Annals of Botany 82: 569576, 1998

Article No. bo980710

Embryogenic Response and Mitotic Instability in Callus Cultures Derived from
Maize Inbred Lines Diering in Heterochromatic Knob Content of Chromosomes
A. FLUMINHAN* and M. L. R. DE AGUIAR-PERECIN
Departamento de GeneTtica, Escola Superior de Agricultura Luiz de QueiroTz, Uniersidade de Sago Paulo,
13400-970, Piracicaba, SP, Brazil
Received: 7 March 1997 Returned for revision: 3 July 1997 Accepted: 8 June 1998
Four families of sister inbred lines derived from a tropical maize variety have been evaluated for their ability to form
callus cultures with a morphogenetic response. Lines were homozygous for heterochromatic knobs at 6L
#
, 6L
$
, 7L
and 8L
"
but diered for the presence or absence of K2L, K3L, K7S and K9S. Clear dierences in embryogenic
response were observed between the families of inbreds. Only one family formed friable, highly embryogenic type II
calli ; the other families formed slow growing non-embryogenic or poorly embryogenic cultures. All the genotypes
screened showed a similar response to the two culture media tested, suggesting that genetic factors are responsible for
the major dierences among the families. Mitotic abnormalities were investigated in Feulgen preparations of most
cultures. Anaphase bridges resulting fromdelayed chromatid separation, typical bridges and fragments were observed.
In a previous study, delayed chromatids were shown to be held together at heterochromatic knob sites, while typical
bridges would be formed by dicentric chromatids arising frombreakage-fusion-bridge cycles initiated by chromosome
arms broken during the primary event. In the present study, the frequency of both types of bridges was not strictly
correlated with the knob content of the genotypes analysed. This suggests that knobs may undergo alterations in
culture leading to mitotic disturbance, and that this response may be genotype dependent.
# 1998 Annals of Botany Company
Key words: Zea mays L., maize, plant tissue culture, somatic embryogenesis, somaclonal variation, heterochromatin,
C-banding, breakage-fusion-bridge cycle.
INTRODUCTION
Plant regeneration from maize embryo-derived callus
cultures was rst reported by Green and Phillips (1975).
Since then, it has become evident that the genotype used is
important for tissue culture response. Somatic embryo-
genesis in compact type I calli derived from several inbreds
and hybrids adapted to temperate regions has been reported
(Lu, Vasil and Ozias-Akins, 1982; Lu, Vasil and Vasil,
1983; Duncan et al., 1985; Tomes and Smith, 1985; for a
review see Phillips, Somers and Hibberd, 1988; Henry,
Vain and De Buyser, 1994). Some maize genotypes of
tropical and subtropical origin have also been shown to
produce embryogenic calli (Prioli and Silva, 1989; Furini
and Jewell, 1994; Bohorova et al., 1995). However, few
genotypes give rise to friable type II calli capable of somatic
embryogenesis.
Nuclear as well as cytoplasmic genetic eects on tissue
culture responses and plant regeneration have been reported
(reviewed by Henry et al., 1994). In the case of maize, there
has been success in breeding lines for improved performance
in tissue culture (Armstrong, Romero-Severson and Hodges,
1992).
Numerous studies have been conducted on the genetic
* Present address: Institute of Physical and Chemical Research
(RIKEN), Wako, Saitama, 351-01, Japan.
For correspondence.
and cytogenetic variation in plants regenerated from maize
tissue culture (reviewed by Lee and Phillips, 1988; Peschke
and Phillips, 1992). Although such variation, termed
somaclonal variation (Larkin and Scowcroft, 1981), may be
useful for crop improvement, it is undesirable when genetic
stability is required. Chromosome breakage associated with
heterochromatin regions is frequently observed in plant
tissue culture (Sacristan, 1971; McCoy, Phillips and Rines,
1982; Lapitan, Sears and Gill, 1984, 1988; Murata and
Orton, 1984; Johnson, Phillips and Rines, 1987; Lee and
Phillips, 1987; Joachimiak et al., 1995). Meiotic studies of
regenerated maize have shown that most breakpoints are on
chromosome arms containing heterochromatic knobs, and
it has been proposed that normally late replicating regions
might replicate even later in the culture environment leading
to the formation of anaphase bridges due to delayed
separation of sister chromatids at knob sites (Lee and
Phillips, 1987). Recently, Fluminhan, Aguiar-Perecin and
Santos (1996) investigated the occurrence of mitotic in-
stability in embryogenic callus cultures derived from maize
inbreds possessing similar knob composition. Bridges
resulting from delayed separation of sister chromatids held
together at knob sites were observed, thus supporting the
hypothesis proposed by Lee and Phillips (1987). Fur-
thermore, the presence of typical bridges with and without
knobs detected by C-banding, and metaphase cells showing
gross aberrations involving chromosome arms possessing
large knobs, suggested the occurrence of breakage-fusion-
0305-7364\98\110569j08 $30.00\0 # 1998 Annals of Botany Company
570 Fluminhan and Aguiar-PerecinCallus Cultures Deried from Maize Inbred Lines
bridge (BFB) cycles initiated by chromosome arms broken
during the primary event.
In the present work, we analysed the frequency of
initiation of embryogenic calli derived from sister inbred
lines diering in their knob composition, derived from a
maize variety of tropical origin. Mitotic anaphase cells were
examined to investigate the presence of bridges and
correlations between their frequency and knob content of
each genotype analysed.
MATERIALS AND METHODS
Plant material
Sister inbred lines (S
(
, S
)
, S
*
) derived from a maize int
variety (Jac Duro, Sementes Agroceres, Brazil) of tropical
origin were used. These lines were grouped in four families
derived from the cross of two plants of an S
#
progeny from
a single S
"
plant. All these lines are homozygous for
heterochromatic knobs at 6L
#
, 6L
$
, 7L and 8L
"
(some lines
of the sub-group 4411 were also shown to have a very small
knob at 8L
#
), but families diered in the presence of knobs
at 2L, 3L, 7S and 9S, as shown in Table 1 (see references in
Aguiar-Perecin and Decico, 1988). Knobs are medium- and
large-sized, except for the small ones at 6L
#
and 6L
$
.
T 1. Chromosome knob composition and embryogenic response of the families of inbred lines analysed
Knobs* Embryogenic response (%)
Families Sub-families Lines K2L K3L K7S K9S MS Medium N
'
Medium Total
1-3 1-3-1 131311\1 (S
*
) 00 00 jj jj 55n17 (32\58) 50n00 (30\60) 52.54 ab
131312\1 (S
*
) 00 00 jj jj 28n95 (11\38) 27n59 (16\58) 28n13 cde
13151\1 (S
)
) 00 00 jj jj 75n00 (15\20) 78n95 (15\19) 76n92 a
13153\1 (S
)
) 00 00 jj jj 54n39 (31\57) 58n18 (32\55) 56n25 ab
1-3-2 132331\1 (S
*
) 00 00 jj jj 53n85 (42\78) 55n32 (52\94) 54n65 ab
1-3-3 13332\1 (S
)
) 00 00 jj 00 42n31 (22\52) 42n00 (21\50) 42n16 bcd
13333\1 (S
)
) 00 jj jj 00 48n39 (15\31) 45n16 (14\31) 46n77 bcd
133131\3 (S
*
) 00 jj jj 00 27n78 (10\36) 27n27 (21\77) 27n43 de
133112\2 (S
*
) 00 00 jj 00 37n50 (15\40) 37n50 (15\40) 37n50 bcde
133112\3 (S
*
) 00 00 jj 00 52n17 (24\46) 42n86 (15\35) 48n15 abc
13351\2 (S
)
) 00 00 jj 00 35n14 (13\37) 36n84 (14\38) 36n00 bcde
133512\2 (S
*
) 00 00 jj 00 33n93 (19\56) 30n56 (11\36) 32n61 cde
2-1 2-1-1 21113\1 (S
)
) 00 jj 00 jj 0.00 (0\92) 0.00 (0\86) 0.00 h
2-1-2 21241\1 (S
)
) 00 jj 00 jj 11.54 (6\52) 10.00 (8\80) 10n61 fg
2-1-3 21311\2 (S
)
) 00 jj 00 jj 0n00 (0\68) 0n00 (0\68) 0n00 h
4-1 4-1-1 41121\1 (S
(
) jj jj 00 jj 6n67 (5\75) 5n26 (3\57) 6n06 gh
41123\2 (S
(
) jj jj 00 jj 20n00 (8\40) 20n00 (8\40) 20n00 ef
4-1-2 41242\1 (S
(
) jj 00 00 jj 10n11 (9\89) 10n00 (2\20) 10n09 fg
41242\2 (S
(
) jj 00 00 jj 7n50 (6\80) 10n00 (6\60) 8n57 fgh
4-4 4-4-1 44114\1 (S
(
) jj jj jj jj 14n81 (8n54) 10n53 (4\38) 13n04 efg
44114\2 (S
(
) jj jj jj jj 41n51 (22\53) 40n00 (14\35) 40n91 bcd
44133\2 (S
(
) jj jj 00 jj 1n79 (1\56) 3n33 (2\60) 2n59 h
* All lines were homozygous for knobs at 6L
#
, 6L
$
, 7L, and 8L
"
(a very small knob at 8L
#
is present in some plants of lines 44114) ; jj,
homozygous for presence; 00, homozygous for absence.
Signicant dierences between the frequencies of embryogenic calli grown on MS and N
'
media were not detected by analysis of variance.
Numbers within parentheses are the number of embryogenic calli scored at the eleventh subculture per number of calli observed 45 d after culture
initiation.
Means with dierent letters dier signicantly at the 5% level, by Tukey test.
The frequency of embryogenic calli was scored at the eleventh subculture and expressed as percent per number of calli observed 45 d after culture
initiation.
Culture conditions and analysis of embryogenic response
Cultures were initiated from immature embryos 1n0
2n0 mm in length, aseptically isolated from surface
sterilized ears of twove plants of each genotype, self-
pollinated in the eld at Piracicaba, SP, Brazil. About 3040
embryos from each ear were cultured.
Approximately equal numbers of embryos were placed in
Petri dishes containing agar solidied medium with MS
(Murashige and Skoog, 1962) or N
'
(Chu et al., 1975)
inorganic components. Both culture media were supple-
mented as previously described (Fluminhan et al., 1996).
Briey, the organic components were 99 mg l

" inositol,
39n4 mg l

" cysteine and vitamins according to Prioli and

Silva (1989), supplemented with 2 mg l

" 2,4-dichloro-
phenoxy-acetic acid (2,4-D), 20 g l

" sucrose, 20 mg l

"
casein hydrolysate and 8n0 g l

" agar adjusted to pH 5n8.

Cultures were maintained by subculturing every 1520 d at
26 mC in the dark.
Calli were scored at each subculture for the presence of
somatic embryoids, for approx. 56 months. Cultures were
classied as non-embryogenic or embryogenic. Calli identi-
ed as type II, i.e. friable calli with well dened somatic
embryoids, and more complex calli with embryo-like
structures or that were apparently organogenic were scored
Fluminhan and Aguiar-PerecinCallus Cultures Deried from Maize Inbred Lines 571
as embryogenic, as recommended by Tomes and Smith
(1985). Embryogenic response is expressed as the percent of
embryogenic calli at the eleventh subculture (approx. 5
months after culture initiation) per total number of calli
formed 45 d after culture initiation.
Mean values of the embryogenic response of the dierent
lines were compared by the Tukey test, after analysis of
variance to test for signicant dierences between culture
media and eects of families, sub-families with dierent
knob contents and lines (data not shown). All percentage
data were transformed to NP%j0n5 (Plpercentage of
embryogenic calli) before analysis (Snedecor and Cochran,
1980).
Cytological analysis
To investigate the eect of knobs on the formation and
frequency of anaphase bridges, Feulgen stained preparations
from cultures derived from fteen lines were examined (see
Table 2). Five calli from each line cultivated on MS medium
were analysed and samples were taken approx. 6 months
after culture initiation. For mitosis preparations, the
procedure described by Fluminhan et al. (1996) was
employed. Samples of globular stage proembryoids were
taken from embryogenic calli 5 d after their transfer to fresh
medium. As for non-embryogenic cultures, actively growing
regions were collected from callus surface.
Approximately 4050 anaphase cells per callus were
analysed. The anaphase congurations examined were
classied as early, mid- and late anaphases. Very early
anaphases were not scored. The total frequency of anaphase
bridges (delayed separating sister chromatidsjtypical
bridges), expressed as a percentage of the total number of
anaphase cells examined, was evaluated by statistical
analysis for comparison between families, sub-families and
lines. Only observations made on early and mid-anaphases
are reported to avoid underestimating the frequency of
bridges in late anaphases (Fluminhan, 1992). Means of
bridge frequencies were compared by the Tukey test, after
analysis of variance.
RESULTS AND DISCUSSION
Embryogenic response
Clear dierences in embryogenic response were observed
between the four families of inbreds, but the frequencies of
embryogenic calli grown on MS and N
'
media did not dier
signicantly by analysis of variance (Table 1). All the lines
belonging to the family 13 had a higher frequency of
immature embryos capable of producing friable calli with
well formed embryos supported by suspensor-like structures
on callus surface (Fig. 1A). These calli were similar to type
II cultures described by Armstrong and Green (1985). The
frequency of embryogenic calli derived from lines of sub-
families 1-3-1 and 1-3-2 ranged from 76n92 to 28n13%
(Table 1). Most of these cultures could be maintained for
more than 18 months, without alteration in their phenotype.
Genotypes of subfamily 1-3-3 produced a rather lower
frequency of embryogenic cultures, ranging from 48n15 to
27n43%, but also formed type II calli. Aconsistent dierence
was observed in family 2-1, which showed a lower ability for
embryogenesis in culture and usually produced translucent,
slow growing calli. Most cultures were non-embryogenic
(Fig. 1B and C). Calli showing a morphogenetic response,
derived from inbred 21241\1 (10n61%) did not have a type
II appearance, but were organogenic. They formed shoot
apices rather than somatic embryos. Similar results were
observed for genotypes 4-1 and 4-4, with frequencies ranging
from 20n0 to 6n06% within family 4-1, and 40n91 to 2n59%
within family 4-4 (Table 1). Sister inbred lines 44114\1 and
44114\2 gave a dierent response. The calli were compact
and non-embryogenic, but in approximately the seventh
subculture they formed friable regions. When subcultured,
the friable tissues formed rapidly growing cultures, with
shoot apices and somatic embryos that diered in ap-
pearance to type II cultures (Fig. 1D).
Comparison of the frequencies of embryogenic calli
showed signicant dierences among lines, between and
within families (Table 1). The dierences between families
suggest that the frequency of embryogenic calli is inuenced
by genetic factors, whereas dierences between lines within
the same family or subfamily may have resulted from
environmental eects, such as the physiological state of the
donor plant or the immature embryo. All the lines of sub-
families 1-3-1, 1-3-2 and 1-3-3 have the ability to form
friable, highly embryogenic calli detectable 4560 d after
culture initiation. Families 2-1, 4-1 and 4-4 gave a lower
embryogenic response, with slow growth rate in most
cultures. Because of this, we adopted the procedure of
expressing the frequency of embryogenic calli scored at the
eleventh subculture. This made it possible to evaluate the
capacity of these slow growing cultures to give a mor-
phogenetic response.
The data suggest that during successive self fertilizations
which gave rise to the inbreds studied, segregation of genetic
factors aecting culture response might have occurred
among the families of lines. Several studies have addressed
the problem of identifying genetic factors inuencing plant
regeneration from maize cultures (Beckert and Qing, 1984;
Duncan et al., 1985; Tomes and Smith, 1985; Armstrong et
al., 1992). There is evidence that the embryogenic response
can be introduced by breeding into agronomically valuable
genotypes. Armstrong et al. (1992) detected, by RFLP
analysis, chromosome regions promoting embryogenic
callus initiation and plant regeneration and observed that
the frequency of initiation of embryogenic callus from
immature embryos of the elite inbred B73 was increased by
introgression of chromosomal segments from the inbred
A188 through backcross breeding. The embryogenic re-
sponse of lines adapted to tropical and subtropical regions
has also been investigated (Prioli and Silva, 1989; Furini
and Jewell, 1994; Bohorova et al., 1995). Prioli and Silva
(1989) found the highest frequency of embryogenic calli
among Cateto inbreds and suggested that the Cateto race
has a high frequency of genes controlling the plant
regeneration response. It is of interest that the Cateto race
is one of the components of the variety used as the source of
inbreds in the present study (U. Ribeiral, Sementes
Agroceres, pers. comm.). This raises the possibility that the
higher incidence of type II calli in lines of family 1-3 might
572 Fluminhan and Aguiar-PerecinCallus Cultures Deried from Maize Inbred Lines
F. 1. Calli derived from immature maize embryos. A, Friable type II, highly embryogenic callus derived from a line (132331\1) of family
1-3. i12; B, non-embryogenic callus derived from a line (21241\1) of family 2-1. i7; C, non-embryogenic slow-growing calli, derived from one
line of family 2-1. i1n5; D, rapid-growing culture, apparently organogenic derived from fragments of a friable sector of a compact culture of line
44114\2. i1n5.
Fluminhan and Aguiar-PerecinCallus Cultures Deried from Maize Inbred Lines 573
reect a higher incidence of Cateto chromosome segments
important for the embryogenic response. A similar ex-
planation might account for the special features of fast
growing cultures derived fromthe two inbreds of family 4-4.
All the genotypes screened showed a similar response to
the two culture media tested suggesting that genetic factors
are responsible for the major dierences among families.
The composition of the culture medium is known to aect
the embryogenic response of maize tissue cultures. Lines
derived from the same maize embryo form embryogenic
type II callus on proline-containing medium and organo-
genic type I callus on the same medium without proline
(Armstrong and Phillips, 1988). The concentration of 2,4-D
and sucrose (Prioli and Silva, 1989) and supplementation of
media with Dicamba (Furini and Jewell, 1994; Bohorova et
al., 1995) have been reported to aect the initiation of
embryogenic calli of tropical and subtropical inbreds. This
suggests that altering medium composition could be used to
improve the response of the embryogenic genotypes of
family 1-3 and to promote a type II response in the 44114
genotypes.
Cytological analysis
The examination of Feulgen stained preparations revealed
anaphase abnormalities similar tothose described previously
for genotypes of family 1-3 (Fluminhan et al., 1996) : (a)
bridges resulting from delayed separation of chromatids
held together at\or near their ends (Fig. 2A) ; (b) one or two
typical bridges (Fig. 2B) ; (c) anaphases with fragments (Fig.
2C) ; and (d) anaphases with broken bridges, identied by
the presence of chromatids with uncoiled ends (Fig. 2D). In
a previous study of C-banded anaphases, delayed separating
chromatids were shown to be held together at knob sites,
resulting in chromosome breakage (Fluminhan et al., 1996).
Chromosome arms broken during this primary event would
initiate BFB cycles, thus resulting in the typical bridges
observed. Depending on the position of breakpoints in both
types of bridges, fragments may result.
Table 2 shows the frequency of anaphase abnormalities
observed in dierent families of inbred lines. Comparison of
these results with the knob contents of lines presented in
Table 1 shows that the frequency of total bridges and knob
content is not always strictly correlated. This correlation is
to be expected if one assumes that delayed separation of
chromatids at knob sites is the primary event leading to
bridge formation. In cultures derived fromsub-families 1-3-1
and 1-3-2 with the same knob composition, the frequency
of bridges ranged from 4n02 to 8n34%, whereas in subfamily
1-3-3, lacking the large knob at 9S, the frequency was rather
lower, ranging from 2n04 to 5n55%. It is of interest that the
frequency of anaphase bridges was not higher in cultures
derived from sister lines 1-3-3, which are homozygous for
the presence of a medium size knob at 3L (13333\1 and
133131\3).
In the cultures derived from lines belonging to families
4-1 and 4-4, the frequency of total bridges ranged from 7n14
to 1n61% and was not signicantly higher than the values
observed in families 1-3 and 2-1 which have lower knob
contents. Of all the material studied, cultures derived from
line 44114\2 exhibited the lowest frequency of bridges.
Moreover, the number of broken bridges, fragments or
anaphase cells with two or multiple bridges was not higher
in this material. Cultures derived from line 44133\2, which
belongs to family 4-4, but lacks the knob at 7S, showed a
higher frequency of bridges than did cultures 44114\2.
Comparison of subfamilies 4-1-1 and 4-1-2 showed that line
41242\2 lacking K3L had a higher frequency of bridges
(7n14%) than did the line 41123\2 (4n21%) with K3L.
The trend of cultures derived from lines 1-3-3 to have
lower frequencies of bridges than cultures 1-3-1 and 1-3-2
could be explained by the absence of the large knob at 9S in
lines 1-3-3. Previously we showed that chromosome 7 is
quite frequently altered in cultures derived from lines
belonging to the sub-groups 1-3-1 and 1-3-2 (Fluminhan et
al., 1996). In contrast, chromosome 9 with a large knob at
the short arm showed a lower occurrence of alterations in a
culture derived from a family 1-3 genotype (Santos and
Aguiar-Perecin, unpubl. res.). Therefore, the bridges found
in the present work might have been induced by delayed
separation of chromosome 7 and\or chromosome 9
chromatids. On the other hand, the observation that cultures
derived from lines of families 4-1 and 4-4 did not have
higher frequencies of mitotic abnormalities, e.g. the lower
occurrence of bridges in genotype 44114\2, suggests that
breakage events resulting from mitotic disturbance at knob
sites may be genotype dependent. Although the lines used
were derived from a single plant, we found that families
diered in embryogenic response and these families also
diered in owering time and seed size (Aguiar-Perecin,
unpubl. res.).
There is considerable evidence that chromosomal break-
age in cultured plant cells is associated with abnormalities in
the delayed replication of DNA characteristic of repetitive
sequences present in heterochromatin (see Peschke and
Phillips, 1992; Nuti Ronchi, 1995). The correlation between
heterochromatin content and occurrence of chromosome
aberrations in regenerated plants appears to depend on
genotype. McCoy et al. (1982) concluded that chromosomal
aberrations found in meiotic cells of regenerated oat plants
arose fromchromosome breakage events in culture resulting
from abnormal replication of pericentromeric hetero-
chromatin. However, they found that the frequency of
cytogenetically abnormal plants diered in the two geno-
types studied.
Bebeli, Karp and Kaltsikes (1990) reported the occurrence
of chromosome variation including translocations, tetra-
ploidy and trisomy in addition to meiotic disturbances in
rye plants regenerated from sister lines diering in their
content of telomeric heterochromatin. Heterochromatin
content and chromosome variability was not strictly
correlated and the mode of regeneration and occurrence of
somaclonal variation was not clearly related. Bebeli,
Kaltsikes and Karp (1993a, b) also found that R
#
families
derived from immature-embryo cultures of triticale and rye
lines diering in heterochromatin content varied in their
incidence of somaclonal variants for agronomic traits.
Triticale lines possessing telomeric heterochromatin released
more variation than those lacking it, but similar results were
not observed in rye.
574 Fluminhan and Aguiar-PerecinCallus Cultures Deried from Maize Inbred Lines
F. 2. Feulgen stained anaphase cells of the callus cultures showing abnormalities. A, Early anaphase showing one chromosome with delayed
separating chromatids (primary event) ; B, late anaphase with two typical bridges (probably originated from dicentric chromatids involved in a
BFB cycle) and two fragments; C, late anaphase with two fragments; D, mid-anaphase with a broken bridge. Bar l10 m.
T 2. Abnormalities in early and mid-anaphase cells in 56 month old callus cultures deried from lines with dierent knob
contents
No. cells
Anaphase cells with abnormalities (%)
Total Total bridges
Lines analysed* DSC 1B 2B Fragm. Others abnorm. (%) (%)
131311\1 174 1n15 2n30 0n57 1n15 0n57 5n74 4n02 abcd
131312\1 96 3n13 4n17 1n04 2n08 1n04 11n46 8n34 d
13151\1 180 1n67 3n33 0n56 0n0 0n0 5n56 5n56 abcd
13153\1 150 1n33 3n33 0n67 0n67 1n33 7n33 5n33 abcd
132331\1 136 2n21 4n41 1n47 1n47 2n94 12n50 8n09 cd
13333\1 129 1n55 2n33 0n78 1n55 0n0 6n21 4n66 abcd
133131\3 140 0n71 2n14 0n0 1n43 0n71 4n99 2n85 ab
133112\1 216 2n31 2n31 0n93 0n93 0n93 7n41 5n55 abcd
13351\2 126 0n79 2n38 0n0 0n79 0n0 3n96 3n17 abc
133512\2 197 0n51 1n02 0n51 1n02 1n02 4n08 2n04 ab
21241\1 227 1n32 1n32 0n88 0n44 0n44 4n40 3n52 abcd
41123\2 190 1n05 2n63 0n53 0n53 1n58 6n32 4n21 abcd
41242\2 140 2n14 4n29 0n71 0n71 0n71 8n56 7n14 bcd
44114\2 248 0n40 1n21 0n0 0n40 0n0 2n01 1n61 a
44133\2 231 1n30 2n16 0n87 0n0 0n0 4n33 4n33 abcd
* Five calli from each line were analysed.
DSC, Delayed separating chromatids; 1B, 2B, respectively, one and two typical bridges (possibly formed by dicentric chromatids) ; Fragm,
fragments; Others, broken bridges or broken bridgesjfragments.
Total bridges lDSCj1Bj2B. Means with dierent letters dier signicantly at the 5% level by Tukey test.
Fluminhan and Aguiar-PerecinCallus Cultures Deried from Maize Inbred Lines 575
Therefore, we suggest that the frequency of bridges
observed in the present study depends not only on the
presence of knobs in chromosome arms, but also on the
genotype and its response to culture environment as well. It
is of interest that Fluminhan and Kameya (1996) found that
the overall frequencies of cells with mitotic abnormalities
were similar in cultures derived fromtwo genotypes diering
in their knob composition. However, the genotype with
higher knob content showed a higher incidence of cells with
abnormalities involving several chromosomes, such as
multiple bridges and fragments. This nding is dierent
from that observed in genotype 44114\2. We did not nd a
correlation between the type of culture and frequency of
mitotic abnormalities. It is of interest that Armstrong and
Phillips (1988) showed that culture medium inuences the
type of callus culture induced and that the frequency of
cytological abnormalities in regenerated plants is higher for
type II than for type I cultures at both 16- and 36-week
regeneration cycles.
In summary, we have identied an experimental system
for selecting maize genotypes yielding rapid-growing
embryogenic cultures with a low incidence of chromosome
instability. Data show that if heterochromatic knobs
undergo alterations in culture leading to mitotic disturbance,
this phenomenon also may depend on the genotype response
to culture environment. Thus, the frequency of chromosome
breakage is not always correlated with the number of knobs.
Among the material we screened, lines that showed high
embryogenic response should be explored in further studies
involving modications of the culture medium; lines
belonging to sub-family 1-3-3, lacking K3L and K9S,
represent the most interesting material for this investigation.
Interestingly, the eect of 2,4-D, a common component of
plant tissue culture media, has been extensively investigated
and controversial results concerning correlations between
the concentration of this hormone, mitotic irregularities and
chromosome aberrations have been reported (see Bayliss,
1980; Singh, 1993). This may suggest that 2,4-Dcould aect
genotypes dierently; this nding requires further investi-
gation.
ACKNOWLEDGEMENTS
This work was supported by FAPESP and CNPq. The
authors are grateful to: Sementes Agroceres SA, for their
support during the nal stages of the work; Professors
C. G. B. Demetrio and J. B. de Miranda-Filho (ESALQ\
USP) for help with statistical analysis; Eng. Agr. Juliana
Decico for collaboration in the determination of the knob
composition of the inbreds used; and to C. A. Verissimo
and S. C. M. Molina, respectively, for their help in tissue
culture and cytological work.
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