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White Paper

Using the Accuri C6 Flow Cytometer ® for Rapid and Accurate Analysis of the Nuclear DNA Contents of Flowering Plants

Summary. The nuclear DNA content (C-value) is a fundamental most convenient and accurate way to determine C-values of plants, but sampling of the ca. 480,000 species of angiosperms reported. For plant species for which C-values are available, these values span an extraordinary range, approximately 1250-fold (0.2 pg to 254.8 pg). For plants whose C-values are the range of possible C-values exceeds the dynamic range of

In determining C-values for new plant species, the Accuri C6 high dynamic range of the ADC (24 bits, providing 16 million range of the described angiosperms. This white paper cellular debris, and includes typical measurements from plant Authors: David W. Galbraith and Georgina M. Lambert University of Arizona, Department of Plant Sciences and Bio5

for the unknowns and the appropriate controls within the empirically, it considerably slows down the sample throughput.

Razor blades are available from VWR (VWR International. The seeds on the plates are vernalized by incubation at 4oC for 2 days before transfer to a Conviron growth chamber under a 12-h day/12-h night illumination regime. Tissues are chopped using a new razor blade for 2-3 minutes. Gibbstown. Catalog number 537059). We recommend the procedures all be done in a walk-in cold room. in 3 inch plastic pots containing Scotts MetroMix 3000 (The Scotts Miracle-Gro Company. West Chester PA.. the Plant DNA Analysis Template is opened. Cincinnati. In this white paper. we germinate seeds and grow plants under sterile conditions as described previously (Zhang et al. The homogenate is filtered through the 30 µm nylon filter. and a stock solution (1 mg/mL) in deionized water is prepared. can provide simultaneous linear estimates of nuclear DNA content spanning at least two orders of magnitude (a range of 0. Catalog number 04-0042-231). Standardization is complicated by base-pair specificity inherent to some fluorochromes (DAPI and Hoechst 33257 being AT-specific (Dolezel et al. and 1. we provide detailed information as to how to use the Accuri C6 Flow Cytometer® for the analysis of plant genome sizes. Finally. Dolezel et al... and a univariate histogram of FL2A. and stored in aliquots at -20°C. and staining in the presence of RNAase eliminates interference by double-stranded RNA. This covers most of the C-values encountered in the flowering plants found across the world. uniquely. Samples are generally taken after four weeks of plant growth. and are emptied or refilled. with an incident light flux of 150 to 175 μm m-2 sec-1 and a temperature of 22oC (day) and 20oC (night). and mithramycin and chromomycin A3 being GC-specific). Catalog number EN0531). Samples are harvested the day of the flow analyses. The fluorescence signals (pulse area measurements) are screened by the following filter configurations: (a) FL-1: a 530/14 nm band-pass filter (b) FL-2: a 585/20 nm band-pass filter. except those of Arabidopsis. and incubated on ice for 15 minutes.™ 1 . in a suitable buffer. rather than within. Propidium iodide is available from Calbiochem (EMD Chemicals. the plant tissues are homogenized by chopping. This template consists of the following histograms: FSC-A vs SSC-A.and 8-peak fluorescent bead mixtures (Spherotech) according to instructions (CFlow User Guide pp. The CFLow® software is started. FL1-A vs FL2-A. 1983). FL3-A vs FL2-A. The plates are kept in a vertical orientation so that the roots grow on the surface of. The razor blade is discarded after a single use. 2007). 1983) or plant standards (Johnston et al. We germinate all seeds.5 mL) is added to a labeled tube containing 2. The seeds mentioned in this paper are available from Lehle Seeds (Round Rock TX 78680-2366 USA) and from J. Materials and Methods Materials. Ohio. the agar medium. NJ. seeds are sterilized by immersion for 5 minutes in a freshly-prepared solution of commercial bleach diluted 1:1 with distilled water containing 10 μL/mL Triton-X100. We purchased plants of Astroemeria aurea (Lily of the Incas) from a local supermarket. of which propidium iodide (PI) is the most popular. plants are watered daily. Plant Production. Flow Cytometry. such as chicken red blood cells (Galbraith et al. A fuller discussion can be found at Galbraith (2009). 1983) is added in the proportion of 1. Performance validation of the Accuri C6 Flow Cytometer is then done using 6.. The Accuri C6 Flow Cytometer is switched on. using a razor blade.. All procedures are done at 4oC or on ice. Marysville. Flow cytometry within reach. see also hyperlink c in Table 1). Dolezel (Olomouc. Galbraith’s buffer supplemented with 0. The fluorescence produced by intercalating fluorochromes. In brief. Preparation of Plant Homogenates and Labeling of Nuclei.5 µL of 10 mg/mL DNAse-free RNAse A. NJ.9 pg).m-2 sec-1.5 mL buffer per 100 mg tissue. MD. 2008).1% w/v Triton X-100 (Galbraith et al. In order to allow the nuclei to pass through the flow cell. After filtration of the homogenate to remove large particulate materials. Each slicing action of the razor blade should cut cleanly and without bruising the tissue. and (c) FL-3: a 670 nm long-pass filter. Propidium iodide is then added to a final concentration of 50 μg/mL. and this instrument.3480. The plants were supplied by InterAmerican Products. 6-7). these have a notional porosity of 30 μm.. The DNA histograms produced by the C6 display excellent peak CVs. the samples are stained with DNAspecific fluorochromes and the fluorescence is quantified. Approximately 50-100 mg of plant tissue is excised and placed in a plastic 60 mm petri dish standing on a pre-chilled metal plate placed on ice in a rectangular plastic tray. 1999. Ohio) in a growth room at 25oC under a 12/12 light dark cycle with a light intensity of 150-175 μ. The homogenate (0.einsteins. are not biased by base-pair composition.Introduction Flow cytometry is the simplest way to determine plant nuclear DNA contents (Galbraith et al. 2007. Catalog number 55411-050). and is stored at 4oC. and allowed to warm up for 15 minutes before analyses are begun. The seeds are then rinsed 3x in sterile distilled water. Czech Republic). Waste and sheath fluid bottles are checked during this time.. Nylon filters are from Partec (Swedesboro. For Arabidopsis. The absolute values for the amounts of the plant nuclear DNA are found by comparison to cells or nuclei of known DNA content.2% agar. and planted on Murashige and Skoog (MS) agar plates supplemented with 2% sucrose. as required. Analysis of the homogenates is based on lightscatter and fluorescence signals produced from 20 mW laser illumination at 488 nm. RNAse A (10 mg/ml in water) is from Fermentas (Glen Burnie. The stained samples are incubated on ice in darkness for 30 minutes prior to analysis.

Dolezelb J. Dolezel for FSC. The tissues of some species can be refrigerated in damp paper towels for up to two days prior to analysis should this be necessary. (2007) provide additional suggestions for sample preparation in these situations. additional populations are seen if somatic endoreduplication occurs. This region will contain two populations of nuclei for nonendoreduplicated species.11 3. The zoom tool is used to locate and isolate the peaks appearing within this univariate 2C DNA contents. The default threshold values on the C6 are set at 2 . Other species can release secondary plant products that reduce fluorochrome fluorescence.html http://lmcc. and one may need to use more than the 200 mg sample size suggested in the protocol to provide sufficient nuclei for analysis. These data are used to calculate the DNA content of the nuclei. Saxa Medicago sativa L. young.19 34. Common name Thale cress Radish Alfalfa Garden pea Rye Wheat Lily of the Incas a b c 2C DNA content (pg) 0.09 16. 2007) Kew C-value Database (Bennett and Leitch. Threshold levels are set empirically to eliminate from detection the large amounts of irrelevant debris that are found in plant homogenates. The flow cytometer is routinely operated at the Slow Flow Rate setting (14 μL sample/ minute). and data acquisition for a single sample should typically occupy 3-5 minutes. Source tissues should be fresh. Personna. Regions of identification are then placed across the lowest peaks to export values representing peak positions and CVs.000.. source of seeds. Dolezelb Lehle Seedsa Inter-American Productsc http://www. 2004) Source of plants and hyperlinks Lehle Seedsa J. and well hydrated. Species Arabidopsis thaliana ecotype Columbia Raphanus sativus cv. Double-edged shaving blades are Table 1. cv.90 DNA content attribution Kew C-value Database (Bennett and Leitch. America Safety Razor Company) which generally should have an edge thickness of 0. cv. 2007) Kew C-value Database (Bennett and Leitch. These values are adjusted while acquiring data and observing the position of the nuclei on the bivariate dot plots such that all the nuclei are on scale with the least amount of debris appearing in these plots. Dolezelb Lehle Seedsa J.32 1. 2004) Olomouc website (Dolezel et al. Many sources exist for satisfactory blades (VWR. 2004) Kew C-value Database (Bennett and Leitch. and attribution.ieb. or chopping with dull razor blades. and univariate histograms of PI fluorescence (FL2-A) are accumulated based on this gate.22 mm or finer. any changes in homogenate color and consistency are contraindications. This is usually due to the source tissue being of poor condition. Dankovske Triticum aestivum L. We set the threshold at 10. The PI-stained nuclei will appear on the bivariate plots as a region of dots clustered around a diagonal line. with a secondary threshold of 1. Occasionally low yields of intact nuclei from the chopping procedure are www. Ctirad Secale cereale L. Names of plant species.65 80. and the RUN tab is clicked in CFlow to initiate data acquisition. 2004) Olomouc website (Dolezel et al. Careful observation of the samples during chopping is recommended. Scientific.Plant homogenate samples are introduced to the SIP.. and this setting will exclude from analysis very small nuclei. A polygonal gate is drawn to enclose the nuclei. 2007) Olomouc website (Dolezel et al. Poor CVs may be the result of excessive chopping. Fisher Scientific. alternatives are to employ different parts of the plant for analysis. Grasses are often problematic. Cimarron Pisum sativum L. line 812 Alstroemeria aurea Grah. Some plants may release mucilaginous compounds into the chopping buffer resulting in slime which should not be run through the instrument. Freezing tissues is not recommended.50 9. and/or to include additives to the chopping buffer to absorb secondary products. Troubleshooting. If these are observed.php?protocols=dna http://www.!cps.interamericanproducts.000 for FL-2. Old or dried-out tissues do not generally provide histograms of adequate quality.

1% (when determined using the CFlow software. so that absolute cell counts are automatically recorded for each experiment. and 2. The second cluster.™ 3 . so that runs from different days can be more easily compared. we have recommended 15 minutes. after manually positioning windows at the 50% points on either side of the peak). but it is possible some species may require longer staining times.065.6% (when determined using SigmaPlot. (B) Uniparametric histogram of FL2-A fluorescence. 1991). boxed in red. In this white paper. This can be easily monitored by flow analysis over time of successive samples taken from a single stained homogenate.ideal. Flow cytometry within reach. with fitting of a Gaussian function to the extracted data).0 fluorescence units (a ratio of 1:1.516. Figure 1A shows the biparametric frequency distribution (FL2-A versus FL3-A) of a PI-stained homogenate prepared from pea (Pisum sativum) leaf tissue. Unstable (drifting) peak positions may be due to insufficient periods of staining. The CVs for the G0/G1 peak are 2. the gated objects provide a very nice uniparametric DNA distribution with clearly-defined G0/G1 and G2 peaks.3 and 1. One. Two clusters are seen within this frequency distribution. Leitch and Bennett at The Royal Botanical Gardens. Flow analysis of homogenates from pea seedling tissue (A) Biparametric dot plot of FL2-A (585/40 nm) versus FL3-A (>670 nm) fluorescence emission. corresponds to subcellular debris. The peak positions in this case are 533.8%) of the objects detected by the flow cytometer. gated on region P1 of panel A. 1983. as analyzed by the C6 Flow Cytometer. A particularly useful resource is the C-value database maintained by Drs. as would be predicted for PIstained nuclei. .. Overall. This histogram is typical of the nuclear DNA contents found within non-endoreduplicated plant tissues (Galbraith et al. No correlation is seen between the FL2 and FL3 signals within this cluster. Kew (Bennett and Leitch. roughly star-shaped cluster (arrow) which comprises most of the objects in the homogenate. 2004).996). forms a very minor proportion (1. along with their 2C DNA content values. Pea has a reasonably large nuclear genome.Associate Professor of Immunology and Pediatrics Figure 1. and is known to produce high quality uniparametric DNA histograms (3). we are extremely pleased with the C6. the six-log scale means that we can collect all data without compensation. and would highly recommend it.898. Objects in this population exhibit a very strong correlation between the FL2 and FL3 signals. The Accuri C6 Cytometer and CFlow Plus have far exceeded our expectations. If you place gates around this population (region P). Results The plants used in this study are identified in Table 1. except that one must find or devise suitable holders for these to avoid inadvertent self-mutilation. Secondly. The C6 provides important additional data that we have previously been missing. One is the highly accurate volume determination.

87703. except using roots. or a 2C value of 0.5 (mean ± S.. Figure 2.). the CVs are also very acceptable ( and 179634.1% and 3.5%. 91698.588.32 pg). the mean FL2-A value of the 2C peak positions was 21.9999). and 171246. 1991). 2. corresponding to an endoreduplicative series (2C. (B) Enlargement of the square region-ofinterest (R1) containing the nuclei. and this produces multiple clusters within the frequency distributions. For seven repetitions on separate days. 4C. except using roots. etc. 16C. and 2. Day-to-day reproducibility of the C-value measurements on the Accuri C6 is excellent. The nuclei comprise a slightly larger proportion of the detected signals (region P1). These clusters fall on the characteristic PI-DNA 4 .8%. except using roots.2% of the detected signals) provides uniparametric distributions with well-defined peaks (Fig. Flow analysis of homogenates from arabidopsis leaf (A-C) and root (D-F) tissues (A) Biparametric dot plot of FL2-A versus FL3-A fluorescence emission. The mean fluorescence values of the peak positions for the nuclei are 24541.4%). which contains one of the smallest genomes of flowering plants (~157Mb (Bennett et al. 44930.2 (r2 = 0. 2003).5. 3. 8C.633. the CVs were 3.4%.6 ± 1. (D) As for panel A.1. Gating on region P1 again provides uniparametric histograms of excellent quality and linearity (Fig. (F) As for panel C. using different Arabidopsis seedlings as the source of nuclei. (C) Uniparametric histogram of FL2-A fluorescence. www. Arabidopsis exhibits endoreduplication within most of its somatic tissues and organs (Galbraith et al. 47446.7%. gated on region P1 of panel B.6%. 2F): the fluorescence values of the peaks are 22773.6.9999). 2E). 2. Similar results are seen for arabidopsis root homogenates (Figs.). 2C) typical of Arabidopsis thaliana (Galbraith et al. which fit a straight line almost perfectly (r2 = 0.9.AccuriCytometers. (E) As for panel B.D.. 2. which becomes particularly obvious following electronic expansion of the region in which it falls (Fig. The nuclei are indicated by polygonal region P1. Gating around this expanded region (P1. 2B)..Figure 2A illustrates the corresponding biparametric frequency distribution obtained from leaf homogenates of Arabidopsis thaliana. 2D. 1991). probably due to the absence of chloroplasts from roots. ~1.

(B) Enlargement of the square region-of-interest (R2) containing the nuclei. 3486157. 2.7.8%. 2.1%. Arabidopsis 8C. Triticum aestivum. (E) As for panel D except employing a log scale.4. 3D. Alstromeria aurea. For Figure 3C. Arabidopsis 4C. 453340.2. Figure 3.8%. Assignment of the six peaks to nuclei of individual species is done from the results of accumulation of histograms produced for analyses of the unmixed homogenates of individual species. then mixed.4%. 3B). Alstroemeria 2C. 1584795. 8C designate the C-values for the individual peaks within these species. the individual peaks and their positions and CVs are: Arabidopsis 2C. Pisum sativum. 77941. but these can be captured using a suitably-placed polygonal window (P2). 2. Abbreviations: At.5.2. filtered. 4C. 4. 2.The next experiment extends the observed linearity of PIbased C-value measurements to a dynamic range that spans most of the values reported for flowering plants. and Alstroemeria aurea). Ta. 40333. Triticum 2C.5%. with identification of the six DNA content peaks. The composite biparametric frequency distribution contains the diagonal region characteristic of PI-stained nuclei (Figs. (D) Plot of DNA content versus the mean fluorescence values of the 2C peak positions for the four species. Aa. r2 >0. Flow cytometry within reach. Triticum aestivum. 3E.2%.99). gated on region P2 of panel B. Pisum 2C. Simultaneous analysis of mixtures of plant homogenates (A) Biparametric dot plot of FL2-A versus FL3-A fluorescence emission. Arabidopsis thaliana. 3C). Ps.™ 5 . 3. The peak position values are strongly correlated with the reported nuclear DNA content values (Figs. Plant tissues are taken from four species (Arabidopsis thaliana. the nuclei are enclosed by polygonal region P2. (C) Uniparametric histogram of FL2-A fluorescence. 20868. chopped. and the resultant FL2-A uniparametric distributions have well-defined peaks corresponding to the nuclei of the different species (Fig. and stained separately.8. 3A. Pisum sativum. it is clear that the alstroemeria nuclei produce FL3-A signals that are off-scale. On magnification of the region. 2C.

this also can obscure the nuclei. Second. but also provides a convenient way to deal with the large amounts of debris released from plant tissues by the chopping process (Galbraith et al. including issues relating to experimental manipulations (the staining time. 3B) does not impede nuclear analysis. 3D.8) and the highest bin value available on the instrument (~16 x 106). and those relating to the C6 itself (deposition of murky substances on the flow cell walls). reflected by the low CVs. generally. 3A.8 pg (Fritillaria assyriaca Baker (Buitendijk et al. Since light scatter signals are. Nevertheless. ploidy. for example).9 pg. it is very simple to define the region of interest within biparametric distributions of FL2-A versus FL3-A since these detection channels roughly split the fluorescence emission of PI-DNA into shorter and longer wavelength spectral components (Figs. This dynamic range is larger than that of the described 2C values for the flowering plants. 6 . This is for two main reasons: first. which is about six-fold smaller than the smallest record for the flowering plants (Bennett and Leitch. C-value measurements are available only for about 2% of the species comprising the angiosperms.000. and the presence of secondary products. We conclude that. The variability in the position of the Arabidopsis 2C peak had a CV of 7.AccuriCytometers. from 0. 2007). 1976. the C6 displays a very high level of reproducibility in day-to-day measurements of the fluorescence of PI-stained plant nuclei. 1998)). 2A. Given the extreme ranges of 2C values encountered in the plant kingdom. Debris from different tissue sources. 2005). for accurate flow analysis of nuclear DNA contents. auto-rescaling obscures the presence of the nuclei on frequency distribution displays. careful adjustment of thresholds is required to allow visualization of the nuclei. 3) around the PI-DNA region downwards below the Arabidopsis 2C peak revealed little noise from debris for FL2-A values as low as 2. Overall. arrests prior to accumulation of data for adequate numbers of nuclei. nevertheless. the C6 employs a 24-bit analog-to-digital converter (ADC) for signal processing. Experience with other flow cytometers indicates data accumulation under conditions of linear signal amplification is preferred over logarithmic amplification. this white paper indicates the C6 Flow Cytometer should be able to measure any plant 2C value without modification to the instrument. The upper limit to C-value measurements using the C6 will be about 370 pg DNA. dynamic range. The Accuri C6 is optimally suited for the flow cytometric determination of plant nuclear DNA contents. based on the observed mean position for the 2C peak of alstroemeria (80. A further. for example shoots (Figs. for routine analysis of plant nuclear DNA contents.2pg (Fragaria viridis Duch. the Hoechst dyes. general problem in C-value determinations relates to the choice of a standard against which to determine the 2C value for the unknown species. in which the object of interest (the cell) represents most of the population. channel 3486157. 3E). based on total counts of scattering particles. 1-3). the linearity of measurement of plant genome sizes based on PI fluorescence extends over a dynamic range from 0. Second. Plant debris both scatters light and can be autofluorescent. it can therefore be tricky to establish appropriate conditions and standards. the Accuri C6 Flow Cytometer provides an excellent measurement platform. Caution should be observed.. but not overlapping. This is the first time this relationship has been demonstrated over such a large range. Extending polygonal gate P2 (Fig. and mithramycin/ chromomycin (Dolezel et al.32 to 80. Correlation between the fluorescence emissions within these two spectral bands produces an angled linear region within the biparametric contour plots containing discrete peaks of fluorescence. or about three decades of DNA content. 2D. six decade. This problem is solved by the Accuri C6 in two ways. In comparison to situations typical in flow analysis of animal cell suspensions. the objects of interest (the nuclei) are a very minor population. or investigations of other issues requiring C-value determinations. was consistently high across the entire range of measurements.3%. Zonneveld et al. ideally using a plant species having a C-value similar to that of the unknown. and mixing homogenates from different plant species (Figs. This avoids base-pair bias inherent to DAPI. This value would correspond to a nuclear 2C DNA content of 0. and without the requirement for even simple adjustments such as insertion of neutral density filters.. used for triggering flow cytometric measurements.) to 254. www. 2B) and roots (Figs. 1-3). the lowest representing the 2C nuclei within G0/G1 cells (Figs. Autofluorescence produced by the photosynthetic organelles of aerial tissues can also overlap the emission of DNA-specific fluorescent signals and.032 pg. Bennett and Leitch 2004). the C6 is equipped with lasers that optimally excite PI for use as a DNA fluorochrome. due to historical performance limitations of the latter. since chloroplasts greatly outnumber nuclei within the cells of green tissue. without appropriate thresholding. 2004.9 pg DNA (Figs. for analysis of C-values using plant homogenates. This essentially obviates the need for internal standardization of DNA content values. 2E) can be readily excluded for consideration. This conventionally has been an internal standard. and sample acquisition. Using the C6. Appropriate controls can readily handle these issues. This exceeds the largest record in the Kew C-value database by a factor of 1.Discussion and General Troubleshooting The nuclear DNA contents (C-values) of higher plants span a remarkable range of values (Bennett and Smith..5-fold. and remarkably. which provides an exceptional. The accuracy of measurement of the nuclear DNA contents. 1983). since a number of trivial issues can alter the fluorescence emission detected by the Accuri.. Gating around this region provides very clean uniparametric histograms.

AZ 85721-0240 USA Tel: Fax: Email: +1 (520) 621-9153 +1 (520) 626-4824 galbraith@arizona. call: 734. 212:87-106. Harkins KR. In the U. Cytometry 75A:692-698. Accuri Cytometers. Harkins KR. and Flow cytometry within reach are registered trademarks of Accuri Cytometers.8000 In Europe. MI 48103 USA CustomerService@AccuriCytometers. Suda J (2007). Inc. Tucson. Maddox . Ives Cambs PE27 3LF UK EuroTech@AccuriCytometers. Zhang CQ. Barthelson RA. go to: www. Knapp S (1991). A. Ann Botany 91:547-557. Plant Syst Evol 1998.. more than 300 www. call: +44 (0)1480 308380 For other countries.AccuriCytometers.994.AccuriCytometers. Greilhuber J. Helen St. Galbraith DW. with the permission of the author. University of Arizona 1657 E. Galbraith DW (2008). Sharma DP. Lambert GM. and (Alstroemeriaceae). (~100 Mb) and Drosophila cytometry show genome size in Arabidopsis to be ~157 Mb and thus MB. Ayres NM.S. Firoozabady Am J Bot ©2009 Accuri Cytometers. Galbraith DW. C-band polymorphism in Alstroemeria Accuri Cytometers (Europe) Ltd 56 Edison Road St. ligtu. Dolezel J. Martina Novotná tel.: +420 731 426 047 e-mail: novotna@biotech. nuclear DNA contents over the full range of described angiosperm 2C values. Systemic endopolyploidy in Arabidopsis thaliana.Notes This white paper is abstracted in part from Galbraith (2009). Galbraith DW (2009). Literature Cited Trans Roy Soc London B 274B:227-274. Plant Physiol 96:985-989. 173 Parkland Plaza Ann Arbor. Ann Botany 96:229-244. Accuri. C6 Flow Cytometer. Inc.