Vol. 55, no. 2: 115-119, 2002
CARYOLOGIA
Maize somatic chromosome preparation: pretreatments and genotypes for obtention of high index of metaphase accumulation MÔNICA 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 optimize 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 higher 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 cytological 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.
INTRODUCTION The identification of maize somatic chromosomes has been successful through the analysis of C-banded metaphases and the characteristic patterns of C-banding has been highly valuable: presence of heavily stained bands corresponding with heterochromatic knobs, and other less stained bands seen at the nucleolus organizer region (NOR) and the centromeres, these ones more conspicuous in prophases and prometaphases (WARD 1980; A GUIAR -PERECIN 1985; AGUIAR-P ERECIN and VOSA, 1985; RAYBURN et al. 1985; JEWELL and ISLAN-FARIDI 1994). Also, the detection of gross chromosome aberrations in somatic chromosomes possessing knobs has been possible by the analysis of C-banded metaphases (FLUMIN* Corresponding author: fax ++55 1934336706; e-mail:
[email protected]
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). However, accurate identification of mitotic chromosomal markers is important for studies involving somatic tissues and even for the evaluation of polymorphisms among maize varieties. Therefore, the obtention of cytological preparations with high frequency of metaphases showing chromosomes with clear morphology is highly desirable. Several pretreatments for metaphase and prometaphase accumulation have been described in plants, such as combinations of mitotic fuse and protein synthesis inhibitors, and meristematic cell synchronization using hydroxyurea 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). HAN
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Fig. 1 – a. Feulgen stained preparation of a root tip meristem of the 441123 x 4443 hybrid treated with 8-hydroxyquinoline 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 after 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 K6L2, K6L3 and K8L1, K8L2.
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MAIZE SOMATIC CHROMOSOME PREPARATION AND METAPHASE ACCUMULATION
In the present study, we report the results of experiments aiming the selection of maize genotypes yielding high mitotic index in root tips, and the evaluation of metaphase and prometaphase accumulation by 8-hydroxyquinoline and combinations of this mitotic fuse inhibitor and cycloheximide, a protein synthesis inhibitor. Two tropical 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 present investigation. Their knob composition, (references in AGUIAR-PERECIN and DECICO 1988) is presented 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 accumulation of metaphases and prometaphases, two types of pretreatments were compared: 8-hydroxyquinoline at 300 ppm for 2.5 hours at 28oC, and a combination of 8-hydroxyquinoline at 300 ppm and cycloheximide at 12.5 ppm for 2.5 hours at 28oC. Then, the root tips were fixed in 3:1 alcohol:acetic acid and then, kept in 70% ethanol at 4oC. Roots to be used for C-banding preparations were stored in the fixative at 4oC. The mitotic index and the effects of pretreatments were evaluated in Feulgen stained preparations. The staining procedure was carried out as previously described (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 60°C, rinsed in deionized water 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 coverslips were removed in liquid nitrogen and after air drying, 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 technique was also employed. A procedure previously described (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 solution. The cover-slips were removed in liquid nitrogen and the preparations were air-dried and kept in absolute ethanol at 4°C, for at least 12 hours. The treatment in 1.5% barium hydroxide was made at 37oC for 20 minutes. The slides were washed in deionized water, transferred to 2 X SSC at room temperature for 5 minutes and then, incubated in the same solution at 60°C for 1 hour. After rinsing in deionized water and alcohol series (70%, 95% and 100%) the preparations were stained in a 1% solution of Gurr’s 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 expressed as a percent of the total number of cells examined) of untreated root tips was determined by scoring 500 randomly selected cells in each root preparation. Five root tips from each genotype were used. The effects of pretreatments were evaluated by determining the frequencies of metaphase cells, designated as metaphase indices (number of metaphases expressed 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 supercontracted by the treatments were also scored, as mentioned 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 pretreated ones. The mitotic index of the lines (6.44% and 7.80%) is quite comparable to the
Table 1 – Designation and constitution of heterochromatic knobs of the genotypes investigated. Genotypes 441123 4443 441123 x 4443
K2L ++ ++ ++
K3L ++ 00 +0
K6L2 ++ ++ ++
K6L3 ++ ++ ++
Knobs* K7S ++ 00 +0
* K = knob; L = long arm; S = short arm; the numbers represent the chromosomes. Homozygous for the presence (++) or absence (00) of knobs.
K7L ++ ++ ++
K8L1 ++ ++ ++
K8L2 ++ ++ ++
K9S ++ 00 +0
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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 441123
Mitotic Index (%) 7.80 (195/2500)
4443
6.44 (161/2500)
441123 x 4443
9.24 (231/2500)
Pretreatments* Control 8-hydroxyquinoline 8-hydroxyquinoline + Cycloheximide Control 8-hydroxyquinoline 8-hydroxyquinoline + Cycloheximide Control 8-hydroxyquinoline 8-hydroxyquinoline + Cycloheximide
Metaphase Index** (%) 1.96 (49/2500) 4.72 (118/2500) 6.20 (155/2500) 1.92 (48/2500) 3.00 (75/2500) 5.56 (139/2500) 2.44 (61/2500) 5.96 (298/5000)*** 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.
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 material 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, resulting in higher mitotic index in the hybrid, may be interesting. The pretreatments resulted in metaphase accumulation and the combination of 8-hydroxiquinoline and cycloheximide was more effective for the three genotypes investigated (Table 2). Fig. 1a shows a sample of a Feulgen stained preparation of a root tip of the hybrid. The higher frequency of metaphase accumulation (metaphase index = 8.84%) observed after the combined treatment, 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 percentage of dividing cells. High indices of metaphase accumulation are due to the presence of prometaphases supercontracted by the cycloheximide, which can be distinguished from metaphases by some aspects: chromosome ends rather uncondensed, mainly in knobless chromosome arms, and sister chromatids partially held together at knob sites (visualized as C-bands). Figs. 1b and 1d show well condensed Feulgen stained and C-banded metaphases with chromatids clearly 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-hydroxiquinoline 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-banded 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 cycloheximide and 8-hydroxiquinoline represent interesting parameters for maize cytogenetic research. The concentration of cycloheximide used was lower than the references reported in the literature (TLASKAL 1980; KINDIGER 1994). Higher concentrations of cycloheximide are appropriated 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 cycloheximide, maize metaphase chromosomes showed an extremely condensed appearance not convenient for research involving physical mapping of chromosomal markers or identification of aberrations. Acknowledgments – Financial support of Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) is acknowledged.
MAIZE SOMATIC CHROMOSOME PREPARATION AND METAPHASE ACCUMULATION
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LEE J.-H., ARUMUGANATHAN K., KAEPPLER S.M., KAEPPLER H. F. and PAPA C.M., 1996 – Cell synchronization and isolation of metaphases chromosomes 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 different levels made feasible by new isolation methods. Chromosoma, 102: 96-101. SCHWARZACHER T., LEITCH A.R. and HESLOP-HARRISON J.S., 1994 – DNA:DNA in situ hybridization - methods for light microscopy. In: Harris N. and Oparka K. J. (Eds.) “Plant Cell Biology: A Practical Approach”, pp. 127-155. Oxford University Press, Oxford. SILVAROLLA M.B. and AGUIAR-PERECIN M.L.R. DE, 1994 – Evaluation of chromosome number stability 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 mitotic chromosomes. Can. J. Genet. Cytol., 22:61-67. Received October 11, 2001; accepted January 9, 2002
