Analysis of Ploidy Level

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:

• Rapidity, precision and convenience (several hundred samples per working day)
• No need for piding cells
• Non-destructive (requires small amount of tissue)
• Analysis of large populations of cells (detection of subpopulations - mixoploidy)

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:

• Control of ploidy stability (e.g. micropropagation in vitro or conformity of seed lots)
• Screening for haploid plants (e.g. production of dihaploids in cereal breeding)
• Screening for diploid plants (e.g. selection of diploid plants after genetic transformation)
• Screening for triploid plants (e.g. hybrid seed production)
• Screening for novel ploidy levels (e.g. production of tetraploids)
• Screening for interspecific hybrids
• Detection of aneuploid plants

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.

• The use of euploid plant of the same species as an internal standard
• The use of a different species as an internal standard

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:

Analysis of Endopolyploidy

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)

• The presence of multiple ploidy levels complicates interpretation of DNA content histograms
• A caution should be taken when using nuclei isolated from differentiated plant tissues for estimation of DNA ploidy and/or detection of mixoploidy

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.

Cell Cycle Analysis

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.

Determination of Nuclear Genome Size

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

Estimation of Nuclear Genome Composition

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:

• Pelipita = A + 2×B = 0.62 pg + 2 ×0.57 pg = 1.76 pg
• Grand Nain = 3×A = 3 ×0.62 pg = 1.86 pg

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

Determination of Sex in Dioecious Plants

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

Estimation of Nuclear DNA Base Content

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

Analysis of Effects of Irradiation

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)

Analysis of Chromatin Structure

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)

Procedure Using LB01 Buffer

Procedure Using Lysis Buffer LB01 (Dolezel et al. 1989)

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.

References

• Dolezel J, Binarova P, Lucretti S. Analysis of nuclear DNA content in plant cells by flow cytometry. Biologia Plantarum 31: 113 - 120 (1989)

Two - Step Procedure

A Two-Step Procedure (Otto 1990, Dolezel and Godhe 1995)

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.

References

• Dolezel J, Gohde W. Sex determination in dioecious plants Melandrium album and M. rubrum using high-resolution flow cytometry. Cytometry 19: 103 - 106 (1995)
• Otto F. DAPI staining of fixed cells for high-resolution flow cytometry of nuclear DNA. In: Crissman HA, Darzynkiewicz Z (eds.). Methods in Cell Biology. Vol. 33., Pp. 105 - 110. Academic Press, New York, 1990.

A Simplified Two - Step Procedure

A Simplified Two-Step Procedure

VERSION A

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.

VERSION B

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.

Procedure Using a Tris-MgCl2 Buffer

Procedure Using Tris-MgCl₂ (Pfosser et al. 1995)

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.

References

• Pfosser A, Amon A, Lelley T, Heberle-Bors E. Evaluation of sensitivity of flow cytometry in detecting aneuploidy in wheat using disomic and ditelosomic wheat-rye addition lines. Cytometry 21: 387 - 393 (1995)

Procedure with Fixed Tissues

Procedure Using Formaldehyde-Fixed Tissues and Cells (Sgorbati et al. 1986)

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.

References

• Sgorbati S, Levi M, Sparvoli E, Trezzi F, Lucchini G. Cytometry and flow cytometry of 4',6-diamidino-2-phenylindole (DAPI)-stained suspensions of nuclei released from fresh and fixed tissues of plants. Physiologia Plantarum 68: 471 - 476 (1986)

Calibration Particles

Calibration Particles

 

Preparation of Fixed Chicken Red Blood Cell Nuclei for Instrument Alignment (Dolezel, unpublished)

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.

Reagents and Solutions

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

• adjust volume to 200 ml 
  
• adjust pH to 7.5 
  
• filter through a 0.22 µm filter; store at 4°C

Lysis buffer LB01 (Dolezel et al., 1989)

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

• adjust volume to 200 ml 
  
• adjust to pH 7.5 with 1N HCl 
  
• add 220 µl ß-mercaptoethanol (15 mM) 
  
• filter through a 0.22 µm filter 
  
• store at -20°C in 10 ml aliquots

Otto Buffer I

0.1M citric acid monohydrate	4.2 g
0.5% (v/v) Tween 20	1 ml

• adjust volume to 200 ml 
  
• filter through a 0.22 µm filter; store at 4°C

Otto Buffer II

0.4M Na2HPO4 .12H2O

28.65 g

• adjust volume to 200 ml 
  
• filter through a 0.22 µm filter 
  
• store at room temperature 
  
• Note: A DNA fluorochrome can be added prior to final volume adjustment. In that case, the solution should be stored in darkness.

Tris buffer

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

• adjust volume to 500 ml 
  
• adjust pH to 7.5 
  
• store at 4°C

Formaldehyde fixative (4% v/v)

Dilute stock formaldehyde (MERCK cat. no. 1.04003.) in Tris buffer to a final concentration of 4% (v/v)

• The formaldehyde fixative should be freshly prepared.

CRBC buffer I

140 mM NaCl	1.637 g
10 mM sodium citrate	588.2 mg
1 mM Tris	24.23 mg

• adjust volume to 200 ml 
  
• adjust pH to 7.1

CRBC buffer II

140 mM NaCl	0.41 g
5% (v/v) Triton X-100	2.5 ml

• adjust volume to 50 ml

CRBC buffer III

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

• adjust volume to 500 ml 
  
• adjust pH to 7.1

DAPI stock solution

0.1 mg/ml DAPI

5 mg

• disolve in 50 ml H₂O 
  
• filter through a 0.22 µm filter to remove small particles 
  
• store at -20°C in 1 ml aliquots 
  
• DAPI is available from Molecular Probes, Inc., (Eugene, Oregon).

Propidoum iodide stock solution

1 mg/ml propidium iodide

50 mg

• disolve in 50 ml H₂O 
  
• filter through a 0.22 µm filter to remove small particles 
  
• store at -20°C in 0.5 ml aliquots 
  
• PI is available from Molecular Probes, Inc., (Eugene, Oregon).

RNase stock solution

1 mg/ml RNase (IIA Sigma)

25 mg

• disolve in 25 ml H₂O 
  
• filter through a 0.22 µm filter to remove small particles 
  
• heat to 90°C for 15 min to inactivate DNases 
  
• store at -20°C in 0.5 ml aliquots

Optical Filter Sets for Arc-Lamp Flow Cytometers

Analysis of DAPI Flurescence

Heat protection	KG1
Excitation filter	BG38 + UG1
Dichroic mirror	TK420
Barrier filter	GG435

Increased sensitivity may be achieved by using a high numerical aperture objective (e.g., Partec 40x1.25 quartz, glycerine).
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

The use of a high numeric aperture objective (e.g., Partec 40x1.25 quartz, glycerine) is highly recommended.
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.

Plant DNA Standards

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: dolezelueb [dot] cas [dot] cz

References

• Dolezel J, Bartos J, Voglmayr H, Greilhuber J. Nuclear DNA content and genome size of trout and human. Cytometry 51: 127 - 128 (2003) Abstract PDF

• Dolezel J, Dolezelova M, Novak FJ. Flow cytometric estimation of nuclear DNA amount in diploid bananas (Musa acuminata and M. balbisiana). Biologia Plantarum 36: 351 - 357 (1994)
• Dolezel J, Greilhuber J, Lucretti S, Meister A, Lysak M A, Nardi L, Obermayer R. Plant genome size estimation by flow cytometry: Inter-laboratory comparison. Annals Botany 82 (Suppl. A): 17 - 26 (1998)
• Dolezel J, Sgorbati S, Lucretti S. Comparison of three DNA fluorochromes for flow cytometric estimation of nuclear DNA content in plants. Physiologia Plantarum 85: 625 - 631 (1992)
• Lysak MA, Dolezel J. Estimation of nuclear DNA content in Sesleria (Poaceae). Caryologia 52: 123 - 132 (1998)
• Tiersch TR, Chandler RW, Wachtel SSM, Ellias S. Reference standards for flow cytometry and application in comparative studies of nuclear DNA content. Cytometry 10: 706 - 710 (1989)

Cell Cycle Synchronisation

These protocols are used to induce high degree of metaphase synchrony in meristem root-tip cells. The procedures use a combination of hydroxyurea, a DNA synthesis inhibitor, and the anti-microtubular drug amiprophos-methyl or oryzalin. The original version of the procedure has been published by Dolezel et al. (1992).

Analysis of Cell Cycle Synchrony

Microscopic Analysis of Cell Cycle Synchrony

This protocol is used to estimate the extent of mitotic synchrony and the frequency of cells in metaphase in synchronized root tips.

Harvest the root tips (1 cm) in deionized H₂O.

1. Fix them in 3:1 fixative overnight at 4°C.
2. Remove fixative with several washes in 70% ethanol.
3. If needed, store the tips in 70% ethanol at 4°C.
4. Wash the tips in several changes of deionized H₂O.
5. Hydrolyse tips in 5N HCl at room temperature for 25 min.
6. Wash in deionized H₂O and incubate in Schiff's reagent for 1 hour.
7. Wash tips in deionized H₂O and macerate for about 1 min in 45% (v/v) acetic acid.
8. Cut off the darkly stained meristem tip and squash it in a drop of fructose syrup under an 18 x 18-mm coverslip.
9. Prepare five different slides. On each slide, analyse at least 1000 cells and determine the proportion of cells in metaphase.

Notes

Squash preparations prepared in fructose syrup can be maintained for few days in a refrigerator but they are not permanent. To make permanent slides, perform the following procedure:

1. Squash meristem tip in a drop of 45% (v/v) acetic acid.
2. Immediately after that, place the slide on a block of dry ice.
3. After freezing, peel off the coverslip.
4. Dehydrate the slide in two changes of 96% ethanol in Coplin jars.
5. Leave to air-dry overnight.
6. Dip the slide in xylene and mount it in a drop of DePeX (Serva).

Flow Cytometric Analysis of Cell Cycle Synchrony

This procedure is used to estimate of cell cycle synchrony induced by hydroxyurea and APM treatments. The protocol is based on the analysis of DNA content of isolated nuclei after staining with 4-,6-diamidino-2-phenylindole (DAPI). This dye is excited at UV and thus the flow cytometer must be equipped with a light source providing excitation in UV. We use BD FACSVantage equipped with a Coherent Innova 305 argon-ion laser.

1. Harvest the root tips (1 cm) in deionized H2O.
2. Immediately transfer the root tips into 25 ml of formaldehyde fixative and fix them at 5°C for 30 min (legumes) or 20 min (cereals).
3. Wash the roots in 25 ml of Tris buffer three times for 5 min at 5°C.
4. Excise root meristems and transfer them in 5-ml polystyrene tube containing 1 ml of LB01 lysis buffer.
5. Isolate nuclei by homogenising at 9500 rpm for 15 sec.
6. Stain the nuclei in suspension by adding DAPI stock solution to final concentration of 2 µg/ml.
7. Filter the suspension through a 50-µm nylon mesh into 5-ml polystyrene tube.
8. Make sure that the flow cytometer is aligned well for univariate analysis and that the band-pass filter 424/44 is placed in front of the fluorescence 1 (FL1) detector.
9. Introduce the sample; let it stabilise at appropriate flow rate (e.g., 200 particles/s).
10. Set a gating region on a dot plot of forward scatter height (FSC-H) and FL1 pulse height (FL1-H) to exclude small debris and large clumps.
11. Adjust photomultiplier voltage and amplification gains so that the peaks corresponding to G1 and G2 nuclei are seen on a histogram of FL1 pulse area (FL1-A).
12. Analyse 10 - 20 thousand nuclei and save the result on a disc.

Notes

Although the nuclei suspension can be stored overnight, it is recommended to perform the analysis on the same day of isolation.

Preparation of Chromosome Suspensions

In this procedure, chromosomes are released from synchronized root tips mechanically after a mild fixation with formaldehyde. Chromosomes are released into a polyamine lysis buffer (LB01) which stabilises their structure. The method has been originally developed for chromosome isolation in field bean (Dolezel et al. 1992). Chromosome suspensions prepared according to this procedure are suitable for flow cytometric analysis and sorting.

Flow Karyotyping and Chromosome Sorting

Univariate Flow Karyotyping and Chromosome Sorting

This protocol describes the analysis and sorting of plant chromosomes stained with 4-,6-diamidino-2-phenylindole (DAPI). This method was originally developed by Lucretti et al. (1993) for field bean and requires a flow cytometer equipped with a light source providing excitation in UV. We use BD FACSVantage equipped with a Coherent Innova 305 argon-ion laser.

Flow Karyotyping

1. Stain a chromosome suspension (approx. 1ml) by adding DAPI stock solution to final concentration of 2 µg/ml.
2. Filter the suspension through a 20-µm nylon mesh.
3. Make sure that the flow cytometer is properly aligned for univariate analysis. Make sure that the band-pass filter 424/44 is placed in front of the fluorescence 1 (FL1) detector.
4. Run a dummy sample (LB01 buffer containing 2 µg/ml DAPI) to equilibrate the sample line.
5. Introduce the sample; let it stabilise at appropriate flow rate (e.g., 200 particles/s). If possible, do not change the flow rate during the analysis!
6. Set a gating region on a dot plot of forward scatter height (FSC-H) and FL1 pulse height (FL1-H) to exclude debris, nuclei and large clumps.
7. Adjust photomultiplier voltage and amplification gains so that chromosome peaks are evenly distributed on a histogram of FL1 pulse area (FL1-A).
8. Analyse 20 - 50 thousand chromosomes and save the result on computer disc.

Chromosome Sorting

1. Make sure that the sorting device is properly adjusted.
2. Run the sample and display the signals on a dot plot of FL1 pulse width (FL1-W) versus FL1-A.
3. Adjust the FL1-W amplifier gain and width offset as needed to achieve optimal resolution of the width signal.
4. Check for stability of the break-off point and of the side streams.
5. Define sorting region for the largest chromosome on the FL1-W versus FL1-A dot-plot.
6. Select 1 drop sort envelope (number of deflected drops) and - counter - sort mode giving the highest purity and count precision.
7. Sort exact number of chromosomes (e.g., 50) on a microscope slide.
8. Check the number of chromosomes using a fluorescence microscope (do not cover the drop with a coverslip!).
9. If the number is not correct, repeat adjustment of the sorting device using fluorescent beads.
10. Define a sorting region for the chromosome to be sorted on the dot-plot of FL1-W versus FL1-A.
11. Select sort mode and sort envelope according to required purity, number of chromosomes to be sorted, and desired volume for the sorted fraction.
12. Sort the required number of chromosomes into a polystyrene tube containing the appropriate amount of the collection liquid. The amount and composition of the collection liquid depends on the number of sorted chromosomes and on their subsequent use. For PCR, use a small amount (20 - 60 µl) of sterile deionized H₂O in 0.5-ml PCR tube).
13. Briefly spin the tube at room temperature.
14. Sort chromosomes onto a microscope slide for estimation of purity using PRINS.

References

Lucretti S, Dolezel J, Schubert I, Fuchs J. Flow karyotyping and sorting of Vicia faba chromosomes. Theoretical and Applied Genetetics 85: 665 - 672 (1993)

Bivariate Flow Karyotyping and Chromosome Sorting

This protocol describes the analysis and sorting of plant chromosomes after dual staining with 4’,6-diamidino-2-phenylindole (DAPI) that binds preferentially to AT-rich regions of DNA, and with mithramycin that binds preferentially to GC-rich regions of DNA. This method was originally developed by Lucretti and Dolezel (1997) for broad bean and requires a flow cytometer equipped with two lasers (one of them UV). We use BD FACSVantage equipped with Coherent Innova 305 and Coherent Innova 70C argon-ion lasers.

Bivariate Flow Karyotyping

1. Add MgSO₄   stock solution to chromosome suspension (approx. 1ml) to a final concentration of 10 mM.
2. Stain the chromosomes in suspension by adding DAPI stock solution to a final concentration of 1.5 µg/ml and mithramycin stock solution to a final concentration of 20 µg/ml. Leave to equilibrate for 30 min on ice.
3. Make sure that the flow cytometer is properly aligned for bivariate analysis. Use a half mirror to split the fluorescence of DAPI to FL1 detector through the 424/44 band-pass filter and mithramycin fluorescence to fluorescence 4 (FL4) detector through a 575/26 band-pass filter.
4. Run a dummy sample (LB01 buffer containing 1.5 µg/ml DAPI and 20 µg/ml mithramycin) to equilibrate the sample line.
5. Filter the sample through a 20-µm nylon mesh.
6. Run the sample; let it stabilise at appropriate flow rate (e.g., 200 particles/s). If possible, do not change the flow rate during the analysis.
7. Set a gating region on a dot plot of FSC-H versus FL1-H.
8. Adjust photomultiplier voltages and amplification gains so that chromosome peaks are evenly distributed on histograms of FL1-A and FL4 pulse area (FL4-A).
9. Display the data on a dot-plot of FL1-A versus FL4-A.
10. Analyse 20 - 50 thousand chromosomes and save the result on a disc.

Chromosome Sorting

1. Make sure that the sorting device is properly adjusted.
2. Run the sample and display the signals on a dot plot of FL1-A versus FL4-A.
3. Check for stability of the break-off point and of the side streams.
4. Define sorting region for the largest chromosome on the FL1-A versus FL4-A dot-plot.
5. Select 1 drop sort envelope (number of deflected drops) and counter sort mode giving the highest purity and count precision.
6. Sort exact number of chromosomes (e.g., 50) on a microscope slide.
7. Check the number of chromosomes using a fluorescence microscope (do not cover the drop with a coverslip!).
8. If the number is not correct, repeat adjustment of the sorting device using fluorescent beads.
9. Define a sorting region for the chromosome to be sorted on a dot plot of FL1-A versus FL4-A.
10. Select sort mode and sort envelope according to required purity, number of chromosomes to be sorted and desired volume for the sorted fraction.
11. Sort the required number of chromosomes into a polystyrene tube containing the appropriate amount of the collection liquid. The amount and composition of the collection liquid depends on the number of sorted chromosomes and on their subsequent use. For PCR, use a small amount (20 - 60 µl) of sterile deionized H₂O in 0.5-ml PCR tube.).
12. Briefly spin the tube at room temperature.
13. Sort chromosomes onto a microscope slide for estimation of purity using PRINS.

References

Lucretti S, Dolezel J. Bivariate flow karyotyping in broad bean (Vicia faba). Cytometry 28: 236 - 242 (1997)

Two-Step Sorting

This protocol is used to sort chromosomes when their frequency in the original suspension is too low. This is frequently the case for large chromosomes, which break more easily than smaller chromosomes during chromosome isolation (Lucretti et al. 1993). During the first sort, the sample is enriched for the required chromosome. During the second sort, the chromosomes are sorted with a high purity. We use this protocol with BD FACSVantage dual laser flow cytometer and sorter.

First Sort

1. Make sure that the sorting device is properly adjusted.
2. Run the sample and display the signals on a suitable distribution.
3. Check for stability of the break-off point and of the side streams.
4. Define sorting region for the largest chromosome on a suitable distribution.
5. Select 1 drop sort envelope (number of deflected drops) and counter sort mode giving the highest purity and count precision.
6. Sort exact number of chromosomes (e.g., 50) on a microscope slide.
7. Check the number of chromosomes using a fluorescence microscope (do not cover with a coverslip!).
8. If the number is not correct, repeat adjustment of the sorting device using fluorescent beads.
9. Select the enrich mode and 3 deflected drops sort envelope that allows for the highest recovery.
10. On a suitable distribution, define a sorting region for the chromosome(s) to be sorted.
11. Sort approximately 105 chromosomes into 400 µl of LB01T into a 1.5 ml sample polystyrene cup (Deltalab).

Second Sort

1. Add fluorescent dye(s) to pre-sorted chromosome suspension to reach recommended final concentration(s).
2. Run the sample and define sorting region for the chromosome to be sorted.
3. Select sort mode and sort envelope. (Sort mode and sort envelope are selected according to the required purity, number of chromosomes to be sorted and desired volume for the sorted fraction.).
4. Sort the required number of chromosomes into a polystyrene tube containing corresponding amount of the collection liquid. (The amount and composition of the collection liquid depends on the number of sorted chromosomes and on their subsequent use). For PCR, use a small amount (20 - 60 µl) of sterile deionized H₂O in 0.5-ml PCR tube).
5. Briefly spin the tube at room temperature
6. Sort chromosomes onto a microscope slide for estimation of purity using PRINS.

Notes  
The actual number of chromosomes that should be sorted depends on the number of chromosomes that will be sorted during the second sort. It is recommended to sort at least five times more chromosomes than the final number required.  
In some cases, it may be practical to enrich the sample for more than one chromosome. Individual chromosomes are sorted during the second sort.

References

Lucretti S, Dolezel J, Schubert I, Fuchs J. Flow karyotyping and sorting of Vicia faba chromosomes. Theoretical and Applied Genetetics 85: 665 - 672 (1993)  

Estimation of Purity of Flow-Sorted Chromosomes

Estimation of Purity of Sorted Chromosomes Using FISH

This protocol describes a procedure that allows precise estimation of the purity of a sorted fraction (Kubalakova et al. 2000). The procedure is based on specific fluorescent labelling of repetitive DNA sequences that show characteristic pattern of distribution among the chromosomes. The sequences are labelled using hybridization of specific probes labelled with biotin or digoxigenin and detected with avidin, streptavidin or anti-digoxigenin antibody conjugated with fluorochromes. The chromosomes are evaluated using a fluorescence microscope.

I. Sort Chromosomes
1.Pipet 10 µl of P5 buffer containing 5% sucrose (w/v) onto a clean microscope slide.
2.Immediately sort 1000 chromosomes into the drop.
II. Perform FISH Reaction
1.Pipette 10µl P5 Buffer onto a clean microscopic slide and immediately sort 1000 chromosomes into the drop
2.Air-dry the slide and store at room temperature
3.Add 25µl Hybridization Mix to the area containing flow-sorted chromosomes, cover with a glass coverslip and seal up with rubber cement.
4.Denature for 45 sec at 80°C by placing the slide on a temperature controlled hot plate.
5.Transfer the slide to a wet chamber and incubate at 37°C overnight.
6.Remove carefully the coverslip and wash in 2 × SSC for 10 min at 42°C
7.Perform a stringent wash in 0.1 × SSC for 5 min at 42°C.
8.Wash again 10 min in 2 × SSC and next 10 min in 2 × SSC decrease gradually temperature to room temperature.
9.Wash in 4 × SSC for 10 min at room temperature.
10.Apply 60µl 1% Blocking Buffer onto the slide, cover with parafilm and incubate for 10 min in at room temperature. Repeat this step two times.
11.Apply fluorescently labeled antibody and/or fluorescently labeled avidin in 1% Blocking Buffer for 1 hour at 37°C
12.Wash the slide three times in 4 × SSC for 5 min at 40°C.
13.Mount the slide with Vectashield containing DAPI.
14.Use fluoresce microscope to identify sorted chromosomes and determine the presence of contaminating chromosomes by evaluating at least 100 chromosomes on three different slides.

References 

Kubaláková, M., Valárik, M., Bartoš, J., Vrána, J., Číhalíková, J., Molnár-Láng, M., Doležel, J.: Analysis and sorting of rye (Secale cereale L.) chromosomes using flow cytometry. - Genome 46: 893-905, 2003. 
Doležel, J., Kubaláková, M., Bartoš, J., Macas, J.: Flow cytogenetics and plant genome mapping. - Chrom. Res. 12: 77-91, 2004.
Šafář, J., Bartoš, J., Janda, J., Bellec, A., Kubaláková, M., Valárik, M., Pateyron, S., Weiserová, J., Tušková, R., Číhalíková, J., Vrána, J., Šimková, H., Faivre-Rampant, P., Sourdille, P., Caboche, M., Bernard, M., Doležel, J., Chalhoub, B.: Dissecting large and complex genomes: flow sorting and BAC cloning of individual chromosomes from bread wheat. – Plant J. 39: 960-968, 2004.
Kubaláková, M., Kovářová, P., Suchánková, P., Číhalíková, J., Bartoš, J., Lucretti, S., Watanabe, N., Kianian, S.F., Doležel, J.: Chromosome sorting in tetraploid wheat and its potential for genome analysis. – Genetics, 170: 823-829, 2005.

Estimation of Purity of Sorted Chrmosomes Using PRINS

This protocol describes a procedure that allows precise estimation of the purity of a sorted fraction (Kubalakova et al. 2000). The procedure is based on specific fluorescent labelling of repetitive DNA sequences that show characteristic pattern of distribution among the chromosomes. The sequences are labelled using a primed in situ labelling (PRINS) and the chromosomes are evaluated using a fluorescence microscope.

I. Sort Chromosomes

1. Pipet 15 µl of LB01 buffer (for barley) or PRINS buffer (for legumes and wheat) containing 5% sucrose (w/v) onto a clean microscope slide.
2. Immediately sort 1000 chromosomes into the drop.
3. Air-dry in an aseptic box for about 1h.

II. Perform PRINS Reaction

1. Stick 'Frame-Seal' incubation chamber to the slide over the specimen area.
2. Pipet 25-µl of PRINS reaction mix into the frame and place a polyester coverslip over the frame.
3. Run the PRINS reaction using the following PCR cycles:

   1 cycle:	5 min	94°C (denaturation)
   	5 min	55°C (annealing)
   	10 min	72°C (extension)
   8 cycles:	1 min	91°C (denaturation)
   	1 min	55°C (annealing)
   	3 min	72°C (extension)
   1 cycle:	1 min	91°C (denaturation)
   	5 min	55°C (annealing)
   	10 min	72°C (extension)

4. Remove the cover, add 100 µl of the stop buffer, and incubate for 2 min at 70°C.
5. Remove the stop buffer and transfer the slide to a 10-cm petri dish.
6. Add 70 µl wash buffer and incubate at room temperature for 5 min. Repeat the washing step twice.

III. Examine Slides

1. Add 70 µl of wash buffer containing DAPI (0.2 µg/ml) to counterstain the chromosomes.
2. Drain excess fluid, but not dry.
3. Add 8 µl of Vectashield antifade solution and cover with a glass coverslip.
4. Gently squeeze out excess solution and seal with rubber cement.
5. Examine the slide with fluorescence microscope.

Notes

The drops are dried within 1 h. However, the PRINS reaction should be performed next day after overnight incubation at room temperature. 
The actual conditions for the PRINS reaction (i.e., denaturation and annealing temperature) must be optimised by for given species and primer pair. 
During slide observation, use a DAPI filter first to localise sorted chromosomes. Avoid prolonged exposure to excitation light that fades rapidly both DAPI and fluorescein.

References 

Kubalakova M, Lysak MA, Vrana J, Simkova H, Cihalikova J, Dolezel J. Rapid identification and determination of purity of flow-sorted plant chromosomes using C-PRINS. Cytometry 41: 102 - 108 (2000) Abstract PDF

Estimation of Purity of Sorted Chrmosomes Using PCR with Chromosome-Specific Primers

I. Sort Chromosomes

1. Prepare PCR tubes containing 19 µl of sterile deionized H2O (final volume after sorting will be approximately 20 µl).
2. Sort 500 chromosomes into each tube.
3. Freeze the tube and store it at -20°C.

II. Perform PCR Reaction

1. Thaw a chromosome fraction.
2. Add 30 µl of PCR premix, vortex and spin briefly.
3. Perform PCR amplification using the following cycles:

   1 cycle:	5 min	denaturation at 94°C
   35-40 cycles:	30 sec	denaturation at 94°C
   	1 min	annealing at the optimised temperature
   	2-3 min	extension at 72°C
   1 cycle:	10 min	extension at 72°C

4. Hold the samples at 4°C.

III. Analyze PCR Products

1. Take equal amounts of PCR products (5 - 10 µl) from each tube and add 1 - 2 µl of loading buffer.
2. Load the samples onto the agarose gel bathed in TAE buffer.
3. Load DNA molecular weight markers.
4. Run electrophoresis; stop when bromophenol blue reaches a point 3 cm from the edge of the gel.
5. Stain the gel with ethidium bromide working solution (0.5 mg/ml).
6. Photograph the gel and analyse the presence of products in individual lanes.

Notes

This protocol was optimised for RFLP-derived markers in barley. For other types of markers and plant species, slight modification might be necessary. 
It is important to freeze the tubes even if the reaction is to be performed on the same day of sorting. 
Annealing temperature must be optimised by for given primer pair and the template. 
The electrophoresis should be run at constant voltage of 4 - 5 V/cm.

References

Lysak MA, Cihalikova J, Kubalakova M, Simkova H, Kunzel G, Dolezel J. Flow karyotyping and sorting of mitotic chromosomes of barley (Hordeum vulgare L.). Chromosome Research 7: 431 - 444 (1999)

Reagents and Solutions

use deionized or double-distilled water in all recipes

---

Hoaglands Nutrient Solution (Gamborg and Wetter 1975)

Solution A

H3BO3	280 mg
MnSO4 . H2O	340 mg
CuSO4 . 5H2O	10 mg
ZnSO4 . 7H2O	22 mg
(NH4)6Mo7O24 . 4H2O	10 mg

Adjust volume to 100 ml with deionized H₂O 
Store at 4°C

Solution B

H2SO4

0.5 ml

Adjust volume to 100 ml with deionized H₂O 
Store at 4°C

Solution C

Na2EDTA	3.36 g
FeSO4	2.79 g

Adjust volume to approximately 400 ml 
Heat the solution to 70°C while stirring until the colour turns yellow-brown 
Cool down, adjust the volume to 500 ml 
Store at 4°C

Hoaglands Stock Solution (10x)

Ca(NO3)2 . 4H2O	4.7 g
MgSO4 . 7H2O	2.6 g
KNO3	3.3 g
NH4H2PO4	0.6 g
solution A	5 ml
solution B	0.5 ml

Adjust volume to 500 ml with deionized H₂O 
Store at 4°C

Hoaglands Nutrient Solution (1x)

10x stock solution	100 ml
solution C	5 ml

Adjust volume to 1000 ml with deionized H₂O 
Prepare just before use

Hoaglands Nutrient Solution (0.1x)

10x stock solution	10 ml
solution C	0.5 ml

Adjust volume to 1000 ml with deionized H₂O 
Prepare just before use

Amiprophos-Methyl Solutions 
Stock Solutions (20 mM)

amiprophos-methyl

60.86 mg

Adjust volume to 10 ml with cold acetone 
Store at - 20°C in 1 ml aliquots 

Treatment Solution 
Prepare the treatment solution by adding specified volume of amiprophos-methyl stock solution to Hoaglands Nutrient Solution

B5 nutrient medium (Gamborg et al. 1968)

Solution A

H3BO3	300 mg
MnSO4 . H2O	758 mg
ZnSO4 . 7H2O	200 mg

Adjust volume to 1000 ml with deionized H₂O 
Store at 4°C

Solution B

Na2MoO4 . 2H2O	25 mg
CoCl2 . 6 H2O	2.5 mg
KI	75 mg
CuSO4 . 5 H2O	2.5 mg

Adjust volume to 100 ml with deionized H₂O 
Store at 4°C

Solution C

thiamine	100 mg
pyridoxine	10 mg
nicotinic acid	10 mg

Adjust volume to 100 ml with deionized H₂O 
Store at 4°C

Solution D

m-inositol

100 mg

Adjust volume to 100 ml with deionized H₂O 
Store at 4°C

Solution E

Na2EDTA	3.36 g
FeSO4	2.79 g

Adjust volume to approximately 400 ml 
Heat the solution to 70°C while stirring until the color turns yellow-brown 
Cool down and adjust the volume to 500 ml 
Store at 4°C

B5 Stock Solution (10x)

KNO3	30 g
MgSO4 . 7H2O	5 g
(NH4)SO4	1.34 g
CaCl2 . 2H2O	1.5 g
NaH2PO4 . H2O	1.5 g
solution A	100 ml
solution B	10 ml

Adjust volume to 500 ml with deionized H₂O 
Store at 4°C

B5 Nutrient Solution (1x)

10x stock solution	100 ml
solution C	10 ml
solution D	10 ml
solution E	5 ml

Adjust volume to 1000 ml with deionized H₂O 
Adjust the final pH to 5.5 
Sterilise by autoclaving

Tris Buffer

10 mM Tris	0.606 g
10 mM Na2EDTA	1.861 g
100 mM NaCl	2.922 g

Adjust volume to 500 ml with deionized H₂O 
Adjust the final pH to 7.5 using 1N NaOH 
Prepare just before use

Formaldehyde Fixative

10 mM Tris	0.303 g
10 mM Na2EDTA	0.931 g
100 mM NaCl	1.461 g
0.1% v/v Triton X-100	250 µl

Adjust volume to 200 ml with deionized H₂O 
Adjust the final pH to 7.5 using 1N NaOH 
Add formaldehyde solution (37%, Catalog No. 1.04003, Merck, Darmstadt, Germany):

Species	Volume	Conc.
Field Bean	27 ml	4%
Pea	20 ml	3%
Chickpea	13.5 ml	2%
Vicia sativa	13.5 ml	2%
Barley	13.5 ml	2%
Oat	13.5 ml	2%
Rye	13.5 ml	2%
Wheat	13.5 ml	2%
Durum Wheat	13.5 ml	2%

Finally, adjust volume to 250 ml with deionized H₂O 
Prepare the fixative just before use

LB01 Lysis Buffer (Dolezel et al. 1992)

15 mM Tris	0.363 g
2 mM Na2EDTA	0.149 g
0.5 mM spermine . 4HCl	0.035 g
80 mM KCl	1.193 g
20 mM NaCl	0.234 g
0.1 % v/v Triton X-100	200 µl

Adjust volume to 200 ml with deionized H₂O 
Adjust the final pH*) 
Filter through a 0.22 ľm filter to remove small particles 
Add 220 ľl ß-mercaptoethanol and mix well 
Store at - 20°C in 10 ml aliquots 
*) Please note that the original LB01 buffer was adjusted to pH 7.5. However, we have found later that the optimal pH differs for different applications. To distinguish the variants, we use the following abbreviations: 
LB01/7.5  pH adjusted to 7.5 using 1N HCl 
LB01/9  pH adjusted to 9 using 1M NaOH

LB01T Lysis Buffer (for sorting)

22.5 mM Tris	0.545 g
3 mM Na2EDTA	0.223 g
0.75 mM spermine . 4HCl	0.052 g
120 mM KCl	1.790 g
30 mM NaCl	0.351 g
0.15% v/v Triton X-100	300 µl

Adjust volume to 200 ml with deionized H₂O 
Adjust the final pH to 7.5 using 1N HCl 
Filter through a 0.22 ľm filter to remove small particles 
Add 330 ľl ß-mercaptoethanol and mix well 
Store at - 20°C in 5 ml aliquots

3:1 Fixative 
Mix 3 volumes of 96% ethanol with 1 volume of glacial acetic acid 
Prepare the fixative just before use

Schiff's reagent

1N HCl	30 ml
Parafuchsin (Serva, C.I.42500)	2 g
K2S2O5	3.8 g
deionized H2O	170 ml

Stir for 2 hours in a tightly closed bottle and leave to stay overnight 
Add 2 g of active charcoal 
mix 1 min and filter through a paper filter moistened with 1N HCl 
Repeat the filtration if the solution is not colourless 
Store in a tightly closed bottle at 4°C

Fructose syrup

fructose	30 g
deionized H2O	20 ml

Incubate the mixture at 37°C overnight 
Add a crystal of thymol 
store at 4°C

5N HCl

HCl - 35% (v/v)

419.8 ml

Adjust volume to 1000 ml with deionized H₂O

45% (v/v) acetic acid

acetic acid - 99% (v/v)

227 ml

Adjust volume to 500 ml with deionized H₂O

DAPI Stock Solution (0.1 mg/ml)

Dissolve 5 mg DAPI in 50 ml deionized H₂O by stirring for 60 min. 
Filter through a 0.22 ľm filter to remove small particles 
Store at - 20°C in 0.5 ml aliquots

Mithramycin Stock Solution (1 mg/ml) 

Dissolve 50 mg mithramycin A in 50 ml deionized H₂O by stirring for 60 min. 
Filter through a 0.22 ľm filter to remove small particles 
Store at - 20°C in 0.5 ml aliquots

Sheath Fluid SF50

50 mM NaCl

7.31 g

Adjust volume to 2500 ml with deionized H₂O 
Sterilise by autoclaving

Magnesium Sulfate Stock Solution (100 mM)

Dissolve 1.23 g of MgSO₄ . 7H₂O in 50 ml of deionized H₂O. 
Filter through a 0.22 ľm filter to remove small particles 
Store at 4°C

PRINS Buffer

10 mM Tris base	0.605 g
50 mM KCl	1.864 g
2mM MgCl2.6H2O	0.203 g

Adjust volume to 500 ml with deionized H₂O 
Adjust the final pH to 8.0 using 1N HCl 
Sterilise by autoclaving 
Store at 4°C

PRINS Reaction Mix

10x DNA polymerase buffer*	5 µl
25 mM MgCl2 (4 mM final)	5 µl
2 mM dCTP, dGTP (0.1 mM each final)	2.5 µl
0.2 mM fluorescein-12-dUTP (2 ľM final)	2 µl
0.2 mM fluorescein-15-dATP (2 ľM final)	2 µl
0.2 mM dTTP (34 ľM final)	4.25 µl
0.2 mM dATP (34 ľM final)	4.25 µl
20 ľM forward primer (2 ľM final)	5 µl
20 ľM reverse primer (2 ľM final)	5 µl
5U/ľl Taq DNA polymerase (3 U / 50 ľl final)	1.5 µl

*) The buffer contains 15 mM MgCl₂ 
Add sterile deionized H₂O to 55 ľl (includes 5 ľl for evaporation) 
Actual composition of the mix (e.g., MgCl₂, concentration and ratio of labelled and unlabeled nucleotides, primer concentration) should be optimised for given primer pair and species

Stop Buffer for PRINS

0.5 M NaCl	2.923 g
50 mM Na2EDTA	1.861 g

Adjust volume to 100 ml with deionized H₂O 
Adjust the final pH to 8.0 using 1N NaOH 
Sterilise by autoclaving 
Store at 4°C

Wash Buffer for PRINS

100 mM maleic acid	1.161 g
150 mM NaCl	0.876 g
0.05% v/v Tween-20	0.5 ml

Adjust volume to 100 ml with deionized H₂O 
Adjust the final pH to 7.5 using 1N NaOH 
Sterilise by autoclaving 
Store at 4°C

PCR Premix

(for a 50 ľl reaction mixture)

10x Taq DNA polymerase buffer*	5 µl
25 mM MgCl2 (1.5 mM final)	3 µl
10 mM dNTPs (0.2 mM each final)	1 µl
50 ľM forward primer (1 ľM final)	1 µl
50 ľM reverse primer (1 ľM final)	1 µl
5U/ľl Taq DNA polymerase (2.5 U/50 ľl final)	0.5 µl
sterile deionized H2O	18.5 µl

*) The buffer does not contain MgCl₂ 
Mix well and centrifuge briefly 
Prepare shortly before the use

TAE buffer 

Stock Solution (50 x)

Tris (2M final)	242 g
glacial acetic acid (1 M final)	57.1 ml
0.5 M EDTA (pH 8.0), (100 mM final)	200 ml

Adjust volume to 1000 ml with deionized H₂O 
Store at room temperature 

Working Solution (1x) 
Dilute TAE stock solution 1:50 in deionized H₂O (final concentrations: 40 mM Tris, 20 mM acetic acid, 2 mM EDTA)

Loading Buffer

0.5M EDTA (pH 8.0) (100 mM final)	2 ml
SDS (1% w/v final)	100 mg
bromophenol blue (0.05% w/v final)	5 mg
xylenecyanol (0.05% w/v final)	5 mg
gycerol (50% v/v final)	5 ml

Adjust volume to 10 ml with deionized H₂O 
Store at room temperature

Ethidium Bromide Solution

Stock Solution (0.5 mg/ml)

Dissolve 5 mg ethidium bromide in 10 ml deionized H₂O by stirring for 60 min

Ethidium Bromide Working Solution (0.5 µg/ml)

Dilute stock solution 1:1000 in deionized H₂O 
The solution may be used several times 
Store at room temperature

References 

• Dolezel J, Cihalikova J, Lucretti S. A high-yield procedure for isolation of metaphase chromosomes from root tips of Vicia faba. L. Planta 188: 93 - 98 (1992) 
• Gamborg OL, Wetter LR. Plant Tissue Culture Methods. Saskatoon: National Research Council of Canada (1975) 
• Gamborg OL, Miller RA, Ojima K. Nutrient requirement of suspension cultures of soybean root cells. Experimental Cell Research 50: 151 - 158 (1968)
  

Suppliers of Reagents, Consumables and Equipment

Plant DNA Standards

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).

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: dolezelueb [dot] cas [dot] cz

References

• Dolezel J, Bartos J, Voglmayr H, Greilhuber J. Nuclear DNA content and genome size of trout and human. Cytometry 51: 127, 128 (2003)  Abstract PDF
• Dolezel J, Dolezelova M, Novak FJ. Flow cytometric estimation of nuclear DNA amount in diploid bananas (Musa acuminata and M. balbisiana). Biologia Plantarum 36: 351, 357 (1994)
• Dolezel J, Greilhuber J, Lucretti S, Meister A, Lysak M A, Nardi L, Obermayer R. Plant genome size estimation by flow cytometry: Inter-laboratory comparison. Annals Botany 82 (Suppl. A): 17 - 26 (1998)
• Dolezel J, Sgorbati S, Lucretti S. Comparison of three DNA fluorochromes for flow cytometric estimation of nuclear DNA content in plants. Physiologia Plantarum 85: 625, 631 (1992)
• Lysak MA, Dolezel J. Estimation of nuclear DNA content in Sesleria (Poaceae). Caryologia 52: 123 - 132 (1998)
• Tiersch TR, Chandler RW, Wachtel SSM, Ellias S. Reference standards for flow cytometry and application in comparative studies of nuclear DNA content. Cytometry 10: 706 - 710 (1989)