INTRODUCTION

The identification of maize somatic chro-

mosomes has been successful through the
analysis of C-banded metaphases and the char-
acteristic patterns of C-banding has been high-
ly valuable: presence of heavily stained bands
corresponding with heterochromatic knobs,
and other less stained bands seen at the nucle-
olus organizer region (NOR) and the cen-
tromeres, these ones more conspicuous in
prophases and prometaphases (WARD 1980;
AGUIAR-PERECIN 1985; AGUIAR-PERECIN and
VOSA, 1985; RAYBURN et al. 1985; JEWELL and
ISLAN-FARIDI 1994). Also, the detection of gross
chromosome aberrations in somatic chromo-
somes possessing knobs has been possible by
the analysis of C-banded metaphases (FLUMIN-
HAN et al. 1996). Recently, most studies of the
molecular organization of maize chromosomes
have reported physical mapping of repetitive
DNA sequences on pachytene chromosomes
(ANANIEV et al. 1998; CHEN et al. 2000). How-
ever, accurate identification of mitotic chromo-
somal markers is important for studies involv-
ing somatic tissues and even for the evaluation
of polymorphisms among maize varieties.
Therefore, the obtention of cytological prepa-
rations with high frequency of metaphases show-
ing chromosomes with clear morphology is high-
ly desirable. Several pretreatments for metaphase
and prometaphase accumulation have been de-
scribed in plants, such as combinations of mi-
totic fuse and protein synthesis inhibitors, and
meristematic cell synchronization using hydrox-
yurea for chromosome sorting, as well. (TLASKAL
1980; PAN et al. 1993; SCHUBERT et al. 1993;
SCHWARZACHER et al. 1994; SILVAROLLA and
AGUIAR-PERECIN 1994; LEE et al. 1996).
CARYOLOGIA Vol. 55, no. 2: 115-119, 2002
Maize somatic chromosome preparation:
pretreatments and genotypes for obtention of high
index of metaphase accumulation
MNICA R. BERTO and MARGARIDA L. R. AGUIAR-PERECIN*
Departamento de Gentica, ESALQ, Universidade de So Paulo, 13400-970, Piracicaba, SP, Brazil.
Abstract - The present paper reports the results of experiments aiming to opti-
mize procedures for maize somatic chromosome preparation, by selecting maize
genotypes yielding high mitotic index in root tips, and evaluating metaphase and
prometaphase accumulation by 8-hydroxiquinoline and a combination of this
mitotic fuse inhibitor with cycloheximide, a protein synthesis inhibitor. Two
tropical inbred lines and their hybrid were used. The values of mitotic index
ranged from 6.44 to 7.80 % in the lines and 9.24 % in the hybrid, a value high-
er than references in the literature. The combination of 8-hydroxiquinoline at 300
ppm and cycloheximide at 12.5 ppm for 2.5 hours was effective for the three
genotypes investigated, resulting in a high index of metaphase and prometaphase
cells per preparation showing chromosomes suitable for identification of cyto-
logical markers, in the hybrid genotype. The hybrid selected and the treatments
employed represent interesting parameters for maize cytogenetic research.
Key words: mitotic index, root meristem cells, somatic chromosomes, Zea mays L.
* Corresponding author: fax ++55 1934336706; e-mail:
mlrapere@carpa.ciagri.usp.br
116 BERTO and AGUIAR-PERECIN
Fig. 1 a. Feulgen stained preparation of a root tip meristem of the 441123 x 4443 hybrid treated with 8-hy-
droxyquinoline at 300 ppm combined with cycloheximide at 12.5 ppm for 2.5 hours, showing accumulation of
metaphase and prometaphase cells. b-e. Aspects of metaphase and prometaphase chromosome morphology af-
ter this treatment: Feulgen stained metaphase (b) and prometaphase (c); C-banded metaphase (d) and
prometaphase (e). Note that the bands in the long arms of chromosomes 6 and 8 correspond to two fused knobs,
respectively K6L
2
, K6L
3
and K8L
1
, K8L
2
.
In the present study, we report the results of
experiments aiming the selection of maize geno-
types yielding high mitotic index in root tips, and
the evaluation of metaphase and prometaphase
accumulation by 8-hydroxyquinoline and combi-
nations of this mitotic fuse inhibitor and cyclo-
heximide, a protein synthesis inhibitor. Two trop-
ical inbred lines and their respective hybrids were
used.
MATERIALS AND METHODS
Material
Sister inbred lines derived from a maize brazilian
flint variety (Jac-Duro, Sementes Agroceres, Brazil)
and their respective hybrid were selected for the pre-
sent investigation. Their knob composition, (refer-
ences in AGUIAR-PERECIN and DECICO 1988) is pre-
sented in Table 1, and represent important markers for
chromosome identification using C-banding method.
Cytological Preparations
To evaluate the mitotic index of the genotypes
used, excised root tips from germinating seedlings
were fixed in 3:1 alcohol:acetic acid. For the accumu-
lation of metaphases and prometaphases, two types of
pretreatments were compared: 8-hydroxyquinoline at
300 ppm for 2.5 hours at 28
o
C, and a combination of
8-hydroxyquinoline at 300 ppm and cycloheximide at
12.5 ppm for 2.5 hours at 28
o
C. Then, the root tips
were fixed in 3:1 alcohol:acetic acid and then, kept in
70% ethanol at 4
o
C. Roots to be used for C-banding
preparations were stored in the fixative at 4
o
C.
The mitotic index and the effects of pretreatments
were evaluated in Feulgen stained preparations. The
staining procedure was carried out as previously de-
scribed (AGUIAR-PERECIN and VOSA 1985), with some
modifications. After pretreatment, the root tips were
rinsed in deionized water for 5 minutes, hydrolised in
1N HCl for 8 minutes at 60C, rinsed in deionized wa-
ter for 5 minutes, stained in leuco-basic-fuchsin for 45
minutes and washed in tap water for 5 minutes. The
root tips were then transferred to 45% acetic acid for
1 to 5 minutes, root caps were removed and the roots
were dissected to release the meristematic cells.
Squashing was made in 1% acetocarmine. The cover-
slips were removed in liquid nitrogen and after air dry-
ing, the preparations were mounted in Canada balsam.
For the analysis of the effects of the pretreatments
of roots of the hybrid genotype, the C-banding tech-
nique was also employed. A procedure previously de-
scribed (AGUIAR-PERECIN 1985) was employed, with
some modifications. Root tips stored in the fixative
were transferred to 45% acetic acid for 1 to 5 minutes,
for maceration, dissected and squashed in the same so-
lution. The cover-slips were removed in liquid nitro-
gen and the preparations were air-dried and kept in
absolute ethanol at 4C, for at least 12 hours. The
treatment in 1.5% barium hydroxide was made at
37
o
C for 20 minutes. The slides were washed in deion-
ized water, transferred to 2 X SSC at room tempera-
ture for 5 minutes and then, incubated in the same so-
lution at 60C for 1 hour. After rinsing in deionized
water and alcohol series (70%, 95% and 100%) the
preparations were stained in a 1% solution of Gurrs
R66 Giemsa, for 2 to 5 minutes, washed in deionized
water, air-dried and mounted in Canada balsam.
Evaluation of the pretreatments
The mitotic index (number of cells in mitosis ex-
pressed as a percent of the total number of cells ex-
amined) of untreated root tips was determined by
scoring 500 randomly selected cells in each root prepa-
ration. Five root tips from each genotype were used.
The effects of pretreatments were evaluated by deter-
mining the frequencies of metaphase cells, designated
as metaphase indices (number of metaphases ex-
pressed as a percent of the total number of cells). The
number of roots and cells examined was the same as
for the untreated roots. Prometaphases supercon-
tracted by the treatments were also scored, as men-
tioned below.
RESULTS AND DISCUSSION
Table 2 shows the values of mitotic index of
untreated root tips and a comparison between the
metaphase frequencies of these roots and the pre-
treated ones. The mitotic index of the lines
(6.44% and 7.80%) is quite comparable to the
MAIZE SOMATIC CHROMOSOME PREPARATION AND METAPHASE ACCUMULATION 117
Table 1 Designation and constitution of heterochromatic knobs of the genotypes investigated.
Genotypes Knobs*
K2L K3L K6L
2
K6L
3
K7S K7L K8L
1
K8L
2
K9S
441123 ++ ++ ++ ++ ++ ++ ++ ++ ++
4443 ++ 00 ++ ++ 00 ++ ++ ++ 00
441123 x 4443 ++ +0 ++ ++ +0 ++ ++ ++ +0
* K = knob; L = long arm; S = short arm; the numbers represent the chromosomes.
Homozygous for the presence (++) or absence (00) of knobs.
values of 4-6% reported in the literature
(TLASKAL 1980; LEE et al. 1996), but lower than
the one found for the hybrid 441123 x 4443
(9.24%), which proved to be an excellent mater-
ial for cytogenetic research. This suggests that
further investigation on the possible occurrence
of gene effects or even their interaction with the
presence of knobs in heterozygous state, result-
ing in higher mitotic index in the hybrid, may be
interesting.
The pretreatments resulted in metaphase ac-
cumulation and the combination of 8-hydrox-
iquinoline and cycloheximide was more effective
for the three genotypes investigated (Table 2). Fig.
1a shows a sample of a Feulgen stained prepara-
tion of a root tip of the hybrid. The higher fre-
quency of metaphase accumulation (metaphase in-
dex = 8.84%) observed after the combined treat-
ment, was found in preparations of the hybrid, but
in some regions of the root tips, metaphase indices
approaching 18% were found and appeared to
correspond to regions containing a higher per-
centage of dividing cells. High indices of
metaphase accumulation are due to the presence
of prometaphases supercontracted by the cyclo-
heximide, which can be distinguished from
metaphases by some aspects: chromosome ends
rather uncondensed, mainly in knobless chromo-
some arms, and sister chromatids partially held to-
gether at knob sites (visualized as C-bands). Figs.
1b and 1d show well condensed Feulgen stained
and C-banded metaphases with chromatids clear-
ly separated, a characteristic effect of treatment by
cycloheximide combined with 8-hydroxiquinoline.
Figs. 1c and 1e show condensed prometaphases of
the hybrid. The combination of 8-hydroxiquino-
line and cycloheximide represents an achievement
for the investigation of aspects of the variability of
arm size of knobbed and knobless maize mitotic
chromosomes, which can be identified in C-band-
ed cells.
The present investigation aiming to optimize
treatments to accumulate metaphase cells, using
genotypes selected for high mitotic index,
showed that the hybrid genotype selected and the
treatment employing a combination of cyclohex-
imide and 8-hydroxiquinoline represent interest-
ing parameters for maize cytogenetic research.
The concentration of cycloheximide used was
lower than the references reported in the litera-
ture (TLASKAL 1980; KINDIGER 1994). Higher
concentrations of cycloheximide are appropriat-
ed for chromosome counting, for prophases are
also highly condensed into a type of metaphase
conformation (TLASKAL 1980; SILVAROLLA and
AGUIAR-PERECIN 1994). In previous experiments
(not shown) using higher concentrations of cy-
cloheximide, maize metaphase chromosomes
showed an extremely condensed appearance not
convenient for research involving physical map-
ping of chromosomal markers or identification of
aberrations.
Acknowledgments Financial support of Con-
selho Nacional de Desenvolvimento Cientfico e
Tecnolgico (CNPq) and Fundao de Amparo
Pesquisa do Estado de So Paulo (FAPESP) is ac-
knowledged.
118 BERTO and AGUIAR-PERECIN
Table 2 Values of mitotic index (percentage of mitotic cells) and metaphase accumulation by the pretreatments (expressed
as metaphase index = percentage of metaphases and prometaphases) of the genotypes investigated
Genotypes Mitotic Index Pretreatments* Metaphase Index**
(%) (%)
441123 7.80 (195/2500) Control 1.96 (49/2500)
8-hydroxyquinoline 4.72 (118/2500)
8-hydroxyquinoline +
Cycloheximide 6.20 (155/2500)
4443 6.44 (161/2500) Control 1.92 (48/2500)
8-hydroxyquinoline 3.00 (75/2500)
8-hydroxyquinoline +
Cycloheximide 5.56 (139/2500)
441123 x 4443 9.24 (231/2500) Control 2.44 (61/2500)
8-hydroxyquinoline 5.96 (298/5000)***
8-hydroxyquinoline +
Cycloheximide 8.84 (442/5000)***
* 8-Hydroxiquinoline at 300 ppm and cycloheximide at 12.5 ppm. Untreated roots were used as control.
** Numbers within parentheses correspond to the original frequencies of cells analysed. Five root tips per treatment were used.
*** Evaluations of 2500 preparations stained by Feulgen + 2500 preparations stained by C-banding.
REFERENCES
AGUIAR-PERECIN M.L.R. DE, 1985 C-banding in
maize. I. Band patterns. Caryologia, 38: 23-30.
AGUIAR-PERECIN M.L.R. DE and DECICO J.U., 1988
Preliminary results on the segregation of knobs
(C-bands) in inbred lines derived from a flint va-
riety. Maize Genetics Cooperation Newsletter,
62: 100.
AGUIAR-PERECIN M.L.R. DE and VOSA C.G., 1985
C-banding in maize. II. Identification of somat-
ic chromosomes. Heredity, 54: 37-42.
ANANIEV E.V., PHILLIPS R.L. and RINES H.W., 1998
A knob-associated tandem repeat in maize ca-
pable of forming fold-back DNA segments: Are
chromosome knobs megatransposons? Proceed-
ings of the National Academy of Sciences USA,
95: 10785-10790.
CHEN C.C., CHEN C.M., HSU F.C., WANG C.J.,
YANG J.T. and KAO Y.Y., 2000 The pachytene
chromosomes of maize as revealed by fluorescence
in situ hybridization with repetitive DNA se-
quences. Theoretical and Applied Genetics, 101:
30-36.
FLUMINHAN A., AGUIAR-PERECIN M.LR. DE and
SANTOS J.A. DOS, 1996 Evidence for hete-
rochromatin involvement in chromosome break-
age in maize callus culture. Ann. Bot., 78: 73-81.
JEWELL D.C. and ISLAM-FARIDI N., 1994 A Tech-
nique for Somatic Chromosome Preparation and
C-banding of Maize. In: M. Freeling and V. Wal-
bot (Eds), The Maize Handbook, pp. 484-
493. Springer-Verlag, New York.
KINDIGER B., 1994 A Technique for the Preparation
of Somatic Chromosomes of Maize. In: M. Freel-
ing and V. Walbot (Eds), The Maize Hand-
book, pp. 481-483. Springer-Verlag, New York.
LEE J.-H., ARUMUGANATHAN K., KAEPPLER S.M.,
KAEPPLER H. F. and PAPA C.M., 1996 Cell syn-
chronization and isolation of metaphases chro-
mosomes from maize (Zea mays L.) root tips for
flow cytometric analysis and sorting. Genome, 39:
697-703.
PAN W.H., HOUBEN A. and SCHELEGEL R., 1993
Highly effective cell synchronization in plant roots
by hydroxyurea and amiprophos-methyl or
colchicine. Genome, 36: 387-390.
RAYBURN A.L., PRICE H.J., SMITH J.D. and GOLD
J.R., 1985 C-band heterochromatin and DNA
content in maize mitotic chromosomes. Amer. J.
Bot., 72: 1610-1617.
SCHUBERT I., DOLEZEL J., HOUBEN A., SCHERTHAN
H. and WANNER G., 1993 Refined examination
of plant metaphase chromosome structure at dif-
ferent levels made feasible by new isolation meth-
ods. Chromosoma, 102: 96-101.
SCHWARZACHER T., LEITCH A.R. and HESLOP-HAR-
RISON J.S., 1994 DNA:DNA in situ hybridiza-
tion - methods for light microscopy. In: Harris N.
and Oparka K. J. (Eds.) Plant Cell Biology: A
Practical Approach, pp. 127-155. Oxford Uni-
versity Press, Oxford.
SILVAROLLA M.B. and AGUIAR-PERECIN M.L.R. DE,
1994 Evaluation of chromosome number sta-
bility in two sugarcane varieties. Brazil. J. Genet.,
17: 237-242.
TLASKAL J., 1980 Combined cycloheximide and
8-hidroxyquinoline pretreatment for study of plant
chromosomes. Stain Technology, 54: 313-319.
WARD E.J., 1980 Banding patterns in maize mitot-
ic chromosomes. Can. J. Genet. Cytol., 22:61-67.
Received October 11, 2001; accepted January 9, 2002
MAIZE SOMATIC CHROMOSOME PREPARATION AND METAPHASE ACCUMULATION 119