- Open Access
A modified method for preparing meiotic chromosomes based on digesting pollen mother cells in suspension
Molecular Cytogenetics volume 8, Article number: 80 (2015)
Meiotic chromosome preparation is a key step in plant meiotic research. Pollen mother cell (PMC) wall elimination is beneficial to cytogenetic experimental procedures. Without wall interference, these procedures are easier and more successful. In existing methods it is difficult to eliminate PMC walls completely and uniformly. In this paper, we present an improved method for digesting PMC walls, and one for providing massive chromosomal spreads on a slide for other cytogenetic experimental procedures.
Three plants were selected to exhibit the modified meiotic chromosome preparation method. PMCs were dispersed as single cells and incubated in a mixed enzyme solution (3 % cellulose + 0.3 % pectinase + 1 % snailase) for 1.5–2.5 h. In total, 28.28 % cells were lost during this process. There were 800–1900 spreads on every slide and no PMC wall interference was found on any of the slides. The spreads were also evenly distributed on the slides. More spreads were obtained when PMC and protoplast densities in the suspension were increased. All three plants’ spreads were successfully used to locate a 5 s rDNA conserved sequence. The Nicotiana hybrid’s spreads were successfully used to identify the hybrid’s parental genome.
This is an alternative method for meiotic chromosome preparation. Through this method, PMC walls can be completely and uniformly eliminated, and hundreds of spreads on every slide can be obtained. These spreads can be successfully used for DNA in situ hybridization.
Meiotic chromosome preparation plays a major role in meiotic research, and many methods have been employed [1, 2]. These methods can be primarily divided into two types. In one type, pollen mother cells (PMCs) are spread without wall elimination. In the other, PMCs are spread after PMC wall elimination.
Originally, PMCs were squeezed on slides and immediately squashed by cover slips. Chromosomes were released by tapping or pressing [3, 4]. The squash-based methods are simple and fast, and many excellent spreads have been obtained using this method. Thus, it has been widely used in plant cytogenetic research [5–7]. Squash-based methods are mainly employed for karyotype analyses, and are used less for in situ hybridization (ISH) . Later, new methods without PMC wall elimination were introduced, but their use was limited [9–12]. Interference from PMC walls and wall fragments limits the use of these methods.
Methods with wall elimination were developed later [13–16]. The methods described by Zhong et al.  and Ross et al.  were successfully used in spread preparation for fluorescence in situ hybridization (FISH). These methods and some modifications have been widely used in the molecular cytogenetic analysis of meiosis in rice (Oryza), Brassica, potato (Solanum tuberosum), cotton (Gossypium), and lily (Lilium) [2, 17–21]. In these methods, whole anthers/buds are incubated in mixed enzyme solutions. Unfortunately, anther and/or bud walls were not permeated by the enzyme solution and PMCs grouped together. Thus, PMC walls could not be totally eliminated using low concentrations of enzymes and short digestion times . Although higher concentration enzymes and longer digestion times may eliminate the walls completely, many PMC protoplasts are also degraded. Thus, PMC walls cannot be digested uniformly by incubating the whole anther in a mixed enzyme solution.
In this paper, we improved a method to digest PMC walls completely and uniformly. The key step is the dispersion of PMCs as single cells incubated in a mixed enzyme solution. Prepared spreads were used in FISH and genomic ISH (GISH). To present this method, three different plants were used as materials. We hope that the method provides a new tool for plant meiotic chromosome preparations in cytogenetic research.
Loss ratio of cells
A Nicotiana hybrid was used to test the cell loss ratio during chromosome preparation using this modified method. There were 115.36 ± 14.65 PMCs per μl suspension when PMCs were squeezed from anthers into 200 μl distilled water at step 2. There were 1,654.67 ± 81.73 meiotic spreads counted, which calculated to a 28.28 % cell loss.
Meiotic spreads prepared by this modified method
Approximately 800–1900 meiotic spreads were obtained on every slide. There were no spreads containing walls or wall fragments, and no wall fragments were found on the slides (Fig. 1a). Thus, all of the PMC walls were completely and uniformly eliminated in the mixed enzyme solution after a 1.5–2.5 h incubation.
More spreads were obtained when the PMC density in suspension was increased at the incubation step. We obtained the same results when the protoplast density was increased at the resuspension step.
Many nice spreads suited to karyotype analyses were obtained. The Nicotiana hybrid’s meiotic chromosomes at different stages during meiosis are clearly shown in Fig. 1 (b–f). Distinguishable spreads of diploid and tetraploid cabbage (Brassica oleracea L.) at different stages are shown in Fig. 2. Maize (Zea mays L.) meiotic chromosomes before the second meiotic division are shown in Fig. 3.
Interference from the cytoplasm in maize spreads was more obvious than that in the other two plants. This may be because the cytoplasm in maize PMCs was originally thick. However, the thick cytoplasm did not affect chromosome recognition. Unfortunately, complete spreads after telophase I were not found on the slides (Fig. 3). This is because PMCs divide into two separate daughter cells within the wall after telophase I, but the two daughter cells move too far away from each other after the wall is digested.
5 s rDNA conserved sequence location on meiotic chromosomes by FISH
A 5 s rDNA conserved sequence was successfully located on meiotic spreads from the Nicotiana hybrid, cabbage and maize using FISH (Figs. 4 and 5, arrows). Obvious signals were detected in cabbage and Nicotiana hybrid spreads (Fig. 4b, f; Fig. 5b, d). Signals were detected on maize spreads even with the interference from the thick cytoplasm (Fig. 4d). In our experiments, 50–200 satisfactory spreads with obvious signals were obtained on every treated slide. The most satisfactory spreads occurred on the Nicotiana hybrid slides. This might be related to the weak interference from the thin cytoplasm.
Identification of the Nicotiana plumbaginifolia genome in the Nicotiana hybrid
Signals from the N. plumbaginifolia genome were detected on almost every spread from the Nicotiana hybrid. More than 80 satisfactory spreads were obtained on every treated slide. As a result, GISH figures from the prophase to tetrad stage of the Nicotiana hybrid were obtained and the N. plumbaginifolia genome was easily distinguished by GISH at the different stages (Fig. 6).
Uniform wall elimination in suspension
Protoplast preparation is widely used  and it was employed using our method. PMCs are loosely connected to each other in the anther . Thus, they are easily squeezed from anthers, and they are easily dispersed in suspension as single cells using a light shock. Lower levels of PMC accumulation occurred when PMCs were incubated in a mixed enzyme solution in bottles with flat bottoms. Shaking at times optimized the method. In this manner every PMC’s wall could be digested completely.
Application prospects for this modified method
Our method was successfully used to prepare meiotic chromosomes from a Nicotiana plant, cabbage, and maize. Nicotiana plants have been usually used for allotetraplod evolutionary research and classification studies based on cytogenetic analyses [24–26]. Cabbage is a plant in Brassica, which is very important for vegetable and oil production. Brassica plants are widely used in cytogenetic research [27–29]. Maize is also a model plant for plant cytogenetic research [30–32]. Meiosis is one topic of cytogenetic research. Thus, this modified method would contribute to the cytogenetic analyses of these widely studied plants.
Possible applications in large-scale cytogenetic analyses
Currently, the heterogeneous PMCs and their daughter cells in many plants, such as some polyploid plants and some interspecific hybrids, have become objects of cytogenetic research [18, 33]. Accurate results should be obtained from the analysis of massive numbers of spreads instead of relying on one or a few cells. Although the existing methods can easily analyze homogeneous cells and are sufficient for simple analyses of heterogeneous cells, they are difficult to apply to the molecular cytogenetic analysis of heterogeneous cells because of PMC wall interference. The interference limits the accumulation of enough satisfactory spreads. Through our modified method, PMC walls were completely and uniformly eliminated, and the spread density can be adjusted. These spreads were successfully used in FISH and GISH processes. This makes it possible to collect massive amounts of information from a few slides. Thus, the modified method could be used in large-scale cytogenetic analyses of heterogeneous PMCs and their daughter cells.
An easily mastered method
There were no technical problems in the modified process. First, PMCs can be easily squeezed from anther. The following steps require accessible materials, such as flat-based bottles, water bath, centrifuge, and alcohol lamp. Only the mouth blowing at the spreading step requires some technical expertise. In our experience, this method can be easily mastered. Even beginners from two of our collaborator’s laboratories obtained nice spreads on the first attempt.
Disadvantages of the modified method
There are still some disadvantages to this modified method. First, it is easier to squeeze PMCs from larger anthers with more PMCs, such as tobacco, than from smaller anthers with fewer PMCs, such as cabbage. Thus, we advise that more anthers could be needed if the anther is small and contains only a few PMCs. Anther wall tissue can remain in the PMC suspension, because somatic cells and PMCs can be easily distinguished. Second, some PMCs with separated protoplasts, such as maize PMCs after telophase II, are not suited to be treated by this method because the integrity of the PMCs will be destroyed when the wall is eliminated. A less severe digestion process may be used to avoid this disadvantage. Third, the chromosome spreading at the last step requires technique. To make the results from different operators more uniform, an instrument, such as an airbrush, may be used as an assistive device. However, the production of a suitable instrument is time consuming. We will test these in future work. Fourth, there were 800–1900 spreads on every slide, but only 50–200 satisfactory FISH spreads were obtained. Thus, the treatments before hybridization need to be improved to obtain more satisfactory FISH images on a slide.
The modified method can eliminate PMC walls completely and uniformly. It produced meiotic chromosome preparations from three widely and well-researched plants. Using this method, massive spreads without PMC wall interference were obtained on one slide. Spreads of all three plants prepared through this method were suitable for the location of a 5 s rDNA conserved sequence by FISH. The Nicotiana hybrid spreads were also suitable for the identification of a parental genome by GISH.
A Nicotiana hybrid (2n = 58), diploid (2n = 2× = 18), and tetraploid (2n = 4× = 36) cabbage (Brassica oleracea L.), and Maize (Zea mays L., 2n = 2× = 10) were used as materials. The Nicotiana hybrid, which was obtained from the hybridization of N. tabacum Lin. octoploid (2n = 8× = 96) and N. plumbaginifolia (2n = 2× = 20), was created in our laboratory. Diploid and tetraploid cabbage plants were from the field of the College of Horticulture and Landscape in Southwest University. Maize was from farmland near Southwest University.
Slide preparation was performed mainly according to Nagata and Takebe , Liang and Li , Zhong et al. , Yang , and Kirov et al. .
1. Flower buds undergoing meiosis were collected at 7:00–9:00, fixed in Carnoy’s fluid [methyl alcohol:acetic acid (V:V = 3:1)] overnight, and stored in 4 °C until the following step.
2. To clear away the Carnoy’s fluid, anthers were washed in distilled water twice after soaking in distilled water for 20 min, cut into two sections, and dipped into 200 μL distilled water in polyethylene centrifuge tubes. Tweezers were used to squeeze PMCs into the distilled water. Then, tubes were lightly shaken on a shaker. Anther walls and other fragments were removed using tweezers and the PMC suspension was obtained. Five Nicotiana hybrid anthers, four maize anthers, and six cabbage anthers were used once.
3. The PMC suspension was centrifuged at 2,000 × g for 3 min, and the supernatant was removed using a pipettor. The precipitate was resuspended in 300 μL mixed enzyme solution (3 % cellulose + 0.3 % pectinase + 1 % snailase, W/V). The suspension was transfer into 5-cm-high tiny cylindrical bottles with flat bottoms and a 0.8-mm radius. Bottles were capped and vertically immersed in a 37 °C water bath for 1.5 h (maize), 2 h (cabbage), or 2.5 h (Nicotiana hybrid), and slightly shaken at times.
4. The suspension was transferred into polyethylene centrifuge tubes and centrifuged at 2000 × g for 3 min. The supernatant was removed and the precipitate was resuspended in 200 μL distilled water. Centrifugation was performed again as in the previous step. The precipitate was resuspended in 100 μL Carnoy’s fluid. Then, 10 min later, 10 μL of suspension was dropped on every greaseless slide, immediately dispersed for a short time by forcefully blow from mouth, and dried rapidly using an alcohol flame. Slides were stained with 5 % Giemsa stain. In total, nine to ten slides were prepared at a time.
Location of the 5 s rDNA conserved sequence by FISH
FISH was performed according to Brammer et al.  with some modifications. Slides were treated with 20 μg/mL pepsin for 8 min (cabbage), 10 min (Nicotiana hybrid), or 50 min (maize) at room temperature. A 20-bp 5 s rDNA conserved sequence (Patent number: CN103409523A) was used as probe. The probe was labeled with 6-carboxytetramethylrhodamine at the 5′-end (Sangon Biotech, Shanghai, China). The probe was dissolved in 2× saline sodium citrate (SSC) as a hybridization solution at a final concentration of 10 ng/μL. The hybridization solution was added to spreads and they were incubated at 37 °C for 4 h. Slides were rinsed in 2× SSC three times for 3 min each time, in 4× SSC (+0.2 % Twain 20 V/V) once for 3 min, and then once in sterile distilled water for 1 min. After air drying, the probe was directly detected under a fluorescent microscope (Olympus, Japan). Photos were taken using a charge-coupled device camera.
Identification of the N. plumbaginifolia genome in hybrid meiotic spreads by GISH
GISH was carried out according to Brammer et al.  with some modifications. Slides were treated with 20 μg/mL pepsin for 10 min at room temperature. Genomic DNA of N. plumbaginifolia was used as the probe, and N. tabacum cv. Yunyan87’s DNA was the blocking DNA. Probes were labeled by random priming using Fluorescein-High Prime (Roche, Mannheim, Germany). The final concentration of the probe in the hybridization mixture was 0.25 ng/μL, and the concentration of the blocking DNA was 2.5 ng/μL. The probe was directly detected under a fluorescent microscope (Olympus). Photos were taken using a charge-coupled device camera.
Genomic in situ hybridization
Fluorescence in situ hybridization
In situ hybridization
Pollen mother cell
Saline sodium citrate
Fukui K, Nakayama S. Plant chromosomes: laboratory methods. Boca Raton: CRC press Inc; 1996.
Pawlowski WP, Grelon M, Armstrong S. Plant Meiosis: Methods and Protocols. New York: Springer; 2013.
Brown SW. The structure and meiotic behavior of the differentiated chromosomes of tomato. Genetics. 1949;34(4):437–61.
Baptista-Giacomelli FR, Pagliarini MS, De Almeida JL. Meiotic behavior in several Brazilian oat cultivars (Avena sativa L.). Cytologia. 2000;65(4):371–8.
Feldman M. The effect of chromosomes 5B, 5D, and 5A on chromosomal pairing in triticum aestivum. Proc Natl Acad Sci. 1966;55(6):1447–53.
Sanamyan M, Petlyakova J, Rakhmatullina E, Sharipova E. Cytogenetic collection of Uzbekistan. In: Abdurakhmonov IY, editor. World cotton germplasm resources. Intech. 2014. p. 247–87.
An HZ, Hu M, Li P, Geng GD, Zhang QQ, Zhang SQ. Chromosomal behavior during meiosis in the progeny of Triticum timopheevii × Hexaploid Wild Oat. PLoS One. 2015;10(5):e0126398.
Gong ZY, Liu XX, Tang D, Yu HX, Yi CD, Cheng ZK, et al. Non-homologous chromosome pairing and crossover formation in haploid rice meiosis. Chromosoma. 2011;120:47–60.
Hizume M, Kondo T, Miyake T. Meiosis in pollen mother cells of 15 Cryptomeria japonica clones with special emphasis on the behavior of the 6th chromosome pair heteromorphic to chromomycin A3 band. Jpn J Genet. 1989;64(4):287–94.
Hizume M, Kaneko K, Miyake T. A method for preparation of meiotic chromosomes of conifers and its applications. Chromosome Bot. 2014;9(3):83–8.
Singha M, Goela S, Meeleyb RB, Dantecc C, Parrinelloc H, Michauda C, et al. Production of viable gametes without meiosis in Maize deficient for an ARGONAUTE protein. Plant Cell. 2011;23:443–58.
Brownfield L, Yi J, Jiang H, Minina EA, Twell D, Köhler C. Organelles maintain spindle position in plant meiosis. Nat Commun. 2015;6:6492.
Zhong XB, De Jong JH, Zabel P. Preparation of tomato meiotic pachytene and mitotic metaphase chromosomes suitable for fluorescencein situ hybridization (FISH). Chromosom Res. 1996;4(1):24–8.
Yang HY, Wang J, Kang XY. Meiotic chromosome preparation techniques of pollen mother cells for laser micro-dissection in Populus spp. Forest Sci Pract. 2010;12(2):74–8.
Yang HY. Studies on Laser Micro-dissection of Chromosome in Populus. Beijing: Beijing Forestry University; 2010.
Ross KJ, Fransz P, Jones GH. A light microscopic atlas of meiosis in Arabidopsis thaliana. Chromosom Res. 1996;4(7):507–16.
Cheng Z, Buell CR, Wing RA, Jiang J. Resolution of fluorescence in-situ hybridization mapping on rice mitotic prometaphase chromosomes, meiotic pachytene chromosomes and extended DNA fibers. Chromosom Res. 2002;10(5):379–87.
Belandres HR, Waminal NE, Hwang YJ, Park BS, Lee SS, Huh JH, et al. FISH karyotype and GISH meiotic pairing analyses of a stable intergeneric hybrid × Brassica oraphanus line BB# 5. Korean J Horticultural Sci Technol. 2015;33(1):83–92.
Tang X, Datema E, Guzman MO, De Boer JM, Van Eck HJ, Bachem CW, et al. Chromosomal organizations of major repeat families on potato (Solanum tuberosum) and further exploring in its sequenced genome. Mol Gen Genomics. 2014;289(6):1307–19.
Wang K, Yang Z, Shu C, Hu J, Lin Q, Zhang W, et al. Higher axial-resolution and sensitivity pachytene fluorescence in situ hybridization protocol in tetraploid cotton. Chromosom Res. 2009;17(8):1041–50.
Khan N. A molecular cytogenetic study of intergenomic recombination and introgression of chromosomal segments in lilies (Lilium). Wageningen: Wageningen University; 2009.
Eeckhaut T, Lakshmanan PS, Deryckere D, Van Bockstaele E, Van Huylenbroeck J. Progress in plant protoplast research. Planta. 2013;238(6): 991–1003.
Goldberg RB, Beals TP, Sanders PM. Anther Development. Basic Principles and Practical Applications. Plant Cell. 1993;5(10):1217–29.
Nakamura R, Kitamura S, Inoue M, Ohmido N, Fukui K. Karyotype analysis of Nicotiana kawakamii Y. Ohashi using DAPI banding and rDNA FISH. Theor Appl Genet. 2001;102(6–7):810–4.
Kitamura S, Inoue M, Shikazono N, Tanaka A. Relationships among Nicotiana species revealed by the 5S rDNA spacer sequence and fluorescence in situ hybridization. Theor Appl Genet. 2001;103(5):678–86.
Kitamura S, Tanaka A, Inoue M. Genomic relationships among Nicotiana species with different ploidy levels revealed by 5S rDNA spacer sequences and FISH/GISH. Genes Genet Syst. 2005;80(4):251–60.
Maluszynska J, Hasterok R. Identification of individual chromosomes and parental genomes in Brassica juncea using GISH and FISH. Cytogenet Genome Res. 2005;109(1–3):310–4.
Howell EC, Kearsey MJ, Jones GH, King GJ, Armstrong SJ. A and C genome distinction and chromosome identification in Brassica napus by sequential FISH and GISH. Genetics. 2008;180(4):1849–57.
Grandont L, Cuñado N, Coriton O, Huteau V, Eber F, Chèvre AM, et al. Homoeologous chromosome sorting and progression of meiotic recombination in Brassica napus: Ploidy does matter! Plant Cell. 2014;26(4):1448–63.
Kanizay LB, Albert PS, Birchler JA, Dawe RK. Intragenomic conflict between the two major knob repeats of maize. Genetics. 2013;194:81–9.
Gaeta RT, Masonbrink RE, Zhao C, Sanyal A, Krishnaswamy L, Birchler JA. In vivo modification of a maize engineered minichromosome. Chromosoma. 2013;122(3):221–32.
Zhang B, Lv Z, Pang J, Liua Y, Guo X, Fu S, et al. Formation of a functional Maize centromere after loss of centromeric sequences and gain of ectopic sequences. Plant Cell. 2013;25(6):1979–89.
Leflon M, Eber F, Letanneur JC, Chelysheva L, Coriton O, Huteau V, et al. Pairing and recombination at meiosis of Brassica rapa (AA) × Brassica napus (AACC) hybrids. Theor Appl Genet. 2006;113:1467–80.
Nagata T, Takebe I. Cell wall regeneration and cell division in isolated tobacco mesophyll protoplasts. Planta. 1970;92(4):301–8.
Liang GL, Li XL. A new method for plant pollen mother cell meiosis chromosome preparation - modified smearing method. J Southwest Agric Univ. 1990;1:72.
Kirov I, Divashuk M, Van Laere K, Soloviev A, Khrustaleva L. An easy “SteamDrop” method for high quality plant chromosome preparation. Mol Cytogenet. 2014;7(1):21.
Brammer SP, Vasconcelos S, Poersch LB, Oliveira AR, Brasileiro-Vidal AC. Genomic in situ hybridization in Triticeae: a methodological approach. In: Andersen SB, editor. Plant breeding from laboratories to fields, Intech. 2013. p. 2–44.
We thank our colleagues at the College of Horticulture and Landscape Architecture in Southwest University for their technical assistance, Dr. Xu Wei in the College of Food Science in SWU for comments on the manuscript and editorial suggestions.
The authors declare that they have no conflict of interests.
Chromosome preparations of Nicotiana hybrid and cabbage were conducted by JD. Maize chromosomes were prepared by QZ, assisted by XY. FISH was carried out by JD and assisted by ZC, QZ, and XY. GISH was carried out by JD. All processes were guided by Pro. SX and Pro. GL. All authors read and approved the final manuscript.
Rights and permissions
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
About this article
Cite this article
Dang, J., Zhao, Q., Yang, X. et al. A modified method for preparing meiotic chromosomes based on digesting pollen mother cells in suspension. Mol Cytogenet 8, 80 (2015). https://doi.org/10.1186/s13039-015-0184-x
- Pollen mother cells
- PMC wall