INTRODUCTION

The identification of maize somatic chro-mosomes has been successful through theanalysis of C-banded metaphases and the char-acteristic patterns of C-banding has been high-ly valuable: presence of heavily stained bandscorresponding with heterochromatic knobs,and other less stained bands seen at the nucle-olus organizer region (NOR) and the cen-tromeres, these ones more conspicuous inprophases and prometaphases (WARD 1980;AGUIAR-PERECIN 1985; AGUIAR-PERECIN andVOSA, 1985; RAYBURN et al. 1985; JEWELL andISLAN-FARIDI 1994). Also, the detection of grosschromosome aberrations in somatic chromo-somes possessing knobs has been possible bythe analysis of C-banded metaphases (FLUMIN-

HAN et al. 1996). Recently, most studies of themolecular organization of maize chromosomeshave reported physical mapping of repetitiveDNA 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 evaluationof 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 metaphaseand prometaphase accumulation have been de-scribed in plants, such as combinations of mi-totic fuse and protein synthesis inhibitors, andmeristematic 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 andAGUIAR-PERECIN 1994; LEE et al. 1996).

CARYOLOGIA Vol. 55, no. 2: 115-119, 2002

Maize somatic chromosome preparation:pretreatments and genotypes for obtention of highindex of metaphase accumulationMÔNICA R. BERTÃO and MARGARIDA L. R. AGUIAR-PERECIN*Departamento de Genética, ESALQ, Universidade de São 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 maizegenotypes yielding high mitotic index in root tips, and evaluating metaphase andprometaphase accumulation by 8-hydroxiquinoline and a combination of thismitotic fuse inhibitor with cycloheximide, a protein synthesis inhibitor. Twotropical inbred lines and their hybrid were used. The values of mitotic indexranged 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 300ppm and cycloheximide at 12.5 ppm for 2.5 hours was effective for the threegenotypes investigated, resulting in a high index of metaphase and prometaphasecells per preparation showing chromosomes suitable for identification of cyto-logical markers, in the hybrid genotype. The hybrid selected and the treatmentsemployed 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 BERTÃO 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 ofmetaphase 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) andprometaphase (e). Note that the bands in the long arms of chromosomes 6 and 8 correspond to two fused knobs,respectively K6L2, K6L3 and K8L1, K8L2.

In the present study, we report the results ofexperiments aiming the selection of maize geno-types yielding high mitotic index in root tips, andthe evaluation of metaphase and prometaphaseaccumulation 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 wereused.

MATERIALS AND METHODS

MaterialSister 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 forchromosome identification using C-banding method.

Cytological PreparationsTo evaluate the mitotic index of the genotypes

used, excised root tips from germinating seedlingswere fixed in 3:1 alcohol:acetic acid. For the accumu-lation of metaphases and prometaphases, two types ofpretreatments were compared: 8-hydroxyquinoline at300 ppm for 2.5 hours at 28oC, and a combination of8-hydroxyquinoline at 300 ppm and cycloheximide at12.5 ppm for 2.5 hours at 28oC. Then, the root tipswere fixed in 3:1 alcohol:acetic acid and then, kept in70% ethanol at 4oC. Roots to be used for C-bandingpreparations were stored in the fixative at 4oC.

The mitotic index and the effects of pretreatmentswere evaluated in Feulgen stained preparations. Thestaining procedure was carried out as previously de-scribed (AGUIAR-PERECIN and VOSA 1985), with somemodifications. After pretreatment, the root tips wererinsed in deionized water for 5 minutes, hydrolised in1N HCl for 8 minutes at 60°C, rinsed in deionized wa-ter for 5 minutes, stained in leuco-basic-fuchsin for 45minutes and washed in tap water for 5 minutes. Theroot tips were then transferred to 45% acetic acid for1 to 5 minutes, root caps were removed and the rootswere 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 pretreatmentsof roots of the hybrid genotype, the C-banding tech-nique was also employed. A procedure previously de-scribed (AGUIAR-PERECIN 1985) was employed, withsome modifications. Root tips stored in the fixativewere 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 inabsolute ethanol at 4°C, for at least 12 hours. Thetreatment in 1.5% barium hydroxide was made at37oC 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 60°C for 1 hour. After rinsing in deionizedwater and alcohol series (70%, 95% and 100%) thepreparations were stained in a 1% solution of Gurr’sR66 Giemsa, for 2 to 5 minutes, washed in deionizedwater, air-dried and mounted in Canada balsam.

Evaluation of the pretreatmentsThe 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 byscoring 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, designatedas metaphase indices (number of metaphases ex-pressed as a percent of the total number of cells). Thenumber of roots and cells examined was the same asfor 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 ofuntreated root tips and a comparison between themetaphase 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 K6L2 K6L3 K7S K7L K8L1 K8L2 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 thanthe one found for the hybrid 441123 x 4443(9.24%), which proved to be an excellent mater-ial for cytogenetic research. This suggests thatfurther investigation on the possible occurrenceof gene effects or even their interaction with thepresence of knobs in heterozygous state, result-ing in higher mitotic index in the hybrid, may beinteresting.

The pretreatments resulted in metaphase ac-cumulation and the combination of 8-hydrox-iquinoline and cycloheximide was more effectivefor 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, butin some regions of the root tips, metaphase indicesapproaching 18% were found and appeared tocorrespond to regions containing a higher per-centage of dividing cells. High indices ofmetaphase accumulation are due to the presenceof prometaphases supercontracted by the cyclo-heximide, which can be distinguished frommetaphases by some aspects: chromosome endsrather 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 stainedand C-banded metaphases with chromatids clear-ly separated, a characteristic effect of treatment bycycloheximide combined with 8-hydroxiquinoline.

Figs. 1c and 1e show condensed prometaphases ofthe hybrid. The combination of 8-hydroxiquino-line and cycloheximide represents an achievementfor the investigation of aspects of the variability ofarm size of knobbed and knobless maize mitoticchromosomes, which can be identified in C-band-ed cells.

The present investigation aiming to optimizetreatments to accumulate metaphase cells, usinggenotypes selected for high mitotic index,showed that the hybrid genotype selected and thetreatment employing a combination of cyclohex-imide and 8-hydroxiquinoline represent interest-ing parameters for maize cytogenetic research.The concentration of cycloheximide used waslower than the references reported in the litera-ture (TLASKAL 1980; KINDIGER 1994). Higherconcentrations of cycloheximide are appropriat-ed for chromosome counting, for prophases arealso highly condensed into a type of metaphaseconformation (TLASKAL 1980; SILVAROLLA andAGUIAR-PERECIN 1994). In previous experiments(not shown) using higher concentrations of cy-cloheximide, maize metaphase chromosomesshowed an extremely condensed appearance notconvenient for research involving physical map-ping of chromosomal markers or identification ofaberrations.

Acknowledgments – Financial support of Con-selho Nacional de Desenvolvimento Científico eTecnológico (CNPq) and Fundação de Amparo àPesquisa do Estado de São Paulo (FAPESP) is ac-knowledged.

118 BERTÃO and AGUIAR-PERECIN

Table 2 – Values of mitotic index (percentage of mitotic cells) and metaphase accumulation by the pretreatments (expressedas 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.

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Received October 11, 2001; accepted January 9, 2002

MAIZE SOMATIC CHROMOSOME PREPARATION AND METAPHASE ACCUMULATION 119