anaphases showed evidences of
the involvement of knobs in the occurrence of bridges.
Table 3 shows the types and frequencies of anaphase bridges
detected in the samples examined. In cells in which delayed
separation of chromatids was observed, chromatids could
be seen held together at the knob site, darkly stained by the
C-banding procedure (Fig. 2A and B). In Fig. 2B the
adherence of sister chromatids at the band corresponding to
K7L is particularly clear; in contrast, the homologous
chromosome shows normal chromatid separation. Typical
bridges with and without C-bands, some of them containing
fragments were observed (Fig. 2C and D; Table 3). In
culture 1-2 one telophase bridge was also observed with a
band and another one with two bridges without bands. The
size of C-bands was variable and in some bridges it was very
tiny. As is discussed below, these typical bridges may have
originated through a breakage-fusion-bridge cycle initiated
by broken arms resulting from delayed separation of
chromatids.
In order to investigate the consequences of these mitotic
abnormalities, C-banded metaphases were analysed. For
comparison, a metaphase of a plant of the original progeny
from which the line 132331 was derived, is shown in Fig. 3A.
All the metaphases and prometaphases examined had a
normal chromosome number. Aberrations involving chro-
mosome 7 were consistently observed in all the culture
samples studied, except for culture 9-4, in which gross
chromosomal aberrations were not detected among nine
Fluminhan et al.Chromosome Breakage in Maize Callus Culture 77
B C
D E
A
F. 3. A, Karyotype of a plant of the original progeny from which 132331 line was derived. B, C, C-banded metaphase cells in culture 14-1
showing amplication of the C-band corresponding to K7L in one of the homologous (B), and both homologous chromosomes (C). D, E,
Metaphases in culture 3-57 (60-week-old subculture) showing a duplication deciency in chromosome 7 short arm, while its homologues has
normal appearance (small arrows). Bars l10 m.
metaphases from a 36-week-old subculture. Some of the
aberrations involving chromosome 7 are seen in Fig. 3.
Amplication of the band corresponding to K7L, in one or
both homologues, was observed in a 48-week-old subculture
of culture 14-1 (Fig. 3B and C). Of eight cells examined, ve
had this aberration in one or both homologues. Metaphases
with one chromosome showing one arm with two bands
were observed in culture 3-57. This chromosome was
observed in ve cells of a 60-week-old subculture (Fig. 3D
and E). In a sample of a 64-week-old subculture, of 27
prometaphases and metaphases examined, 25 had this
chromosome aberration, while all the other chromosomes
of the karyotype appeared to be unaltered. Figure 4A shows
one karyotype found in this subculture. In early metaphases,
an additional euchromatic segment was observed at the end
of the altered arm (Figs 3E and 4A), while in well condensed
metaphases this was not detected (Fig. 3D). As its long arm
appeared to be an unaltered 7L, we hypothesized that there
is a duplication in chromosome 7 short arm and also a
deciency of a distal piece of K7S, resulting from a
breakage-fusion-bridge cycle involving this chromosome, as
discussed below. In the 64-week-old subculture, two
metaphases were also observed with another aberration
involving chromosome 7, also suggesting to be an dupli-
78 Fluminhan et al.Chromosome Breakage in Maize Callus Culture
A
B
F. 4. Karyotypes exhibiting structural aberrations involving chromosome 7 short arm in a 64-week-old subculture of culture 3-57. A, Early
metaphase showing a duplication deciency with two C-bands (K7S) and distal euchromatic segment ; part of an overlapping chromosome is seen
on one of the homologues of chromosome 3 and 5. B, Duplication deciency with K7S at subterminal position. Bar l10 m.
cation deciency involving chromosome 7 short arm, but
without an extra knob (Fig. 4B). In culture 1-2, an aberrant
chromosome, rather similar to the one observed in Fig. 4B,
was observed in 17 prometaphases and metaphases of a 36-
week-old subculture.
DISCUSSION
The present study showed interesting aspects of the
involvement of knobs in the formation of chromosome
bridges leading to chromosome breakage in cells of
proembryoids of maize callus cultures. C-bands were
unequivocally observed at the point where sister chromatids
are held together in anaphase congurations showing
delayed separating chromatids. This event is particularly
clear in Fig. 2B, in which the separation of the chromatids
of chromosome 7 is delayed at K7L. This observation gives
support to the hypothesis previously proposed to explain
the frequent presence of knobs in chromosome arms
involved in rearrangements observed in pachytene chromo-
somes of maize regenerated plants (Lee and Phillips, 1987;
Benzion and Phillips, 1988). So, the occurrence of delayed
or partially suppressed replication of heterochromatic knobs
would explain the origin of this kind of bridge. On the other
hand, the presence of typical bridges observed in Feulgen
and C-banding preparations suggests that after chromosome
breakage resulting from failure of knob replication, the
chromatid type of breakage-fusion-bridge (BFB) cycle
described by McClintock (1939, 1941, 1942) could have
been initiated by broken chromosomes in subsequent
mitoses. Typical bridges without and with bands, some of
them very tiny, observed in C-banding preparations, can be
taken as evidence that breakpoints must have occurred
between knobs and the centromere or at knob sites, thus
reducing their size.
The chromosome aberrations involving chromosome 7,
observed in some cultures, could also be taken as evidence
for the occurrence of BFB cycles in the callus cultures
studied. Therefore, we suggest a mechanism (Fig. 5) to
explain the presence of cells showing a chromosome with
one arm possessing two bands, seen in culture 3-57 (Figs
3D, E and 4A). If the replication of K7S was partially
suppressed or delayed and the breakpoint on the resulting
bridge occurred at the knob site, a decient chromatid with
a reduced knob would be formed. After this primary event,
the chromatid type of BFB cycle could have given origin,
after two mitotic divisions, to a duplication in chromosome
7 short arm and also to a deciency for a distal segment of
K7S. The detection of such a chromosome in several cells of
two subcultures, suggests that this aberration was trans-
mitted over cell generations and that the broken end of this
chromosome healed. We can also interpret that due to its
small size, the additional euchromatic segment present at
the end of the altered arm, can only be detected in early
metaphases and not in highly condensed metaphase chromo-
somes. Certainly, new events of delayed separation of
chromatids could produce changes in this chromosome and
this seems to be the case of the altered chromosome 7 shown
in Fig. 4B and observed in the same sample of the subculture
analysed. In culture 1-2, the altered chromosome rather
similar to the one shown in Fig. 4B may also have originated
through the same primary event hypothesized in Fig. 5,
followed by a BFB cycle and healing of the broken end,
after some cell divisions.
The enlarged band on 7L observed in metaphase cells of
culture 14-1 could also have arisen by a similar mechanism
to that suggested in Fig. 5, but due to a failure of replication
at K7L, and to a breakpoint at\or adjacent to the knob,
between it and the telomere. If this broken chromatid
initiated a BFBcycle, a dicentric chromatid with an enlarged
knob would be produced. If a new break occurred between
this knob and one of the centromeres, a chromosome with
a duplication of the knob and of a proximal euchromatic
segment adjacent to it, would be produced. This chromo-
some would also be decient for the distal euchromatin,
between the knob and the telomere. On the other hand, as
the morphology of such a chromosome seems to be
unaltered, this could also suggest that the enlarged band is
the result of the amplication of knob DNA sequences, as
shown in wheatrye hybrids (Lapitan et al., 1988) and rye
(Karp et al., 1992). However, the conguration seen in Fig.
2B and the presence of bridges in all the subcultures studied,
supports the idea that BFB cycles can be a frequent event
giving origin to duplication deciencies in the cultures
studied.
The anaphase congurationshowing twoknobbed bridges
quite similar one to another (Fig. 2C), could be the result
of the fusion of a broken chromosome 7 and another
Fluminhan et al.Chromosome Breakage in Maize Callus Culture 79
Fai l ure of
repl i cati on
at K7S
Anaphase wi th
del ayed separati ng
chromati ds
Breakage at knob
regi on
Repl i cati on and fusi on
of broken arms
Anaphase
Breakage
Anaphase bri dge
resul ti ng from the
l argest di centri c
chromati d
Repl i cati on and fusi on
of broken ends
Breakage
F. 5. Diagram of the mechanism that possibly gave origin to the duplication deciency (Fig. 4A) in chromosome 7 observed in culture 3-57.
If delayed or partially suppressed replication occurred at K7S, chromosome breakage might result from bridge formation. Then, a chromatid type
of BFB cycle would be initiated by broken chromosome arms and if chromosome healing occurred after some cell divisions, the duplication
deciency originated could be maintained over cell generations. Small arrows indicate hypothetical breakpoints at anaphase bridges.
chromosome, perhaps a knobless one, resembling the
chromosome type of BFB cycle described by McClintock
(1942).
A well known case of bridge formation is the one reported
by Rhoades and Dempsey (1972, 1973), to explain the
elimination of chromatin from knob bearing chromosomes,
that occurs when the replication of knobs is presumably
delayed or suppressed in the presence of B chromosomes at
the second microspore division. The BFB cycle initiated by
broken chromosome arms has been detected in successive
gametophyte mitoses and in the endosperm. Instead, healing
of broken chromosome ends has been reported to occur in
the zygote (McClintock, 1941, 1942; Rhoades and Dempsey,
1973). It is interesting to note that in further studies,
McClintock (1978) reported the occurrence of the BFB
cycle during initial stages of plant development, concluding
that it had ceased in surviving seedlings, thus pointing to the
possibility of BFB cycles to occur in early stages of
development. Also, Rhoades and Dempsey (1972) men-
tioned that all knobbed chromosomes tested might undergo
chromatin loss due to failure of replication at knob sites, but
that chromosome arms possessing large knobs were lost
more frequently. This could also be the case of chromosome
7 in the present study. This chromosome with knobs at both
arms (K7L is a large knob and K7S a medium size one),
would be more prone to be aected during DNA replication
in callus culture.
As mentioned above, the investigation of chromosome
variation through meiotic analysis of regenerated plants of
several maize genotypes has shown that most aberrations
found involved chromosome arms containing knobs (Lee
and Phillips, 1987; Benzion and Phillips, 1988). No
evaluation of gross chromosome aberrations revealed by the
C-banding technique in callus cultures themselves has ever
been made. In the present study, the analysis of C-banded
metaphases of samples of three cultures suggested that
chromosome 7 was more frequently aected and this was
quite clear in culture 3-57. Samples of this vigorously
80 Fluminhan et al.Chromosome Breakage in Maize Callus Culture
growing culture were then subcultured on several Petri
dishes, and a further analysis of 2 to 3-year-old subclones
were carried out, providing new evidence of the occurrence
of BFB cycles and healing of broken ends, involving
chromosomes 7 and 9, possessing large knobs. The results
of the biometrical analysis of the karyotypes found in this
long-term culture and in another one derived from a related
genotype with dierent knob composition and lower
frequency of mitotic abnormalities and chromosome aber-
rations, will be reported elsewhere (Santos and Aguiar-
Perecin, unpubl. res.). It is also interesting to emphasize that
a meiotic analysis of regenerated plants derived from these
cultures is a next step, important for the understanding of
the degree of transmission of chromosome aberrations from
cultures to regenerants and also, to evaluate the eciency of
the investigation of maize chromosome abnormalities
through the analysis of somatic C-banded chromosomes.
Some studies reporting or inferring the appearance of
BFB cycles in itro are found in the literature. Murata and
Orton (1984) found a high frequency of cells with structural
changes involving chromosome fusion in a 6-month-old
suspension culture of celery highly associated with hypo-
diploidy and involving late-replicating telomeric hetero-
chromatin. They proposed that these aberrations might be
the primary source of other chromosome changes, that
would have been produced by BFB cycles. This assumption
was supported by their observation of anaphase bridges
(17%) and would explain their previous observation of
multiconstrictional chromosomes in a 1n5-year-old cell
suspension culture (Murata and Orton, 1983). Stelly et al.,
1989) found a high frequency of tertiary monosomy among
35 regenerated plants derived from 18-month-old callus
cultures of two cultivars of cotton. They hypothesized that
this type of aberration could be derived from BFB cycles
arising in itro, through mechanisms not detected by the
authors. Basically, they proposed that a dicentric chromatid
could be formed by the fusion of two nonhomologous BFB-
cycle chromatids. Functional monocentricity of this chro-
matid could result from the accumulation of BFB cycles and
loss of intercentromeric chromatin or through permanent
inactivation of one centromere. The possibility of occurrence
of BFB cycles in maize tissue cultures has been discussed but
not reported. In a callus culture derived from mesocotyl
tissue, Balzan (1978) found a high frequency of tetraploid
cells and also detected mitotic abnormalities such as
anaphase bridges and\or lagging chromosomes. Dicentric
chromosomes were found in hypertetraploid cells, but BFB
cycles were not reported. Lee and Phillips (1987) identied
changes in chromosome structure, such as interchanges,
intercalary deciencies and heteromorphic pairs (deciencies
or duplications) in 91 of 189 regenerated plants derived
from 8 to 9-month-old callus cultures of an Oh43-A188
genetic background. To explain the origin of these aberra-
tions, they hypothesized: (a) occurrence of anaphase bridges
owing to late-replication of knobs; (b) occurrence of
simultaneous breakage in homologous chromosomes lead-
ing to duplications and deciencies, whereas simultaneous
breakage in nonhomologous chromosomes could lead to
reciprocal interchanges. Plants from the same culture were
found carrying an identical aberration. As dicentric chro-
mosomes were not detected through meiotic analysis, they
concluded that there was no evidence of the occurrence of
BFB cycles, and that an aberration might trace to a single
event that would have occurred early during culture
initiation.
Our results provide evidence for the occurrence of BFB
cycles in the callus cultures investigated and raises some
interesting questions regarding the eect of culture age on
the frequency of mitotic abnormalities such as anaphase
bridges, and occurrence of chromosome healing. So, if
healing occurs after some cell divisions, then we should not
expect an accumulation of cells undergoing BFB cycles. In
this connection, Stelly et al. (1989) assumed that multiple
BFB cycles would accumulate within cotton cell lines and
would account for the predominance of tertiary monosomy
among the regenerated plants studied. One of the aspects
mentioned in their discussion is that this would be limited
by chromosome healing. Also, it is interesting to note that
as demonstrated by McClintock (1978), BFB cycles may
induce new breakages in other chromosomes not involved in
the cycle, or activate silent transposons. So, if this happened
in a cell culture, breakage events could be accumulated with
age. In the present study, we examined cultures with
dierent ages with the purpose of documenting the presence
of anaphase bridges in dierent samples. This experiment
was not outlined to study age eects, however, the problems
raised are interesting for further investigation.
If delayed separation of chromatids is the primary event
causing bridge formation, then we could expect a lower
frequency of bridges in genotypes with low knob contents.
This was investigated using 5 to 6-month-old cultures
derived from families of inbred lines diering in their knob
content and the results will be reported elsewhere, together
with an account of the morphogenetic response of these
lines. One of the most interesting aspects observed was that
a strict correlation between the frequencies of anaphase
bridges and knob content was not always found, and that
some inuence of the genotype must be important for the
occurrence of mitotic abnormalities (Fluminhan and
Aguiar-Perecin, unpubl. res.).
The processes leading to the occurrence of disturbance in
the cell cycle have been discussed in the literature and
changes in DNA methylation could be one of the mech-
anisms involved in the unusually later replication of
heterochromatin (see Peschke and Phillips, 1992). Both
methylation increases and decreases have been detected in
regenerated plants (Brown and Lorz, 1986; Brown, 1989;
Kaeppler and Phillips, 1993). Kaeppler and Phillips (1993)
observed a trend toward decreasing methylation in progeny
lines derived from tissue culture, concluding that this could
lead to various eects, including chromosome breakage. In
this connection, it is interesting that Neves et al. (1992)
showed that the epigenetic process by which somatic cells of
rye maintain the inactivity of genes responsible for B
chromosome nondisjunction, between fertilization and
meiosis is mediated through DNA methylation. Thus, it is
possible that the unusually later replication of knobs in
culture may be an outcome of methylation decrease.
Some of the problems raised in the present study are
interesting phenomena to be further investigated for a better
Fluminhan et al.Chromosome Breakage in Maize Callus Culture 81
understanding of the causes of chromosome instability in
maize tissue culture and selection of genotypes and culture
conditions to achieve low levels of chromosome variation.
ACKNOWLEDGEMENTS
This work was supported by FAPESP and CNPq. The
authors are grateful to Eng. Agr. J. U. Decico for the
collaboration in the determination of the knob composition
of the inbreds used in this study and for Fig. 3A; and to
C. A. Ver!ssimo and S. A. Gaziola for technical help in
tissue culture work.
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