Protocol


Nature Protocols 2, 2233 - 2244 (2007)
Published online: 6 September 2007 | doi:10.1038/nprot.2007.310

Subject Categories: Genetic analysis | Isolation, purification and separation | Model organisms | Plant biology

Estimation of nuclear DNA content in plants using flow cytometry

Jaroslav Doležel1,2, Johann Greilhuber3 & Jan Suda4,5

Flow cytometry (FCM) using DNA-selective fluorochromes is now the prevailing method for the measurement of nuclear DNA content in plants. Ease of sample preparation and high sample throughput make it generally better suited than other methods such as Feulgen densitometry to estimate genome size, level of generative polyploidy, nuclear replication state and endopolyploidy (polysomaty). Here we present four protocols for sample preparation (suspensions of intact cell nuclei) and describe the analysis of nuclear DNA amounts using FCM. We consider the chemicals and equipment necessary, the measurement process, data analysis, and describe the most frequent problems encountered with plant material such as the interference of secondary metabolites. The purpose and requirement of internal and external standardization are discussed. The importance of using a correct terminology for DNA amounts and genome size is underlined, and its basic principles are explained.

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Introduction

Flow cytometry (FCM) is an excellent method to analyze optical properties such as fluorescence and light scatter of microscopic particles in liquid suspension. Cells and cellular organelles are individually measured at high speed, typically 100–1,000/s, which facilitates representative sampling and detection of subpopulations. Some instruments can sort simultaneously up to four different subpopulations for further analysis and use. These features explain why FCM has become widespread in biomedical research and practice. Although less frequently employed in plant science and industry in the past, FCM is being increasingly used in a broad range of applications1. The estimation of nuclear DNA content is by far the most popular of these applications, with more than 800 publications to date2.

Why nuclear DNA content?

The plant cell nucleus carries most of the hereditary material and, as such, has always been the subject of intensive studies. Estimation of DNA quantity (C-value)3 in absolute units (DNA picograms and number of base pairs) has led to the discovery of more than 2,000-fold variation in the genome size in plants, with the smallest known genome of 63 Mbp found in Genlisea margaretae (Lentibulariaceae)4 and the largest genome of 127 Gbp in the tetraploid Fritillaria assyriaca (Liliaceae)5. Although biological meaning of the variation remains obscure (cf. the C-value paradox/enigma)6, a number of observations correlate genome size with cell cycle duration and cell size, and characters such as life cycle, weediness, threat of extinction, dry mass production, ecological requirements, phenology, seed mass and others (i.e., the nucleotypic theory)7, 8. The availability of data on genome size is critical for many fields of research, including taxonomy and evolutionary changes9. Its knowledge is essential for planning gene cloning and genome sequencing projects10.

Estimation of DNA content in relative units concerns equally important applications. The relationship between ploidy and nuclear DNA content makes the assay suitable for the determination of ploidy level, detection of mixoploidy and, under certain conditions, also aneuploidy. The applications range from taxonomy and population biology to breeding and quality control in seed production11. Simultaneous estimation of DNA content in seed embryo and endosperm makes it possible to determine the genetic origin of a seed, that is, establish its sexual or apomictic origin and gametic ploidy12. More specialized applications include the presence and extent of endopolyploidy13, 14 and the estimation of DNA base content15, 16. As the nuclear DNA content undergoes characteristic changes during passage through the cell cycle, it is also possible to determine the position of a cell within the cell cycle17. Some studies have employed simultaneous analysis of nuclear DNA contents and the amounts of other cellular components such as RNA, proteins, secondary metabolites and/or expression of heterologous proteins18, 19. Due to space limitations these methods are not included in this protocol, which deals exclusively with the estimation of nuclear DNA content.

Why FCM?

FCM has become the method of choice because it is convenient, fast and reliable. Sample preparation usually occupies only a few minutes and does not require expensive reagents. Analysis is rapid, and representative numbers of nuclei can be measured in a short time. Samples are typically prepared from only a few tens of milligrams of plant tissues; hence the method typically is nondestructive and suitable for the analysis of small individuals. An important advantage for ploidy estimation is that tissues containing dividing cells are not required. The fact that the nuclei are measured individually facilitates the analysis of samples with asynchronously dividing cells and the detection of subpopulations with different DNA contents. Nuclear DNA amounts can be analyzed with a high precision, with coefficients of variation (CV) of the DNA peaks generally ranging from 1 to 5% (ref. 2), while CVs of the means of replicate measurements are typically lower than 1% (personal observations).

The widespread adoption of DNA FCM was also stimulated by the availability of simple flow cytometers at affordable prices, sometimes capable of measuring only one fluorescence parameter. While static cytofluorometry was never widely adopted, Feulgen microspectrophotometry (or Feulgen densitometry) was the method of choice before it was supplanted by FCM, that is, until the late 1990s. While it measures individual nuclei, Feulgen densitometry offers similar advantages to FCM. Moreover, it is possible to select the nuclei for analysis and/or for visual inspection after analysis (but note that some current flow cytometers can also record images of measured particles). An advantage of Feulgen densitometry over FCM is that plant samples can be stored for prolonged periods before their processing and analysis (including the estimation of nuclear DNA content in absolute units). The major disadvantage of microspectrophotometry is that sample preparation is more laborious, and that the analysis is much slower. Similar advantages and disadvantages characterize image densitometry; however, until now this method has not been used widely in plant sciences, most probably because reliable software is not available commercially. Laser scanning cytometry combines the speed of FCM, its ability to measure multiple parameters and the possibility of visually inspecting measured particles20. However, due to the higher price of these instruments, this method will probably never be routinely used in laboratories where only DNA content is analyzed.

What are the options for sample preparation?

A sample for FCM is necessarily a liquid suspension of single intact particles. The use of intact cells for the estimation of DNA content is not recommended, because their size can approach or exceed the diameter of flow chamber orifices. Moreover, plant cells can disturb laminar flow due to their irregular shape and consequently interact with the excitation light beam differently, giving rise to variation in fluorescence and light scatter signals. Although this can be ameliorated by removing the cell walls to obtain spherical protoplasts, accurate analysis is still compromised by the unpredictably acentric position of the nuclei within the cell. Other drawbacks of measuring DNA within intact cells and protoplasts include the low permeability of the plasma membrane to most DNA fluorochromes, their nonspecific binding, autofluorescence coming from various pigments and interference of secondary metabolites with DNA staining. These difficulties can be partially overcome by treating protoplasts by a fixative (e.g., methanol) to permeabilize the plasma membrane and remove autofluorescent compounds such as chlorophyll. Provided the experiment does not require simultaneous analysis of nuclear DNA with other targets in cytoplasm, a viable option is to isolate intact nuclei.

How to isolate intact nuclei?

One way to isolate intact nuclei is to lyse protoplasts in a hypotonic buffer. In this manner, the nuclei are gently released from cells, and the procedure usually provides histograms of DNA content with a little background debris and DNA peaks having very low CVs21. However, protocols for protoplast isolation are laborious, and are available only for a limited number of species and for particular tissues at certain developmental stages. A universally accepted method for the preparation of nuclear suspensions for DNA FCM was developed by David W. Galbraith in his laboratory22. In this protocol, cell nuclei are released from a small amount of fresh plant tissue by chopping it with a sharp razor blade in an appropriate buffer solution. After filtration of the homogenate through a nylon mesh and addition of a DNA fluorochrome, the sample is ready for analysis. The method is rapid, convenient and suitable for many (though not all) plant species, organs and tissues.

Variations of Galbraith's protocol have been introduced, which differ in a way the sample is homogenized and by the composition of nuclear isolation buffers (see REAGENT SETUP for the composition of the four most popular buffers). Protocol modifications, including those suitable for nuclei isolation from recalcitrant plant tissues, such as pollen grains, are discussed elsewhere9. Recent options to release nuclei from various tissues include the so-called bead beating method, which involves shaking the tissues in a screw-capped tube containing zirconia/silica or glass beads (A.V. Roberts, personal communication). Release of nuclei from seed tissues has also been described by crushing between two sheets of fine sandpaper12.

Although originally developed for fresh plant tissues, Galbraith's protocol seems suitable for the estimation of relative nuclear DNA content in dry herbarium and silica vouchers23. This advance expands the potential of DNA FCM to applications in taxonomy, population biology and ecology. A requirement for fresh samples evidently limits the application of DNA FCM in some research areas such as field botany, where samples are often collected in remote areas and cannot be immediately analyzed. Analysis of field-collected seeds is a useful option24, but the results should be considered with caution as the origin of male gametes and hence the genotype of seeds is not known. Freezing would be a convenient way of storing plant samples. Although a few studies have demonstrated the feasibility of this approach25, analysis of samples from frozen tissues usually results in lower resolution of histograms of DNA content and excessive background from debris (J.D. and J.S., unpublished observations).

Are fixed plant samples suitable?

The search for alternative methods of nuclei isolation coupled to the need to store samples before analysis has led to the development of methods in which fixed plant samples are analyzed. In fact, the first report on DNA content analysis in plants described the isolation of nuclei from acetic ethanol-fixed root tips, by digesting these tissues with pectinase followed by mechanical disintegration26. The potential of this approach is limited by the need to optimize the enzymatic treatment for different species and tissues, and by the difficulty of isolating single cells from differentiated tissues. A more universal method involved the mechanical release of intact nuclei from formaldehyde-fixed tissues27. Fixation increases nuclei yield and makes them suitable for immunofluorescent staining28. Fixation of isolated nuclei in 70 or 95% ethanol has also been used with some success29. While fixation can facilitate the storage of tissues and nuclei suspensions, it is known to modify chromatin structure and the accessibility of DNA to fluorochromes30. Hence, fixed plant samples cannot be recommended for the estimation of DNA content in absolute units until the reliability of such protocols is demonstrated.

And how to stain the nuclear DNA?

A fluorochrome used to estimate nuclear DNA content must bind selectively and stoichiometrically to DNA. A broad range of fluorochromes are known to specifically bind to DNA31, but only a few are suitable for the quantification of DNA content in plants (Table 1). Ethidium bromide and propidium iodide (PI) intercalate into double-stranded DNA (and double-stranded RNA! (dsRNA)) without base-dependent bias, and thus are suitable for the estimation of DNA content in absolute units, provided the RNA is removed with RNase32, 33, 34. Fluorochromes binding preferentially to AT- and GC-rich regions of DNA are suitable for the estimation of DNA content in relative units when large differences in genomic base content are not expected, such as in autopolyploid series, and are necessary for DNA base content estimation. Out of them, DAPI (4′,6-diamidino-2-phenylindole) has been the most popular, as it provides DNA content histograms with high resolution, uses readily available excitation wavelengths and does not require RNase treatment.


How to interpret the measurements?

FCM records relative fluorescence intensities. Interpretation of results in terms of ploidy and genome size requires a reference standard (i.e., sample with known ploidy level and/or nuclear DNA content). The standardization may either be external or internal. The former involves successive analysis of the sample and the standard, whereas the latter involves simultaneous isolation, staining and analysis of nuclei both from sample and standard. A routine estimation of ploidy can be done using external standard although more precise ploidy analysis, and especially the detection of aneuploidy, requires internal standardization35. An individual belonging to the same plant species is usually employed in ploidy analysis, but animal species such as chicken red blood cells have been used, too. The estimation of DNA content in absolute units requires internal standardization, and animal standards are not recommended34. Table 2 lists plant DNA standards with a range of genome sizes that are available free of charge from the corresponding author.


Is there a terminology to follow?

In order to communicate results, current terminology3 should be used and attention paid to the life cycle-dependent nuclear phase of the material. For example, haplophasic tissues (with chromosome number n) dominate in bryophytes, whereas the majority of the tissues of tracheophytes are in diplophase (with a 2n chromosome number). An unreplicated nucleus of an individual in haplophase has a 1C DNA content, the letter 'C' standing for 'constant' and symbolizing the DNA amount of a 'holoploid genome' with chromosome number n. After DNA replication, the haplophasic nucleus has a 2C DNA content, the same as that of an unreplicated nucleus of an individual in diplophase. The degrees of endopolyploidy are usually best provided in C-levels. The DNA amount of a monoploid genome (i.e., monoploid genome size) with basic chromosome number x is given as its Cx-value. Thus, in somatic tissues of a hexaploid plant, such as bread wheat, Triticum aestivum (2n = 6x = 42), the 2C-value is equivalent to the 6Cx-value. The terms 'haploid', 'diploid', etc., which refer to chromosome numbers, should not be used to indicate DNA contents. Also ploidy data gained using FCM should be distinguished from that obtained via chromosome counting. If ploidy/aneuploidy is inferred from nuclear DNA content but is not confirmed by chromosome analysis, a prefix 'DNA' (i.e., 'DNA ploidy', 'DNA aneuploidy') should be used36.

Is it really so easy?

Sometimes it is not, and the standard methods may fail with specific samples for a variety of reasons. The first trouble may be a difficulty to isolate sufficient intact nuclei for analysis. Such an inconvenience is often linked to the mechanical and chemical structure of the sample. For example, nuclei may be entrapped inside the cells or may stick to mucilaginous compounds37. Aggregation of fluorescent debris particles to the surfaces of isolated nuclei, i.e., so-called 'debris coatings'34, is another phenomenon that results in decreased resolution of DNA content histograms38. A third, particularly acute problem concerns the interference of cytosolic compound(s) with the fluorescent staining of DNA, which usually results in decreased fluorescence intensity. Because the nuclei of the sample and the standard may be affected differentially38, estimation of DNA content in absolute units may be compromised, as evidenced, for example, by poor reproducibility of results. Testing variants of protocols for nuclei isolation and different nuclei isolation buffers is advisable. The buffers can further be modified by manipulating the concentration of detergents and adding compounds counteracting the negative effects of cytosol (see the list of buffer compositions provided by Greilhuber and co-workers34). If the experiment does not require the analysis of nuclei from specific tissues, a useful strategy may be to identify less problematic tissues (e.g., embryo hypocotyl from mature dry seeds)24.

Experimental design

This protocol describes the preparation of nuclear suspensions from fresh leaf tissues, in vitro cultured calli, protoplasts, mature seeds, herbarium vouchers and silica-dry materials, and flow cytometric estimation of DNA content in relative and absolute units (genome size). Particularities of FCM analysis of dehydrated plant tissues and mature seeds are described in detail elsewhere39, 40. For the sake of clarity, protocols involving tissue and nuclei fixation are not covered here.


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Materials

Reagents

  • Plant sample (fresh somatic plant tissue, in vitro cultured calli, living protoplasts, dry mature seeds, silica-dry material and herbarium vouchers) (see REAGENT SETUP)
  • DNA reference standard (living plant somatic tissue); see Table 2 for a list of recommended standards
  • LB01 lysis buffer (see REAGENT SETUP)
  • Galbraith's buffer (see REAGENT SETUP)
  • Tris.MgCl2 buffer (see REAGENT SETUP)
  • Otto I solution (see REAGENT SETUP)
  • Otto II solution (see REAGENT SETUP)
  • PI stock solution (Sigma-Aldrich, cat. no. P4170; Molecular Probes, cat. no. P-3566) (see REAGENT SETUP)
    Caution A mutagen and is moderately toxic. It may be harmful if inhaled, swallowed or absorbed through the skin. Use gloves when handling it. Be careful of particulate dust when weighing out the dye.
  • RNase stock solution (Sigma-Aldrich, cat. no. R5000) (see REAGENT SETUP)
  • DAPI stock solution (Sigma-Aldrich, cat. no. D9542; Molecular Probes, cat. no. D-1306) (see REAGENT SETUP)
    Caution A possible carcinogen. It may be harmful if inhaled, swallowed or absorbed through the skin. It may also cause irritation. Use gloves when handling it. Be careful of particulate dust when weighing out the dye.
  • Calibration particles: fluorescent beads (Molecular Probes, cat. no. A-7304—UV, Partec, cat. no. 05-4020—UV, Partec, cat. no. 05-4006—green) or stained trout red blood cells (Partec, cat. no. 05-7302—DAPI, Partec, cat. no. 05-7303—PI)
  • Cleaning and decontamination solutions for flow systems (Becton Dickinson, cat. no. 340346; Partec, cat. nos. 04-4009 and 04-4010, weak solution of sodium hypochlorite, e.g., household bleach diluted 1:5 in distilled water)
  • Optional: chemical compounds counteracting negative effects of cytosol such as reducing agents (e.g., β-mercaptoethanol) and phenolics-binding compounds (e.g., PVP-10, PVP-40)
  • Tween 20 (Sigma-Aldrich, cat. no. P2287)
  • NaOH
  • Hydrochloric acid (HCl)

Equipment

  • Plastic Petri dishes (5.5 cm diameter)
  • Razor blades (double-edged) and razor blade holder (e.g., model GSM Mini-Glass Scraper; Allway Tools)
  • Nylon mesh (42-μm pore size, squares 2 × 2 cm2); uncut mesh ( Uhelon mesh; Silk and Progress; Moravská Chrastová, CZ); Nytex mesh (Tetko Inc., Elmsford, NY). Alternatively, disposable filters (e.g., Partec, cat. nos. 04-0042-2316 or 04-0042-2317) may be used
  • Polystyrene sample tubes suitable for the flow cytometer (e.g., BD Falcon, cat. no. 352008 for Becton Dickinson instruments and Sarstedt, cat. no. 55.484 for Partec instruments)
  • Sample tube holder
  • Ice container
  • Air displacement pipettes and appropriate tips (1 ml, 200 μl)
  • Flow cytometer with light source suitable for excitation of the DNA fluorochrome used in the study (e.g., diode-pumped solid-state laser emitting at 532 nm or argon-ion laser tuned to 488 or 514.5 nm for samples stained with PI, and a mercury arc lamp or laser tuned to 340–380 nm for samples stained with DAPI). While a one-parameter instrument is sufficient to measure DNA content, the ability to evaluate light scatter properties assists in assessing the possible negative effects of cytosolic compounds on sample quality38. In addition, doublets may be detected on a fluorescence/side scatter cytogram (see EQUIPMENT SETUP)
  • Appropriate software for the evaluation of flow cytometric data
  • Nitrile or latex gloves, safety glasses

Reagent setup

  • Plant material If living plant tissues are used, they should be fresh, ideally sampled from intact, sufficiently watered plants, immediately before analysis. It may be important to choose appropriate developmental stage. Young, rapidly growing leaves usually give the best results; note that differentiated and senescent tissues may comprise a high proportion of endopolyploid nuclei.

    Tissue samples can often be preserved for up to several days if kept at 4 °C in a Petri dish or plastic box, (partially) wrapped in a moistened paper tissue. The period for which a tissue can be stored differs in individual species and should be determined empirically. In some applications (e.g., DNA ploidy estimation), silica-dry materials, herbarium vouchers and mature seeds can be used. However, to avoid unspecified effects on chromatin and DNA staining due to drying or fixation, only fresh living tissues are recommended for high-quality estimation of DNA contents in absolute units.

    In any case, materials potentially colonized by pests (fungi, insects and tissues with insect eggs laid on) harbor risk of biological contamination and accumulation of defence substances, and should therefore be avoided.

    If protoplasts are used, they should be freshly isolated (to avoid regeneration of cell wall) and viable (greater than 90%). Nuclei cannot be isolated from 'collapsed' protoplasts, and it is recommended that protoplasts are purified (e.g., by gradient centrifugation) before use. Protoplast viability can be determined, for example, using fluorescein diacetate41.

    In vitro cultured calli are suitable if they are sufficiently compact and actively growing. Senescent and dying tissues (brown/black) should be avoided. Friable calli that completely disperse to single cells and small cell clusters after immersing in the nuclear isolation buffer are less appropriate since they are difficult to homogenize. Their suitability should be tested empirically.
  • LB01 buffer 15 mM Tris, 2 mM Na2EDTA, 0.5 mM spermine tetrahydrochloride, 80 mM KCl, 20 mM NaCl, 0.1% (vol/vol) Triton X-100. Adjust to pH 7.5 with 1 M NaOH. Filter through a 0.22-μm filter. Add β-mercaptoethanol to 15 mM. Store at −20 °C in 10 ml aliquots. Do not refreeze after thawing.
    Caution β-mercaptoethanol may be fatal if inhaled or absorbed through the skin, and is harmful if swallowed. High concentrations are destructive to the skin, eyes, mucous membranes and upper respiratory tract. Wear gloves and safety glasses and work in a chemical fume hood when handling concentrated solutions.
  • Galbraith's buffer 45 mM MgCl2, 20 mM MOPS, 30 mM sodium citrate, 0.1% (vol/vol) Triton X-100. Adjust pH to 7.0 with 1 M NaOH, filter through a 0.22-μm filter and store at −20 °C in 10 ml aliquots. Do not refreeze after thawing.
  • Tris.MgCl2 buffer 200 mM Tris, 4 mM MgCl2·6H2O, 0.5% (vol/vol) Triton X-100. Adjust pH to 7.5 with 1 N HCl, filter through a 0.22-μm filter and store at 4 °C.
  • Otto I solution 0.1 M citric acid, 0.5% (vol/vol) Tween 20. Filter through a 0.22-μm filter and store at 4 °C.
    Critical It is essential to use cell culture–tested Tween 20 (Sigma-Aldrich, cat. no. P2287). Tween 20 for molecular biology (Sigma-Aldrich, cat. no. P9416) is not suitable for FCM and yields DNA content histograms with lower resolution.
  • Otto II solution 0.4 M Na2HPO4·12H2O. Filter through a 0.22-μm filter and store at room temperature (18–25 °C). Dissolving can be speeded up by heating. Note that a fluorochrome can be added before final volume adjustment. In that case, the solution should be stored in darkness.
  • DAPI stock solution 0.1 mg ml−1 DAPI. Filter through a 0.22-μm filter to remove small particles. Store at −20 °C in 1 ml aliquots. Do not refreeze after thawing.
  • PI stock solution 1 mg ml−1 PI. Filter through a 0.22-μm filter to remove small particles. Store at −20 °C in 1 ml aliquots. Do not refreeze after thawing.
  • RNase stock solution 1 mg ml−1 RNase. Heat to 90 °C for 15 min to inactivate DNases. Filter through a 0.22-μm filter. Store in 1 ml aliquots at −20 °C. Do not refreeze after thawing.

Equipment setup

  • Flow cytometer Check fluidics for proper function and make sure that the tubing is clean, free of air bubbles and that in-line filters are free of any blocking particles such as algae. Note: sodium azide (e.g., 0.02% wt/vol) may be added to the sheath fluid to prevent microbial growth in the reservoir. The optical filter set should be compatible with the spectral properties (i.e., excitation and emission spectra) of the DNA fluorochrome used. Align the instrument using appropriate calibration particles to obtain the lowest coefficient of variation of the fluorescent peaks (light scatter peaks if applicable) and lowest background, and check the instrument linearity (the 4C/2C peak ratio should be in the range of 1.98–2.02). The use of fluorescent beads is advisable as their quality is known and their analysis reflects the instrument behavior. While simple instruments do not require extensive adjustment of many parameters, more sophisticated cytometers may require setting appropriate threshold levels, gating windows and other parameters. In some instruments, fluorescence pulse parameters may be selected, such as pulse height and area. Although, in principle, pulse area should be selected, we recommend consulting manufacturer's instructions for appropriate setting (e.g., pulse height is the standard option in Partec instruments due to the large diameter of the excitation light beam).
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Procedure

  1. Preparing suspensions of intact nucleiNuclear suspensions can be prepared either according to the one-step protocol (option A) or the two-step protocol (either option B or option C). While the one-step protocol presented here works with most plant species, for some of them the two-step protocol results in histograms with higher resolution of DNA peaks. If preparing nuclear suspensions from protoplasts, follow option D.
    1. Preparation of nuclear suspension using one-step protocolTiming: 5 min or more
      1. Place a small amount of plant tissue (typically 20 mg) in the center of a plastic Petri dish.
        Critical step The amount of material used should be determined empirically with respect to the concentration of nuclei in the sample and the amount of debris background on the histogram of DNA content. For internal standardization, add leaf tissues of an appropriate reference standard. The ratio sample:standard tissue amount should be determined empirically, so that their G1 peaks are of similar height on the histograms of DNA content.
      2. Add 1 ml ice-cold nuclei isolation buffer (LB01, Galbraith's buffer, Tris.MgCl2 buffer) to the Petri dish.
        Critical step The choice of nuclear isolation buffer is empirical. It is recommended to test several buffers and select the best performing one for a given plant species and tissue.
      3. Chop the tissue immediately in the buffer with a new razor blade or a sharp (disposable) scalpel.
        Critical step It is important that the razor blade or scalpel is sharp, and the material is chopped into very fine slices and not just squeezed (use each edge of the razor blade only once). The extent of chopping should be determined empirically with respect to the concentration of nuclei in the sample and the amount of debris background on the histogram of DNA content. Dispersal and drying out the buffer during chopping should be avoided. Tilting the Petri dish so that the buffer occupies a small surface is helpful. Chopping the tissue in a walk-in cold room (with all components chilled to 4 °C) may be helpful in recalcitrant plant samples.
      4. Mix the homogenate by pipetting up and down for several times (avoid air bubbles).
      5. Filter the homogenate through a 42-μm nylon mesh into a labeled sample tube. Note that due to losses of the solution during chopping and filtration, the volume of the filtrate is typically approximately 0.5 ml. If critical, sample loss can be reduced by presoaking the nylon mesh with nuclear isolation buffer shortly before filtration. Check visually that the filtrate is free of any particles that may cause instrument clogging.
      6. Add stock solution of a DNA fluorochrome and shake gently. DAPI is typically used at 4 μg ml−1; PI is typically used at 50 μg ml−1 simultaneously with RNase at 50 μg ml−1.
        Critical step As PI binds also to dsRNA, it needs to be used simultaneously with RNase.
      7. Incubate the sample on ice before analysis (a few minutes to 1 h), with occasional shaking.
        Critical step The optimal staining period for a given plant species and tissue should be determined experimentally.
      8. Proceed to Step 2 to analyze nuclear DNA content.
    2. Preparation of nuclear suspension using a two-step protocolTiming: 15 min or more
      1. Follow Step 1A(i).
      2. Add 1 ml ice-cold Otto I solution to the Petri dish.
      3. Follow Step 1A(iii–v).
      4. Pellet the nuclei (150 g/5 min).
        Critical step As the isolated nuclei are stable in the Otto I solution, it is possible to prepare several samples in advance and centrifuge them simultaneously. The relative centrifugal force and duration of centrifugation may require adjustment for certain materials and should be verified empirically.
      5. Carefully remove the supernatant, leaving approximately 100 μl of the liquid above the pellet. Take care not to remove the pelleted nuclei.
      6. Resuspend the nuclei by gentle shaking (flicking the bottom of the tube using a finger may help), and add 100 μl fresh ice-cold Otto I solution.Pause Point Store the suspension at room temperature or at 4 °C until the next step, shaking occasionally. The samples can be kept at this stage for up to several hours. This allows the preparation of many samples before the analysis and their transfer to a FCM laboratory if the samples cannot be prepared on site.
      7. Add 1 ml Otto II solution to the nuclear suspension.
        Critical step The addition of the Otto II solution should raise pH of the nuclear solution to approximately 7.3 (citric acid + phosphate buffer) and increase its salt concentration. To maintain these parameters within a reasonable range, the amount of the added Otto II should be approximately fourfold that of the nuclear solution. The sample should be analyzed within a few minutes of adding Otto II solution. Isolated nuclei may not be stable for prolonged periods after this step.
      8. Add stock solution of a DNA fluorochrome and shake gently. DAPI is typically used at 4 μg ml−1; PI is typically used at 50 μg ml−1 simultaneously with RNase at 50 μg ml−1. Alternatively, the nuclear stains (and RNase) can be included in the Otto II solution.
      9. Incubate the sample for a few minutes, with occasional shaking.
        Critical step The optimal staining period for a given plant species and tissue should be determined experimentally. Short incubations often give the best results.
      10. Proceed to Step 2 to analyze nuclear DNA content.
    3. Preparation of nuclear suspension using a simplified two-step protocolTiming: 5 min or more
      1. Perform Step 1B(i–iii).
      2. Add 2 ml of Otto II solution to the nuclear suspension.
      3. Follow Step 1B(viii–x).
    4. Preparation of nuclear suspension from protoplastsTiming: 10 min or more
      1. Add freshly isolated and viable protoplasts to ice-cold nuclear LB01 isolation buffer to a concentration of 105–106 per ml.
        Critical step In order to improve the release of nuclei from protoplasts, the concentration of detergent (Triton X-100) in the LB01 buffer should be increased to 0.5–2% (vol/vol). Suitable concentrations should be determined empirically. Note: Extremely high detergent concentrations may cause nuclear instability.
      2. Incubate for 5–15 min on ice with occasional shaking of the tube.
      3. Follow Step 1A(v–viii).
  2. Analysis of nuclear DNA contentFCM measures the relative fluorescence of the stained nuclei. The basic protocol for sample analysis is presented below (option A). Options B and C describe ploidy estimation using external and internal standards, respectively, whereas option D describes the analysis of genome size in absolute units. For the sake of simplicity, only one-parameter analyses are considered.
    1. Measurement of relative nuclear DNA fluorescence intensityTiming: 5 min
      1. Introduce the suspension of stained nuclei into the flow cytometer and run it for a few seconds.
        Critical step This period is required to stabilize sample rate and to equilibrate the concentration of dye bound to sample nuclei and to the line tubing.
      2. Adjust the sample rate. Due to rather low concentration of nuclei in the sample, the sample rate usually does not exceed 20–50 nuclei/s.
        Critical step The concentration of nuclei in the sample varies greatly depending on the quantity of source tissue and its type. It may be as low as 2 × 103 nuclei ml−1 in samples prepared from a small amount of silica-dried material, and reach 200 × 103 nuclei ml−1 in samples prepared from seeds and protoplasts. Excessive sample delivery rates may result in broadening DNA peaks and higher peak CVs. Note that some instruments only have a few pre-set sample delivery rates (e.g., slow, medium and fast). In that case, changing sample rate between samples is usually not necessary.
      3. Position DNA peak(s) to the required position on the abscissa by adjusting the instrument gain settings (photomultiplier voltage, amplifier gain or both). A threshold for cutting-off undesirable low-channel signals coming from cell debris and/or autofluorescent compounds may be required.
        Critical step The gain should be kept within the range recommended by the manufacturer to avoid signal anomalies due to saturation effects and other nonlinearities.
      4. Measure 5,000 particles and save the data.
      5. Use appropriate software to assess the quality of analysis by estimating the proportion of debris background, which should be as low as possible, by checking peak symmetry and by evaluating the distribution of fluorescence intensities (width of DNA peaks), expressed as coefficient variation (CV% = SD of the peak/mean channel position of the peak × 100). CVs below 3% are considered good, whereas measurements having CVs more than 5% are considered not acceptable. It should be noted that CVs below 5% cannot be achieved with some materials (e.g., samples rich in polyphenolics, with very small genome size, some herbarium vouchers). Consider that CVs can only be calculated for the measurements done in linear scale (CV values are meaningless for peaks on logarithmic scale).
        Critical step This analysis is critical especially when a new material is measured as the data will be used to choose the optimal methodology.
    2. Ploidy analysis with external standardTiming: 5 min
      1. Run a sample prepared from a (reference) plant with known ploidy (chromosome number) following Step 2A(i and ii).
        Critical step The reference plant must belong to the same species as the unknown sample(s). The plant may not necessarily be diploid. However, this needs to be considered when estimating DNA ploidy of unknown sample(s).
      2. Using instrument gain (photomultiplier voltage, amplifier gain or both), position the peak of G1 nuclei of a diploid reference plant on approximately one-fifth of the linear abscissa (e.g., channel 200 on a 1,024 scale).
        Critical step Do not change instrument settings after this step. It is advisable to run new samples from the reference plant approximately each hour and re-establish its G1 peak position.
      3. Analyze unknown samples following Step 2A(i and ii) and 2A(iv and v).
        Critical step Due to variation in sample preparation, staining and analysis, the peak position may not exactly reflect the ploidy. While in low polyploids a small shift in peak position usually does not compromise reliability of ploidy estimates, attention should be paid to the analysis of high polyploids (e.g., greater than 6x) because relative differences in nuclear DNA content between neighboring cytotypes decrease with increasing ploidy. Reliable estimation of ploidy levels may then require an internal standard (see option C). The use of internal standards is recommended whenever there are doubts about the results, and especially when novel and/or unexpected ploidy is found.
      4. Determine the mean channel number of the G1 sample peak.
      5. Calculate the DNA ploidy of the unknown sample as follows:

        Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com


        Critical step Note that the assay with external standard is not sufficiently precise for aneuploidy detection. This should be done with internal standardization (see option C).Pause Point Optionally, histogram evaluation (Step 2A(v)) can be postponed and performed off-line after data acquisition.
    3. Ploidy analysis with internal standardTiming: 5 min
      1. Prepare the sample by simultaneous chopping and staining tissues from a reference plant with known ploidy (chromosome number) and from the unknown plant sample as described earlier.
        Critical step The reference plant should belong to the same species as the unknown sample(s). Because of this, nuclear DNA may be stained either by PI or DAPI, although the latter option may result in higher resolution of histograms of DNA content. The use of DAPI is also recommended if aneuploidy detection is attempted.
      2. Perform sample analysis following Step 2A(i–v). Positioning the standard peak on an appropriate channel on the abscissa is usually done only once and the following samples are measured under the same instrument settings.
      3. Determine the DNA ploidy of the unknown sample following Step 2B(iv and v).
        Critical step Perfect overlap of peaks of the reference plant and the unknown sample indicates the same ploidy. Bifurcated or nonsymmetrical (skewed) peaks imply aneuploidy. However, aneuploidy may only be reliably identified using simultaneous analysis of a reference and aneuploid plant if the CVs of the DNA peaks are lower than half of the difference between the DNA contents of both plants34, 42. If such a precision cannot be achieved, one solution is to use nuclei of a different plant species with an appropriate genome size as a reference point. In that case, aneuploidy identification requires replicate measurements35. Finally, it is always advisable to accompany unexpected flow cytometric results by conventional chromosome counts.
    4. Estimation of nuclear DNA content in absolute units (genome size)Timing: 5 min
      1. Select a suitable internal DNA reference standard by individually analyzing the DNA contents of the unknown sample and potential DNA reference standards.
        Critical step Choose a reference standard whose genome size does not differ from that of the sample more than twofold. This reduces the risk of errors due to instrument nonlinearity. On the other hand, standards with too similar nuclear DNA content should not be used to avoid peak overlapping (this depends on the resolution of the DNA content histograms, the smallest reasonable difference being 15–20%). It is advisable to use one of the established plant DNA reference standards43, 44 (see Table 2).
      2. Confirm the suitability of the reference standard by analyzing a sample prepared from both tissues simultaneously (see also Box 1).
        Critical step This point provides the opportunity to detect the negative effects of cytosol on DNA staining.
      3. Prepare the sample by simultaneously chopping and staining tissues from a DNA reference standard and unknown sample.
        Critical step The protocol for sample preparation should be suitable for both DNA reference standard and unknown sample. Chopping should be done in a sandwich-like fashion rather than sequentially to make the effects of cytosolic compounds as equable as possible (J. Loureiro, personal communication). Because the standard and the sample may differ in DNA base content (AT/GC ratio), samples for the estimation of genome size should be stained by PI, the binding of which to DNA is not influenced by base composition.
      4. Perform sample analysis following Step 2A(i–v). Positioning the standard peak on an appropriate channel on the abscissa is usually done only once, and the following samples are measured using the same instrument settings.
      5. The amount of nuclear DNA of the unknown sample is calculated as follows:

        Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com


        Critical step Take care of the C-levels of DNA peaks used for calculation. For example, in bryophyte gametophytes and in angiosperm haploids the first peak seen at the left of the histogram usually is in 1C.
      6. Repeat the analysis on at least two other plants from the same accession. Alternatively, three replicates on the same plant may be performed if additional individuals are not available.
        Critical step At least three different measurements should be done on different days to compensate for instrument drifts. The artifactual variation between particular FCM assays is usually below 2%, but may exceed this value in recalcitrant plant species.
      7. Calculate the average 2C nuclear DNA content and perform statistical analysis as required. Mean DNA values should be accompanied by a measure of the spread of individual measurements (e.g., s.d., s.e.m. or maximum/minimum range) and the sample size.
        Critical step Absolute differences between particular measurements (i.e., maximum/minimum value) should not pass the 2% threshold. In case of higher data variability, a possibility of plant material inhomogeneity should be checked (e.g., occurrence of chromosomally aberrant individuals, infected material). Outliers should be removed only upon a sound statistical justification, and their removal should be reported in a publication. Consequences of the removal for statistical significances must be carefully considered.
      8. If needed, convert DNA mass in picograms to the numbers of base pairs or vice versa as follows45: 1 pg DNA = 0.978 × 109 bp.
        Critical step Plant species are often polyploid and therefore a proper terminology for presenting genome size should be followed to avoid incorrect data interpretation3 (see INTRODUCTION). Note that, in molecular biology, DNA quantity is often given in mega base (= DNA length) instead of mega base pair (= DNA mass), which can lead to a twofold error in converting DNA amounts to other units. Another source of potential twofold error may arise from the fact that molecular biologists commonly cite genome sizes in C-values, whereas cytometrists cite genome sizes in 2C-values.Troubleshooting
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Troubleshooting

Troubleshooting advice can be found in Table 3. Box 1 discusses the possible solutions for situations where the cytosol has a negative effect on the estimation of nuclear DNA content.


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Anticipated results

The assay should result in a histogram of fluorescence intensities, which correspond to nuclear DNA contents. Generally, the histograms contain only DNA peaks representing interphase nuclei. S-phase nuclei are rare in samples prepared from leaves, which is the most frequently used tissue. However, they are frequent in tissues with cycling cells such as root tip meristems. The interphase nuclei of cycling cells may be either in G1 or in G2 phases of the cell cycle, but the cycling cells may temporarily or irreversibly leave the cycle in G1 or G2 and enter quiescent states G1/0 and G2/0, respectively. As the nuclei of cycling and quiescent interphase cells have the same DNA amounts, and as it is not trivial to discriminate between quiescent and cycling nuclei, in the following we only refer to the G1 and G2 phases.

The number of DNA peaks on a histogram and their height depend primarily on the sample type. Analysis of freshly growing leaves typically results in histograms with a single prominent DNA peak representing nuclei in G1 phase of the cell cycle and a minor, sometimes undetectable, peak representing G2-phase nuclei (Fig. 1a). In certain instances, the G1 peak may be undetectable and only the G2 peak may be seen. The analysis of older and dormant leaves may reveal a large peak of nuclei arrested in G2 phase (Fig. 1b). However, similar results may also be obtained in a mixoploid plant consisting of diploid and tetraploid cells (Fig. 1c). Many plant species exhibit endopolyploidy, and the presence of endopolyploid nuclei is demonstrated by additional peaks with 8C, 16C, 32C and even higher DNA levels (Fig. 1d). While the DNA content analysis is typically done using linear amplification (Fig. 1a–c), analysis of higher levels of endopolyploidy may be facilitated by using a logarithmic scale for the abscissa (Fig. 1d).

Figure 1: Histograms of relative fluorescence intensities (relative nuclear DNA contents) obtained after the analysis of isolated plant nuclei.
Figure 1 : Histograms of relative fluorescence intensities (relative nuclear DNA contents) obtained after the analysis of isolated plant nuclei.

All samples were prepared according to simplified two-step method (Step 1C), stained with 4′,6-diamidino-2-phenylindole and measured using a Partec PAII flow cytometer equipped with mercury arc lamp as the excitation light source. (a) Analysis of nuclei isolated from a leaf of Pisum sativum yielded a histogram with a single dominant peak of G1-phase nuclei. (b) The analysis of an older leaf of Zea mays revealed a higher proportion of nuclei within G2 phase. (c) A similar histogram is obtained after analyzing a mixoploid plant of Kochia scoparia (2n = 2x + 4x) with diploid and tetraploid cells. (d) The analysis of endopolyploidy in Senecio rowleyanus was facilitated by the use of a logarithmic abscissa, which permitted visualization of higher numbers of endopolyploidy levels.

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Ploidy screening is one of the most frequent applications of DNA FCM. The assay may be performed either with external (Fig. 2a and b) or internal standards (Fig. 2c and d). While the former is suitable for large-scale screening, the latter is more precise since it eliminates most of the sources of variation. External standardization involves the analysis of a plant with known ploidy (Fig. 2a). The analysis of an unknown conspecific plant with the same instrument settings results in a histogram of DNA content whose relative G1 peak position indicates the plant ploidy (Fig. 2b). Simultaneous analysis of a reference and unknown plant (internal standardization) of different ploidy results in a histogram having two G1 peaks. The ratio of the G1 peak positions reflects the ratio of ploidy levels (Fig. 2c). Provided the resolution of a DNA content histogram is sufficiently high, it is possible to discriminate the peaks representing a diploid standard and an aneuploid plant (Fig. 2d).

Figure 2: Flow cytometric ploidy analysis.
Figure 2 : Flow cytometric ploidy analysis.

Ploidy was analyzed in Hieracium echioides using external (panels a and b) and internal (panels c and d) standards. (a) A diploid plant (2n = 2x = 18) was used as an external reference standard and its G1 peak was positioned at channel 200. (b) The G1 peak of the unknown sample appeared on channel 296, and its ploidy was thus estimated as 3x. (c) This conclusion was confirmed after simultaneous analysis of the reference diploid plant and the triploid sample, when the ratio of G1 peak positions was 1.506. (d) Aneuploidy status of a Hieracium hybrid plant (An) was suggested after its simultaneous analysis with an euploid hybrid of the same origin (2n = 45; Eu). Subsequent karyological analyses of the unknown sample revealed 43 chromosomes in the somatic tissues. The samples in panels (ac) were prepared according to Step 1A and using LB01 buffer; sample (d) was prepared according to the standard two-step method (Step 1B). Samples were stained with 4′,6-diamidino-2-phenylindole (DAPI) and measured on a Partec PAII flow cytometer equipped with a mercury arc lamp as the excitation light source.

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Estimation of genome size in absolute units requires internal standardization. The reference standard may have a genome size reasonably larger (Fig. 3a) or smaller (Fig. 3b) than the unknown sample. In both cases, the histograms of DNA content should comprise two large G1 peaks representing nuclei of the reference and the sample. The ratio of their positions determines the genome size of the unknown sample.

Figure 3: Estimation of nuclear DNA content in absolute units.
Figure 3 : Estimation of nuclear DNA content in absolute units.

Examples are given for the analysis with internal reference standards having larger (a) and smaller (b) genome sizes as compared with the unknown sample. (a) The genome size of hexaploid Curcuma angustifolia was 0.847-fold that of the standard (Glycine max 'Polanka'; 2C = 2.50 pg DNA), and its 2C-value was estimated as 2.12 pg DNA. (b) The genome size of tetraploid Hieracium aurantiacum was 1.440-fold of that of the standard (Zea mays 'CE-777'; 2C = 5.43 pg DNA), and its 2C-value was determined as 7.82 pg DNA. The samples were prepared according to the two-step method (Step 1B), stained with propidium iodide (+RNase) and measured on a Partec CyFlow cytometer equipped with a Cobolt diode-pumped solid-state laser emitting at 532 nm.

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Acknowledgments

J.D. was partially supported by the Ministry of Education, Youth and Sports of the Czech Republic (grant awards ME 884 and LC06004), and J.S. by the Ministry of Education, Youth and Sports of the Czech Republic (project no. MSM 0021620828), and the Academy of Sciences of the Czech Republic (no. AV0Z60050516). We thank David W. Galbraith (Tucson, AZ) for revision of the text, and an anonymous reviewer for useful comments.

Competing interests statement: 

The authors declare no competing financial interests.

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  1. Laboratory of Molecular Cytogenetics and Cytometry, Institute of Experimental Botany, Sokolovská 6, CZ-77200 Olomouc, Czech Republic.
  2. Department of Cell Biology and Genetics, Palacký University, Šlechtitelů 11, CZ-78371 Olomouc, Czech Republic.
  3. Department of Systematic and Evolutionary Botany, Faculty of Life Sciences, University of Vienna, Rennweg 14, A-1030 Vienna, Austria.
  4. Department of Botany, Faculty of Science, Charles University in Prague, Benátská 2, CZ-12801 Prague, Czech Republic.
  5. Institute of Botany, Academy of Sciences of the Czech Republic, Průhonice 1, CZ-25243, Czech Republic.

Correspondence to: Jaroslav Doležel1,2 e-mail: dolezel@ueb.cas.cz