Protocols for preparations of suspensions of intact nuclei sutiable for flow cytometric analysis of nuclear DNA content are described here. These protocols are routinely used in this laboratory. The procedure with LB01 buffer (Dolezel et al. 1989) works with most plants species and tissues. LB01 buffer is stored frozen at -20°C in aliquots for convenient use. For a very few species, the rezolution of DNA content histograms is not satiafactory using LB01. In those cases, a modification of a procedure originally developed by Otto (1990) can provide improved resolution (Dolezel and Göhde 1995). If neither of these buffers work, we recomend use of Tris-MgCl2 buffer (Pfosser et al. 1995). This buffer gives very good resolution with Arabidopsis thaliana tissues.
Nuclei can be released into cell homogenates by chopping or by lysis of protoplasts. Intact plant tissues should be disease- and stress-free. Young, rapidly growing leaves usually give the best results. Leaves may be transported or sent by post wrappedin moistened paper tissue and enclosed in a plastic bag. High temperatures should be avoided during transportation.
Because the nuclear DNA content of G1 nucleus reflects the ploidy of a cell, estimation of DNA content is frequently used for ploidy determination.
Table 2. Relation between the ploidy and DNA content of G1 phase nuclei
| Ploidy | DNA Content (G1phase) |
| n | 1C |
| 2n | 2C |
| 4n | 4C |
Flow cytometric analysis involves the estimation of DNA content and not microscopic evaluation of chromosome number. Thus, the terms Ploidy and DNA ploidy should be used to distinguish between karyotype and DNA content analysis, respectively.
Main advantages of flow cytometric assay are:
DNA ploidy analysis using external standard
The instrument is calibrated using nuclei isolated from a plant with known ploidy, e.g. 2n (the position of the G1 peak is recorded). All other samples are characterized by the relative position of their G1 peaks. Units are thus "C-values".
Figure 12. Histograms of relative nuclear DNA content of nuclei isolated from young leaves of Cassava plants (untreated control and plants regenerated from in vitro culture after treatment with a polyploidizing agent)
DNA ploidy analysis using internal standard
The nuclei of the standard with known ploidy and the nuclei of the sample are isolated, stained and analysed simultaneously. The DNA ploidy of the sample is then estimated using the ratio of G1 peaks (units are "C-values").
Internal standardization eliminates the risk of error due to variations in sample preparation and instrument instability. It is recommended for precise DNA ploidy estimation (especially when aneuploidy is suspected).
Research and industrial applications of flow cytometric DNA ploidy analysis include:
Screening for novel ploidy levels
Figure 13. A complete system for production of tetraploids in Musa sp. Flow cytometry is employed to screen ploidy level of plants regenerated after a treatment with polyploidizing agent in vitro. Solid tetraploids are selected at early stage of growth.
Screening for interspecific hybrids
When parental species differ enough in their nuclear DNA content, flow cytometric analysis can detect interspecific hybrids according to their intermediate DNA values. This application of flow cytometry permits screening of large numbers of progenies at early stage.
Figure 14. Identification of hybrid plants obtained after crossing Lolium multiflorum with Festuca arundinacea. Histograms of relative DNA content were obtained after analysis of nuclei isolated from leaf tissues. Chicken red blood cell nuclei (CRBC) were used as an internal reference standard.
Detection of aneuploid plants
The probability of detecting an aneuploid plant depends critically on the precision of the measure (characterized by coefficient of variation of G1 peaks) and on the difference in DNA content between aneuploid and diploid plant. Basically, there are two approaches for detection of aneuploid plants using DNA flow cytometry. In both cases, internal standards should be used.
Figure 15. If nuclei of euploid (E) and aneuploid (A) plants are analysed simultaneously, only analysis resulting in coefficient of variation (CV) lower than half of the difference between their DNA contents will allow detection of aneuploid G1 nuclei. In wheat, an average chromosome represents only 2.4% of the total DNA content. Thus a high resolution analysis resulting in CVs lower than 1.2% is needed to detect aneuploids in wheat using euploid wheat as internal standard.
Figure 16. The use of a different species as an internal standard (this approach is less sensitive to the precision of the measure). Here, an example is given for detection of aneuploids in hexaploid wheat using hexaploid triticale (T) as an internal standard. After calculating G1 peak ratios (sample/standard), the relative difference in DNA content (D) between euploid (E) and aneuploid (A) plant can be calculated:
In some plant species (e.g. in most of angiosperms), cell differentiation may be accompanied by endopolyploidization either viaendomitosis or endoreduplication. Thus, in addition to diploid cells in G1-, S-, or G2- phase of the cell cycle, differentiated plant tissues may contain endopolyploid cells whose DNA content ranges from 4C to 128C or even more.
Figure 17. Schematic representation of the cell cycle with endomitosis (EM) and endoreduplication (ER) pathways.
Figure 18. Changes of nuclear DNA content during one endoreduplication cycle (shaded area)
Figure 19. Histogram of relative DNA content of nuclei isolated from a parenchymatic tissue of a cactus plant. Note the presence of peaks representing endopolyploid nuclei with DNA content up to 32C.
Since the nuclear DNA content reflects the position of a cell within a cell cycle, flow cytometric analysis of nuclear DNA content is suitable for cell cycle analysis. In order to determine the fraction of cell population in the G1, S and G2 phases, a DNA content distribution must be deconvolved (most conveniently using a dedicated computer software).
Figure 20. The distribution of nuclear DNA content of nuclei isolated from broad bean meristem root tip cells. A non-parametric curve-fitting method was used for histogram decomposition for cell cycle phases. G1 and G2 peaks are represented by a Gaussian function, and the S phase is represented by a second degree polynomial, broadened with Gaussian function.
In addition to monoparametric (DNA content) analysis, more sophisticated (biparametric) methods have been developed. These are mostly based on incorporation of 5-bromo-2-deoxyuridine into newly synthesized DNA and its detection using a monoclonal antibody or Hoechst fluorescence quenching.
Comparison of relative positions of G1 peaks corresponding to the sample nuclei and the nuclei isolated from a plant with known DNA content, respectively, permit accurate determination of the "unknown" DNA content:
Figure 21. Relative DNA content distribution of nuclei isolated from young leaves of Musa acuminataerrans and Glycine max cv "Polanka" (2C = 2.50 pg DNA). The ratio of G1 peak means (Glycine /Musa) = 1.984. Thus the 2C nuclear DNA content of M. acuminata errans is equal to 2.50 / 1.984 = 1.26 pg DNA.
Absolute values in pg DNA can be converted to the number of base pairs. The conversion factor is 1 pg = 965 million base-pairs (Mbp).
Table 3. Nuclear genome size of Musa genotypes estimated by flow cytometry*
| Genotype | Genome | 2C DNA Content (pg) | 1C DNA | |
| Mean | CV[%] | (Mbp) | ||
| M. balbisiana | BB | 1,14 | 2,57 | 552 |
| M. acuminata | AA | 1,23 | 1,48 | 593 |
| Pisang Mas (Austria) | AA | 1,25 | 1,62 | 605 |
| M. acuminata errans | AA | 1,26 | 1,72 | 606 |
| Pisang Mas (Malaysia) | AA | 1,26 | 1,14 | 607 |
* Glycine max cv. "Polanka" (2C DNA content = 2.50 pg) was used as an internal reference standard
In allopolyploid species whose basic genomes are known to be of different size, it is possible to use flow cytometric measurement of nuclear DNA content for estimation of their genome composition.
Figure 22. Flow cytometric estimation of nuclear genome composition in two triploid clones of Musa. In both cases, nuclei of Musa (M) and Lycopersicum esculentum (L, 2C = 1.96 pg DNA), which was used as an internal standard, were isolated, stained and analysed simultaneously.
Interpretation of histograms shown in Figure 22:
Table 4. Genome constitution of some Musa clones
| Genotype | Type | Ploidy | Constitution |
| Pisang Mas | Dessert Banana | 2n | AA |
| Gros Michel | -"- | 3n | AAA |
| Cavendish | -"- | 3n | AAA |
| Breed Clones | -"- | 4n | AAAA |
| Lady's Finger | -"- | 2n | AB |
| Mysore | -"- | 3n | AAB |
| "French" Plantain | Plantain | 3n | AAB |
| "Horn" Plantain | -"- | 3n | AAB |
| Bluggoe | Cooking Banana | 3n | ABB |
| Pisang Awak | -"- | 3n | ABB |
If sex chromosomes differ sufficiently in size (and hence in DNA content), or in DNA base composition, flow cytometric analysis of isolated nuclei may be used for early screening of sex in non-flowering individuals.
Figure 23. Histogram of relative nuclear DNA content obtained after simultaneous analysis of nuclei isolated from male (M, 2n = 24, XY) and female (F, 2n = 24, XX) plant of Melandrium album. Compared to the X chromosome, the Y chromosome has approximately two times higher DNA content. Thus, the G1 and the G2 peaks corresponding to both sexes are clearly separated
Base composition of nuclear DNA can be estimated by separate analyses using fluorochromes showing preference to AT- or GC- rich regions of DNA relative to a known standard.
Figure 24. Histograms of relative nuclear DNA content in maize (M) and human leukocytes (L) obtained after staining with fluorescent dyes differing in DNA base preference. Human leukocytes were used as an internal standard. (FR = ratio of G1 peak means of leukocyte and maize nuclei)
Table 5. Estimation of DNA base composition in six plant species using flow cytometry. Human leukocytes (AT = 59,5%) were used as a reference standard.
| Species | 2C (pg) | AT (pg) | AT (%) | GC (pg) | GC (%) |
| R. sativus | 1.11 | 0.78 | 46.99 | 0.88 | 53.01 |
| L. esculentum | 1.96 | 1.51 | 50.17 | 1.5 | 49.83 |
| Z. mays | 5.72 | 2.5 | 44.88 | 3.07 | 55.12 |
| H. sapiens | 7 | 4.17 | 59.5 | 2.83 | 40.5 |
| P. sativum | 9.07 | 5.97 | 61.42 | 3.76 | 38.58 |
| V. faba | 26.9 | 18.32 | 62.31 | 11.08 | 37.69 |
| A. cepa | 34.76 | 29.88 | 69.85 | 12.9 | 30.15 |
| 2C = 2C nuclear DNA content AT sample = AT reference ×fluorescence ratio GC sample = GC reference ×fluorescence ratio AT (%) = AT / (AT + GC) ×100 GC (%) = GC / (AT + GC) ×100 |
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Structural chromosome aberrations in dividing cells lead to unequal distribution of DNA into daughter cells. This is reflected by the increase in variation of nuclear DNA content (increase of coefficient of variation of G1 and G2 peaks). In addition, higher doses may result in changes of cell cycle kinetics.
Figure 25. Histograms of nuclear DNA content in a Chinese hamster cell line exposed to increasing doses of X-rays, with CVs of the G1 peaks shown. The analysis was performed 24 h after irradiation (Otto and Oldiges, 1980)
Chromatin structure (e.g. heterochromatin content) may be evaluated by flow cytometric analysis of nuclei stained by fluorochromes which bind to DNA by different modes (e.g. intercalation / binding to major or minor groove of DNA molecule).
Figure 26. Linear regression of Propidium Iodide (PI) / DAPI fluorescence ratio versus percentage heterochromatin in maize lines. (Rayburn et al., 1992)
1a. Chop a small amount of plant material (typically 20 mg) with a new razor blade or a sharp scalpel in 1ml of ice-cold LB01 in a petri dish. It is preferable to include a DNA fluorochrome (DAPI or propidium iodide) in the buffer prior to chopping. Alternatively, this compound may be added immediately after the filtration (step 2). The stains are used in the following concentrations: DAPI, 2 µg/ml; propidium iodide, 50 µg/ml + RNase, 50 µg/ml.
The actual quantity of plant material to be used for nuclei isolation depends both on the type of tissue and on the species, and must be determined experimentally (higher quantities are usually needed of callus or cultured cells).
1b. As an alternative, protoplasts can be prepared and resuspended in ice-cold LB01 to a concentration of 105 - 106/ml. The concentration of detergent (Triton X-100) in LB01 buffer should be increased to 0.5 % (v/v); this improves the release of the nuclei from the protoplasts.
Nuclei cannot be released from "collapsed" protoplasts, hence protoplast viability is an important consideration. Typically the protoplasts should be 90-100% viable as determined using FDA.
2. Filter the suspension through a 42 µm nylon mesh.
3. Store on ice prior to analysis (a few minutes to one hour).
4. Analyse relative DNA content of isolated nuclei.
1a. Chop a small amount of plant material (typically 20 mg) with a new razor blade or a sharp scalpel in 1ml of ice-cold Otto I buffer in a petri dish.
1b. As an alternative, protoplasts can be prepared, and resuspended in ice-cold Otto I buffer to a concentration of 105 - 106/ml.
2. Filter the suspension through a 42 µm nylon mesh.
3. Pellet the nuclei (150g/5 min).
4. Remove the supernatant, leaving about 100 µl of the liquid above the pellet.
5. Resuspend the nuclei by gentle shaking. Add 100 µl of fresh Otto I buffer.
6. Incubate for 10 - 60 min, depending on species, at room temperature, shaking occasionally. Select the incubation period that gives the lowest background and CV.
7. Add 1 ml of Otto II buffer. It is preferable to include DAPI (or propidium iodide + RNase) in the Otto II buffer. Alternatively, these compounds can be added to the sample after the addition of Otto II buffer. The stains are used at the following concentrations: DAPI, 4 µg/ml; propidium iodide, 50 µg/ml + RNase, 50 µg/ml.
8. Store at room temperature, analyzing within 5 - 15 min.
9. Analyse relative DNA content of isolated nuclei.
Large numbers of samples can be prepared and simultaneously centrifuged (step 3). If necessary, the samples can be kept at room temperature for prolonged periods of time after step 5 (the addition of fresh Otto I buffer) prior to the addition of Otto II buffer and analysis.
Browning due to phenolic compounds may be inhibited by adding 2 µl/ml ß-mercaptoethanol to Otto II buffer prior its use.
In some species, a simplified procedure may be used.
1. Chop a small amount of plant material (typically 20 mg) with a new razor blade or a sharp scalpel in 0.5 ml of ice-cold Otto I buffer in a petri dish.
2. Add 2 ml of Otto II buffer. It is preferable to include DAPI (or propidium iodide + RNase) in the Otto II buffer. Alternatively, these compounds can be added to the sample after the addition of Otto II buffer. The stains are used at the following concentrations: DAPI, 4 µg/ml; propidium iodide, 50 µg/ml + RNase, 50 µg/ml.
3. Mix well with a pipette.
4. Filter the suspension through a 42 µm nylon mesh.
5. Store at room temperature, analyzing within 5 - 15 min.
6. Analyse relative DNA content of isolated nuclei.
Browning due to phenolic compounds may be inhibited by adding 2µl/ml ß-mercaptoethanol to Otto II buffer prior its use.
This procedure gives good results only with some species. If the results are not satisfactory, it is recommended to test a standard two-step procedure.
1. Chop a small amount of plant material (typically 20 mg) with a new razor blade or a sharp scalpel in 0.5 ml of ice-cold Otto I buffer in a petri dish.
2. Add 0.5 ml of ice-cold Otto I buffer, mix well with a pipette.
3. Filter the suspension through a 42 µm nylon mesh.
4. Incubate for 1 - 5 min, shake occasionally.
5. Add 2 ml of Otto II buffer. It is preferable to include DAPI (or propidium iodide + RNase) in the Otto II buffer. Alternatively, these compounds can be added to the sample after the addition of Otto II buffer. The stains are used at the following concentrations: DAPI, 4 µg/ml; propidium iodide, 50 µg/ml + RNase, 50 µg/ml.
6. Store at room temperature, analyzing within 5 - 15 min.
7. Analyse relative DNA content of isolated nuclei.
If necessary, the samples can be kept at room temperature for prolonged periods of time after step 4 ( prior to the addition of Otto II buffer).
Browning due to phenolic compounds may be inhibited by adding 2µl/ml ß-mercaptoethanol to Otto II buffer prior its use.
This procedure gives good results only with some species. If the results are not satisfactory, it is recommended to test a standard two-step procedure.
1a. Chop a small amount of plant material (typically 20 mg) with a new razor blade or a sharp scalpel in 1ml of ice-cold Tris-MgCl2 buffer in a petri dish. It is preferable to include a DNA fluorochrome (DAPI or propidium iodid) in the buffer prior to chopping. Alternatively, this compound may be added immediately after the filtration (step 2). The stains are used in the following concentrations: DAPI, 4 µg/ml; propidium iodide, 50 µg/ml + RNase, 50 µg/ml.
The actual quantity of plant material to be used for nuclei isolation depends both on the type of tissue and on the species, and must be determined experimentally (higher quantities are usually needed of callus or cultured cells).
1b. As an alternative, protoplasts can be prepared and resuspended in ice-cold Tris buffer to a concentration of 105 - 106/ml.
Nuclei cannot be released from "collapsed" protoplasts, hence protoplast viability is an important consideration. Typically the protoplasts should be 90-100% viable as determined using FDA.
2. Filter the suspension through a 42 µm nylon mesh.
3. Store on ice prior to analysis (a few minutes to one hour).
4. Analyse relative DNA content of isolated nuclei.
In some cases, it is not possible to analyze the material immediately after collection. Then a procedure is necessary which permits storage of material for analysis at later date. This is especially critical in experiments involving the measurement of cell cycle kinetics, when samples have to be collected and analyzed at specific time intervals.
Due to changes in chromatin structure, nuclei isolated from fixed tissues are not recommended for determination of DNA content in absolute units (genome size).
1. Fix plant tissues (10 - 100 mg) by addition of 20 ml of formaldehyde fixative for 10 min at 5°C.
The optimal concentration of formaldehyde and the duration of fixation should be determined empirically for given material (to achieve the lowest backround and the highest possible resolution of peaks in the DNA content histograms).
2. Wash out formaldehyde fixative by three changes of Tris buffer, each for 10 min at 5°C.
3. The fixed tissues can be stored at 4°C for up to several days.
4. Homogenize the tissues by crushing with a glass rod in 1ml of ice-cold Tris buffer or LB01 in a petri dish.
Alternatively, nuclei can be isolated by chopping the tissues with a new razor blade and/or scalpel. It is also possible to release the nuclei using a motorized homogenizer (e.g. Polytron PT 1200). In this case, fixed tissues are transferred to a 12 x 75mm polystyrene tube containing ice cold LB01 buffer. This approach is especially convenient for isolation of nuclei from very small root tips and/or small amounts of cultured cells.
5. Filter the suspension through a 42 µm nylon mesh.
6. Store the nuclei at 4°C prior to analysis.
Fixed nuclei can be stored for more than a week. If prolonged storage is required, isolated nuclei should be employed, rather than fixed tissues.
7. Add DAPI to a final concentration of 2 µg/ml.
Binding of propidium iodide to DNA in formaldehyde-fixed chromatin is impaired and the use of DAPI for DNA staining is recommended. Alternatively, the negative effect of the fixation may be reversed by heating (Overton and McCoy 1994) or by acid hydrolysis (unpublished).
8. Analyze relative DNA content of isolated nuclei.
1.Mix 1 ml fresh chicken blood with 3 ml of CRBC buffer I. Centrifuge at 50g for 5 min.
2.Discard the supernatant, resuspend the pellet in 2 ml of CRBC buffer I and mix gently. Centrifuge at 50g for 5 min.
3.Discard the supernatant, resuspend the pellet in 2 ml of CRBC buffer II, and vortex briefly. Immediately add 2 ml of CRBC buffer III, and mix briefly.
4.Centrifuge at 250g for 5 min. (Alternatively, centrifuge at 200g for 5 min).
5.Discard the supernatant, add 2 ml of CRBC buffer III, and mix gently.
6.Centrifuge at 120g for 5 min. (Alternatively, centrifuge at 100g for 5 min).
7.Discard the supernatant, add 2 ml of CRBC buffer III and mix gently.
8.Filter the suspension through a 42 um nylon filter into clean tube, to remove large clumps. Centrifuge at 90g for 5 min. (Alternatively, centrifuge at 70g for 5 min).
9.Discard the supernatant, add 2 ml of CRBC buffer III, mix gently and centrifuge at 90g for 5 min. (Alternatively, centrifuge at 70g for 5 min).
10.Discard the supernatant, vortex the pellet gently, and add 2 ml of cold fresh fixative (ethanol:acetic acid, 3:1). Vortex briefly.
11.If the large clumps occurred in the suspension, filter it through a 42 um nylon filter into clean tube.
12.Leave overnight at 4°C, do not shake!
13.Gently remove the fixative, and vortex the pellet gently.
14.Add 6 ml of ice cold 70% ethanol (if the pellet is weak, add 3 ml of ice cold 70% ethanol), vortex briefly, and syringe through a 30G needle for three times.
15.Finally, filter the nuclear suspension through a 42 um nylon filter to remove large clumps.
16.Store at -20°C.
17.If the concentration of the nuclei is too high, dilute it using ice cold 70% ethanol.
use deionized or double-distilled water in all recipes
Tris-MgCl2 buffer (Pfosser et al. 1995)
0.2 M Tris
4.84 g
4 mM MgCl2 . 6H2O
162.64 mg
0.5% Triton X-100
1 ml
| 15 mM Tris | 363.4 mg |
| 2 mM Na2EDTA | 148.9 mg |
| 0.5 mM spermine tetrahydrochloride | 34.8 mg |
| 80 mM KCl | 1.193 g |
| 20 mM NaCl | 233.8 mg |
| 0.1% (v/v) Triton X-100 | 200 µl |
| 0.1M citric acid monohydrate | 4.2 g |
| 0.5% (v/v) Tween 20 | 1 ml |
| 0.4M Na2HPO4 .12H2O | 28.65 g |
| 100 mM NaCl | 1.461 g |
| 10 mM Na2EDTA | 930.6 mg |
| 0.1% (v/v) Triton X-100 | 250 µl |
| 10 mM Tris | 302.85 mg |
Dilute stock formaldehyde (MERCK cat. no. 1.04003.) in Tris buffer to a final concentration of 4% (v/v)
| 140 mM NaCl | 1.637 g |
| 10 mM sodium citrate | 588.2 mg |
| 1 mM Tris | 24.23 mg |
| 140 mM NaCl | 0.41 g |
| 5% (v/v) Triton X-100 | 2.5 ml |
| 320 mM sucrose | 54.77 g |
| 15 mM MgSO4 . 7 H2O | 1.85 g |
| 15 mM ß-mercaptoethanol | 530 µl |
| 1 mM Tris | 587.3 mg |
| 0.1 mg/ml DAPI | 5 mg |
| 1 mg/ml propidium iodide | 50 mg |
| 1 mg/ml RNase (IIA Sigma) | 25 mg |
Increased sensitivity may be achieved by using a high numerical aperture objective (e.g., Partec 40x1.25 quartz, glycerine). The use of a high numeric aperture objective (e.g., Partec 40x1.25 quartz, glycerine) is highly recommended. Analysis of DAPI Flurescence
Heat protection
KG1
Excitation filter
BG38 + UG1
Dichroic mirror
TK420
Barrier filter
GG435
If needed, excitation light intensity may be decreased by placing a neutral density filter (e.g., NG5) between the UG1 and TK420. This may be necessary when non-linearity of DNA content distributions is observed.Analysis of Propidium Iodide Flurescence
Heat protection
KG1
Excitation filter
BG38 + EM520
Dichroic mirror
TK560
Barrier filter
RG4590
If needed, excitation light intensity may be decreased by placing a neutral density filter (e.g., NG5) between the EM520 and TK560. This may be necessary when non-linearity of DNA content distributions is observed.
It is generally agreed that flow cytometric estimation of nuclear DNA amount in absolute units should be performed using internal standard (the nuclei of a standard are isolated, stained and analysed simultaneously with the nuclei of a sample). DNA content of the sample is then calculated:
C DNA Amount (pg) = sample G1 peak mean × standard 2C DNA amount (pg) standard G1 peak mean
To estimate nuclear DNA content in plants, most laboratories prefer to use plant DNA standards. When choosing a standard, it is advisable to select a taxon whose DNA amount is not very different from that of a sample (this will decrease a risk of errors due to nonlinearity of the instrument). Several species are thus needed to cover the large range of genome size known for plants. Unfortunately, there is no general agreement on DNA standards for plant flow cytometry. This laboratory has been using the following plant cultivars:List of DNA standards suitable for plant DNA flow cytometry
Species
Cultivar
2C DNA Content (pg)*
1C Genome Size (Mbp)**
Reference
Allium cepa
Alice
34.89
17 061
Dolezel et al. (1998)
Vicia faba ssp. faba var.equina
Inovec
26.90
13 154
Dolezel et al. (1992)
Secale cereale
Dankovske
16.19
7 917
Dolezel et al. (1998)
Pisum sativum
Ctirad
9.09
4 445
Dolezel et al. (1998)
Zea mays
CE-777
5.43
2 655
Lysak and Dolezel (1998)
Glycine max
Polanka
2.50
1 223
Dolezel et al. (1994)
Lycopersicon esculentum
Stupicke polni tyckove rane
1.96
958
Dolezel et al. (1992)
Raphanus sativus
Saxa
1.11
543
Dolezel et al. (1998)
*) Nuclear DNA content was established using human male leukocytes (2C = 7.0 pg DNA; Tiersch et al. 1989) as a primary reference standard.
**) 1 pg DNA = 978 Mbp (Dolezel et al. 2003)
All cultivars fulfil the criteria for plant DNA standards as they are: a) suitable for flow cytometric analysis of DNA content; b) seed propagated; c) available as elite lines from breeders. Upon request, this laboratory can provide reasonable amounts of seeds free of charge. Please, send your requests to: dolezel
ueb [dot] cas [dot] cz
