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Researchers involved in the early work with plant FCM isolated protoplasts with hydrolytic enzyme mixtures, lysed the protoplasts and stained them with a fluoro-chrome, mainly DAPI. Dolezel et al. (1989) give examples of Zea mays (Poaceae) and Medicago sativa (Fabaceae) callus and leaf material.

Today, the method of preparing a suspension of nuclei for measurement follows the ingeniously simple procedure of Galbraith et al. (1983). It consists basically of (i) chopping up the plant material with a sharp razor blade to release nuclei into isolation buffer or buffer component, (ii) sieving the homogenate to remove large particles, and (iii) staining the nuclei in (buffered) suspension with the fluorochrome of choice. RNase should be added, if intercalating dyes such as ethidium bromide (EB) or propidium iodide (PI) rather than the base-specific minor grove-binding Hoechst dyes and DAPI (AT specific), or mithramycin, olivo-mycin and chromomycin (GC specific) are used. It is important to use PI or EB to quantify the DNA content without biasing the results with the base content (Do-lezel et al. 1992). A saturation curve of PI is shown in Fig. 4.2, indicating that PI concentrations between 50 and 150 mg l_1 are appropriate. A similar result was obtained by Loureiro et al. (2006a) for Pisum sativum isolated with four different buffers. The steps can be carried out in sequence or can be combined so that chopping, staining and RNase digestion are completed in one or two steps (i.e. the chopping buffer also contains the RNase, or in addition the dye). RNase addition may often show no effect due to the low RNA content, in leaves for instance, and thus may seem dispensable, but is essential with tissues rich in RNA such as meristems and seeds, and is also for principal reasons an established step in the procedure. It should be noted that chopping up the tissue in the stain solution, as

Fig. 4.2 Propidium iodide saturation curve. Nuclei were isolated from co-chopped leaves of Pisum sativum ''Kleine Rheinianderin'' and Secale cereale ''Elect'' in Otto buffer component I. The isolate was divided into 0.4-ml aliquots, which were treated with RNase at 37 0C for 30 min and immediately stored in the refrigerator. The aliquots were then stained with Otto buffer component II supplemented with 0.5, 5, 25, 50, 250 and 500 mg l_1 propidium iodide and measured with a flow cytometer (Partec PA II) after a 1-h incubation at 7 0C. (Original by E. M. Temsch).

Fig. 4.2 Propidium iodide saturation curve. Nuclei were isolated from co-chopped leaves of Pisum sativum ''Kleine Rheinianderin'' and Secale cereale ''Elect'' in Otto buffer component I. The isolate was divided into 0.4-ml aliquots, which were treated with RNase at 37 0C for 30 min and immediately stored in the refrigerator. The aliquots were then stained with Otto buffer component II supplemented with 0.5, 5, 25, 50, 250 and 500 mg l_1 propidium iodide and measured with a flow cytometer (Partec PA II) after a 1-h incubation at 7 0C. (Original by E. M. Temsch).

is sometimes practised, increases the likelihood of skin and laboratory contamination of the sample and also increases the number of disposables that would need to be treated as toxic waste. Also RNase spills can be problematic in some laboratories. It should therefore be carefully considered whether a small gain in time outweighs laboratory safety (but note the recommendations on work with dry material, see above).

4.4.2.1 Isolation Buffers and DNA Staining

Various isolation buffers are used in plant FCM (Table 4.3). Staining is carried out at neutral or slightly basic pH and there is some detailed information available on the effect of pH on DNA specificity for the stain Hoechst 33258. Hilwig and Gropp (1975) showed that in cytological preparations at pH 2, nucleoli and cytoplasm, probably the RNA, are stained as well as chromatin DNA, while at pH 7 only chromatin is stained. Slides stained at pH 2 lost the non-specific DNA staining if mounted with pH 7 buffer, and did not regain it at pH 2 unless re-stained. Other proton concentrations were not tested. For DAPI even less information is available, despite its wide use in cytogenetics and its high level of biochemical evaluation (Kapuscinski 1995). In chromosome cytology, DAPI staining of DNA is generally carried out at pH 7, and this is also the case in plant FCM. However, Wen et al. (2001) in a study on dye concentration and pH in biomedical DNA measurements, found in tumor and mouse cell lines the best CVs (coeffi-

Table 4.3 Ten most popular non-commercial nuclear isolation buffers in plant DNA flow cytometry. Buffers are arranged in decreasing order of preference according to the FLOWer database (see Chapter 18).

Buffer

Composition^

References

Galbraith's 45 mM MgCl2; 30 mM sodium citrate; 20 mM MOPS; 0.1% (v/v) Triton X-100; pH 7.0

MgSO4 9.53 mM MgSO4.7H2O; 47.67 mM KCl; 4.77 mM

HEPES; 6.48 mM DTT; 0.25% (v/v) Triton X-100; pH 8.0

LB01 15 mM Tris; 2 mM Na2EDTA; 0.5 mM spermine.4HCl;

80 mM KCl; 20 mM NaCl; 15 mM b-mercaptoethanol; 0.1% (v/v) Triton X-100; pH 7.5

Otto'sM Otto I: 100 mM citric acid monohydrate; 0.5% (v/v)

Otto II: 400 mM Na2PO4.12H2O (pH approx. 8-9)

Tris.MgCl2Icl 200 mM Tris; 4 mM MgCl2.6H2O; 0.5% (v/v) Triton X-100; pH 7.5

Baranyi'slbl Baranyi solution I: 100 mM citric acid monohydrate; 0.5% (v/v) Triton X-100

Baranyi solution II: 400 mM Na2PO4.12H2O; 10 mM sodium citrate; 25 mM sodium sulfate

Bergounioux's ''Tissue culture salts'' supplemented with 700 mM sorbitol; 1.0% (v/v) Triton X-100; pH 6.6

Rayburn's 1 mM hexylene glycol; 10 mM Tris; 10 mM MgCl2;

Bino's 200 mM mannitol; 10 mM MOPS; 0.05% (v/v) Triton

X-100; 10 mM KCl; 10 mM NaCl; 2.5 mM DTT; 10 mM spermine.4HCl; 2.5 mM Na2EDTA.2H2O; 0.05% (w/v) sodium azide; pH 5.8

De Laat's 15 mM HEPES; 1 mM EDTA Na2.2H2O; 0.2% (v/v)

Triton X-100; 80 mM KCl; 20 mM NaCl; 15 mM DTT; 0.5 mM spermine.4HCl; 300 mM sucrose; pH 7.0

Galbraith et al. (1983)

Arumuganathan and Earle (1991)

Dolezel et al. (1989)

Otto (1990), DoleZel and Gohde (1995)

Pfosser et al. (1995)

Baranyi and Greihuber (1995)

Bergounioux et al. (1986)

Rayburn et al. (1989)

de Laat and Blaas (1984)

a Final concentrations are given. MOPS, 4-morpholinepropane sulfonate; DTT, dithiothreitol; Tris, tris-(hydroxymethyl)-amino-methane; EDTA, ethylenediaminetetraacetic acid; HEPES, 4-(hydroxymethyl)piperazine-l-ethanesulfonic acid. For details on the buffer preparation see the original reference(s). b pH of the buffers is not adjusted.

c The original recipe and reference for Tris.MgCl2 is presented. Several minor modifications have been made so far, nonetheless, the basic composition remains stable.

cients of variation) and least debris at pH 6, while at pH 8 the histograms had already collapsed. At pH 7, in the mouse cell line MAT-B1 the histogram was still highly resolved, while in the line P388/R84 a significant decay in quality was observed. This is difficult to explain and stands in contradiction to the results of Otto et al. (1981). Studies on the effects of pH on staining intensity, histogram quality and DNA specificity in plant FCM are thus urgently required.

PI and EB stain DNA above pH 4, with some increase at higher pH as shown for EB by Le Pecq and Paoletti (1967). The buffer should also provide ionic strength for PI and EB to stain the nucleic acid quantitatively (Le Pecq and Paoletti 1967). If nuclei are isolated at acidic pH in citric acid plus detergent (Otto procedure; Otto et al. 1981), the dye must be added in basic solution (Na2HPO4) so that a final neutral pH is achieved (first used with unfixed plant nuclei by Dolezel and Gohde (1995), then slightly modified by Baranyi and Greilhuber (1995), and later called the ''two-step procedure'' by Dolezel et al. (1998)).

Isolation buffers, in addition to releasing nuclei from the cytoplasm in sufficient quantities, must also maintain nuclear integrity throughout the experiment, protect DNA from degradation by endonucleases and permit stoichiometric DNA staining. From about 26 different isolation formulas described, six are commonly used in plant DNA flow cytometry (Loureiro et al. 2006a; Table 4.3). Their usual components include: (i) organic buffer substances (e.g. Tris, MOPS and HEPES) to stabilize the pH of the solution (usually set between 7.0 and 8.0, which is compatible with common DNA fluorochromes); (ii) non-ionic detergents (e.g. Triton X-100, Tween 20) to release and clean nuclei, and decrease the aggregation affinity of nuclei and debris (note that ionic detergents such as sodium dodecyl sulfate would change the fluorescence properties of the dye molecule; Kapuscinski 1995); (iii) chromatin stabilizers (e.g. MgCl2, MgSO4, spermine); (iv) chelating agents (e.g. EDTA, sodium citrate) to bind divalent cations, which serve as nuclease cofactors; and (v) inorganic salts (e.g. KCl, NaCl) to achieve proper ionic strength (Dolezel and Bartos 2005).

''Otto's buffer'', which is in fact the well-known Mcllvaine's buffer system (e.g. Rauen 1964, pp. 92, 95) plus detergent, was first introduced to FCM in combination with DAPI by Otto et al. (1981) for ethanol-fixed mouse cells, which were resuspended in 0.2 M citric acid plus 0.5% Tween 20, adjusted to pH 7.4 and stained. With regard to this technique Otto et al. (1981) refer to Pinaev et al. (1979), who isolated non-fixed HeLa chromosomes in 0.1 M citric acid plus 0.1 M sucrose plus 0.5% Tween 20. Ulrich and Ulrich (1991) used Otto's buffer for nuclei isolation from living plant tissue, but fixed the nuclei in acetic ethanol; staining and analysis was again carried out in Otto's buffer with very narrow CVs obtained. Otto's buffer system plus DAPI was first used for unfixed plant nuclei by Dolezel and Gohde (1995) for sex identification in Melandrium (Caryophyl-laceae) and basically (with minor modification) also by Baranyi and Greilhuber (1995) to demonstrate the lack of variance of genome size in Pisum sativum (Faba-ceae). This buffer system was obviously the essence of a commercial Partec buffer (solutions A and B) with proprietary composition in the early 1990s. It consists of two components, citric acid plus detergent (''Otto I'') for nuclei isolation, and the basic Na2HPO4 plus fluorochrome (''Otto II''), which is added to the isolate for staining at neutral pH. Baranyi and Greilhuber (1996) first modified and applied this system for EB and PI staining (with some non-essential additions; J. Greilhuber and E. M. Temsch, unpublished data). Otto's buffer differs essentially from other buffers, because the first step combines isolation of nuclei with mild fixation and possibly some histone removal.

The other buffers (Table 4.3) work a priori at near-neutral pH and are based on popular organic buffer substances such as MOPS (Galbraith et al. 1983), Tris (Dolezel et al. 1989; Pfosser et al. 1995) and HEPES (Arumuganathan and Earle 1991). With these buffers it is intended to keep the nuclei in an intact or even sub-vital state. Chromatin stabilizers such as Mg2+ (Galbraith et al. 1983) or spermine (Bino's buffer, Dolezel's LB01 buffer) are added. Mannitol and sucrose are used to provide isotony. Chelators such as EDTA bind metal ions and thus block DNase activity (DNases need Mg2+ and Mn2+). Citrate acts as a chelator as well. Thus, Mg salts as stabilizers combined with chelators as DNase inhibitors seems to make little sense. Some buffers contain mercaptoethanol, sulphite, ascorbic acid and dithiothreitol as reductants, and PVP to bind tannins (see below).

The different buffer characteristics and the cytosolic compounds released upon chopping up the tissue can affect sample and measurement quality. Comparative analyses of buffers are therefore required, but such studies have seldom been undertaken.

Recently, Loureiro et al. (2006a) compared four common and chemically different lysis buffers, namely Galbraith's buffer (Galbraith et al. 1983), LB01 (Dolezel et al. 1989), Otto's buffer (Dolezel and Gohde 1995) and Tris.MgCl2 (Pfosser et al. 1995), taking into consideration the following parameters: fluorescence yield of nuclei in suspension, CVs of G1 peaks, forward and side scatter, amount of debris, and the number of particles released from the sample tissue. Samples were prepared from fresh leaf tissue of seven plant species covering a wide range of genome sizes (1.30-26.90 pg/2C), differing in tissue structure and being either easy to prepare (Pisum sativum, Vicia faba and Lycopersicon esculentum) or more challenging (Oxalis pes-caprae, Oxalidaceae, complicated by acidic cell sap; Celtis australis, Ulmaceae, complicated by mucilage, Festuca rothmaleri, Poaceae, complicated by xeromorphic, and Sedum burrito, Crassulaceae, complicated by succulent leaves).

The buffers performed differently, although with acceptable results in most cases. Excellent results (high fluorescence yield, high nuclei yield, low CV, little debris) were obtained only with some buffers for some species. Oxalis pes-caprae with very acidic cell sap worked only with Otto's and Galbraith's buffer. Spermine (in LB01) seems to be a better chromatin stabilizer than MgSO4, and MOPS (in Galbraith's buffer) seems to be a better buffer substance than Tris (evident in the acidic O. pes-caprae). A higher concentration of detergent (0.5% Triton X-100) was essential for the improved performance of Tris.MgCl2 buffer in Celtis australis which contains a high level of mucilage. Generally, the results obtained with Otto's buffer were excellent (nuclei had high relative fluorescence intensity and the lowest CV values) in many species. An exception was the grass Festuca roth-

maleri, a technically difficult taxon to work with, which produces less satisfactory results with Otto's buffer and Tris.MgCl2. Loureiro et al. (2006a) even found that for a given species the analysis of scatter properties (FS and SS) of nuclei provides a "fingerprint" of each buffer.

The finding that LB01 buffer, which contains Tris as the buffer substance, performed very well while Tris.MgCl2 buffer yielded the least satisfactory results (with exceptions), shows that it is probably not the buffer substance itself which makes a good isolation buffer, but its concentration and the additives such as chromatin stabilizers and antioxidants, ionic strength, and detergent concentration.

Which buffer is preferable? Loureiro et al. (2006a) showed that of the four lysis buffers used, none gave consistently good results with all seven species tested. Although LB01 and Otto's buffer are recommended as the first choice, it is worthwhile testing various buffers to identify the best one for a given material. Notably, Loureiro et al. (2006a) also documented some slight differences in relative fluorescence yield depending on which buffer was used. This would mean that it may be the buffer which causes some divergence between laboratories in the estimation of genome size of the same material. The reasons for this divergence are therefore unclear and deserve investigation.

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