AOBPreview published online on October 18, 2007
Annals of Botany, doi:10.1093/aob/mcm250
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Cytochemistry and C-values: The Less-well-known World of Nuclear DNA Amounts
Department of Systematic and Evolutionary Botany, Faculty of Life Sciences, University of Vienna, Rennweg 14, A 1030 Vienna, Austria
* E-mail johann.greilhuber{at}univie.ac.at
Received: 25 May 2007 Returned for revision: 10 July 2007 Accepted: 14 August 2007
 |
ABSTRACT |
|---|
 TOP ABSTRACT A TRIBUTE TO THE...FEULGEN DENSITOMETRYFLOW CYTOMETRYSTAINING INHIBITORSCONCLUSIONSAPPENDIXACKNOWLEDGEMENTSLITERATURE CITED |
|---|
Background: In the plant sciences there are two widely applied technologies for measuring nuclear DNA content: Feulgen absorbance cytophotometry and flow cytometry (FCM). While FCM is, with good reasons, increasingly popular among plant scientists, absorbance-cytophotometric techniques lose ground. This results in a narrowing of the methodological repertoire, which is neither desirable nor beneficial. Both approaches have their advantages, but static cytophotometry seems to pose more instrumental difficulties and material-based problems than FCM, so that Feulgen-based data in the literature are often less reliable than one would expect.
Scope: The purpose of this article is to present a selective overview of the field of nuclear DNA content measurement, and C-values in particular, with a focus on the technical difficulties imposed by the characteristics of the biological material and with some comments on the photometrical aspects of the work. For over 20 years it has been known that plant polyphenols cause problems in Feulgen DNA cytophotometry, since they act as major staining inhibitors leading to unreliable results. However, little information is available about the chemical classes of plant metabolites capable of DNA staining interference and the mechanisms of their inhibition. Plant slimes are another source of concern.
Conclusions: In FCM research to uncover the effects of secondary metabolites on measurement results has begun only recently. In particular, the analysis of intraspecific genome size variation demands a stringent methodology which accounts for inhibitors. FCM tests for inhibitory effects of endogenous metabolites should become obligatory. The use of dry seeds for harvesting embryo and endosperm nuclei for FCM and Feulgen densitometry may often provide a means of circumventing staining inhibitors. The importance of internal standardization is highlighted. Our goal is a better understanding of phytochemical/cytochemical interactions in plant DNA photometry for the benefit of an ever-growing list of plant genome sizes.
Key words: C-values, Feulgen densitometry, Drosera rotundifolia, flow cytometry, genome size, Pisum sativum, polyphenols, staining inhibitors, standardization, tannins
|
A TRIBUTE TO THE LITERATURE |
|---|
|
TOPABSTRACTA TRIBUTE TO THE...FEULGEN DENSITOMETRYFLOW CYTOMETRYSTAINING INHIBITORSCONCLUSIONSAPPENDIXACKNOWLEDGEMENTSLITERATURE CITED |
|---|
After 40 years of looking at cell nuclei and 30 years of publishing on DNA C-values (Greilhuber, 1977) it seems worthwhile to overview those papers, out of the many on DNA amounts in my files, which appear to have been of particular importance for my own work. In chronological order, the paper by Feulgen and Rossenbeck (1924), in which the famous Nuclealreaktion (later Feulgen reaction) was described, was impressive because of the stringency of the experimental approach based on known facts, their own research results (about Schiff's reagent, aldehydes, bases, sugars, effect of hydrolysis) and the application of these findings to plant cells on the microscope slide. The clarity with which Robert Feulgen had foreseen the importance of his innovative discovery for quantitative work (which began decades later) is impressively evident from a sentence towards the end of the paper (in my free translation): ‘One works with thermometer and watch, standardizes the reactions and lets them do their job’. Ten years later about 400 papers on the Nuclealreaktion had already been published (Milovidov, 1938). A paper by Milovidov (1936), a scientist of Ukrainian origin in Prague, came relatively late to my attention (see Greilhuber, 1997), but must be mentioned as the first explicit demonstration of the staining inhibition by tannins, a finding which was initially rejected by others (discussed in Greilhuber, 1997) and widely acknowledged in cytophotometry only since my studies on conifers (Greilhuber, 1986) and other plants (Greilhuber, 1988a, b). The paper by Swift (1950), in which the constancy of genome size within an organism was established, may be seen as the birth of modern research in plant nuclear DNA content. Here also the symbol C (derived from ‘constant’) for the DNA content of the characteristic chromosome set of an organism was introduced, which would later characterize a whole research discipline (see Bennett and Smith, 1976; Greilhuber et al., 2005). Swift (1950) had already applied a qualified mode of DNA photometry, in which nuclear punctual absorbance and area were considered, so avoiding, to some degree, the distributional error (see below). Patau (1952) and Ornstein (1952) independently provided the world with the two-wavelength method, an ingenious way to correct for the distributional error in static microscope photometry, with which I worked for more than a decade on the Leitz MPV2 cytophotometer. While doing so, the papers by Evans et al. (1966) and Evans (1968) on unorthodox C-value variation in flax and by Miksche (1968, 1971) on geographical genome size variation in conifers were much debated in our laboratory, leading later to a reappraisal of the data. Around that time Fox (1969) published a highly qualified paper which later became influential to me with respect to the ‘cold’ hydrolysis approach, stringent temperature control and the use of formaldehyde as a fixative. The fundamental papers on the nucleotype theory by Bennett (1971, 1972) became very influential when the chromosomes of the genus Scilla shaped my work from 1973 onwards and a Leitz MPV2 cytophotometer (without scanning device) later became available. These and subsequent papers, such as Bennett et al. (1983), Bennett (1987) and Bennett et al. (1998), provided the theoretical superstructure of my work on genome size and also shaped teaching. The list of ‘Nuclear DNA Amounts in Angiosperms’ by Bennett and Smith (1976) became my favourite paper. Not only was this a summary of all C-value literature and data, previously published or made available for the first time, but it also contained a number of criticisms of the then-current practice of measuring DNA amounts and made important suggestions to avoid these shortcomings. For instance, it strongly advocated the inclusion of an ‘internal’ biological standard (Greilhuber et al., 2007) in the experiment. Note that, much later, Bennett et al. (2003) reported a 1C-value of 157 Mbp for Arabidopsis thaliana in an important investigation, making a big step forward towards the better standardization of plant genome size measurements.
Papers on unorthodox C-value variation in Hedera helix (Schäffner and Nagl, 1979) and other plants, presented at a symposium at Kaiserslautern, Germany, in 1978, prompted me at last to re-investigate this problem. These re-investigations and those of the variation in conifers led to the discovery (or rediscovery) of the tannin error (Greilhuber, 1986) and finally to a rejection of the then prevailing concept of the ‘plastic genome’ (König et al., 1987; Greilhuber, 1998, 2005). Methods available at the time were Feulgen scanning cytophotometry (Leitz MPV2 system from approx. 1985 on) and, a decade later, Feulgen DNA image cytometry (CIRES system, Kontron) and FCM (Baranyi and Greilhuber, 1995) (Partec CA II). A paper on infraspecific genome size variation in pea (Cavallini and Natali, 1990) triggered a number of papers on infraspecific genome size variation (or stability as it transpired) in cultivated plants (see Greilhuber, 1998, 2005). Since then, FCM and DNA image analysis have been used in parallel up to the present, with international co-operation being important. Inter-laboratory studies, such as by Dole
el et al. (1998) and Vilhar et al. (2001), testified to the reliability of the Feulgen densitometric method in comparison with FCM. The lists of DNA amounts by Bennett and co-workers and finally the electronic database (Bennett and Leitch, 2005) were an invaluable source of knowledge and inspiration. Nevertheless, a number of erratic papers in the literature have also provided the fuel for some papers in my publication list. In a sense, I am grateful to these authors as well. It seems to me that the material-dependent sources of error, neglected widely in the seventies and eighties, are now being taken much more seriously. In the following, I will outline problems in measuring plant DNA contents, which for me as a practicising photometrist seem to be relevant for best practice (see Appendix for some notes on the values presented).
|
FEULGEN DENSITOMETRY |
|---|
|
TOPABSTRACTA TRIBUTE TO THE...FEULGEN DENSITOMETRYFLOW CYTOMETRYSTAINING INHIBITORSCONCLUSIONSAPPENDIXACKNOWLEDGEMENTSLITERATURE CITED |
|---|
Measurement principles
Only a little theory is necessary for the worker to understand the essentials of Feulgen DNA scanning and image densitometry. With both, the field of measurement, in which a Feulgen-stained nucleus is placed, is divided into very small and thus optically fairly homogeneous areas (pixels), and for any pixel the intensity of monochromatic light, which has passed the spot, I, is measured. The suitable light intensity I0 for the background, as defined by the producer of the system, has been set before. With scanning machines the chosen wavelength used should be in the range of maximum extinction, i.e. yellow, but can also be shifted towards green. With image analysis systems, the green channel of the camera is used for measurement and a green interference filter is also used. With image densitometry, the chromophore amount is calculated via grey values (grey values, usually 255, are directly and linearly proportional to light intensity, I, and transmission, T), while with scanning photometry the light intensity is measured from the light beam which arrives at the photomultiplier. A level of light intensity can be set, below which a pixel is associated with the nucleus. Any of the pixel intensities I is converted to extinction E according to the formulae E = logT–1; T = I x I0–1. Extinction (= absorbance) is directly and linearly proportional to the amount of chromophore in the pixel. All E values of the pixels associated with the nucleus are summed. This gives a number which is directly proportional to the chromophore content. Scanning cytophotometers such as the MPV2 used measurement diaphragm (pinhole) diameters corresponding to about 0·5 µm, while image analysis systems provide a much finer resolution.
Workers on video-based systems should pay attention to the algorithm used by the software for calculating the integrated optical density (IOD) of the nuclei. There are systems that use an incorrect or over-simplified way of calculating IODs (as noted by Vilhar and Dermastia, 2002). Although information on ploidy level may be obtained, the data are worse than necessary (e.g. Muñoz et al., 2006; compare Vilhar et al., 2001; Hardie et al., 2002; Vilhar and Dermastia, 2002). As grey values (GV; 0 = black, 255 = white) are directly proportional to intensity of light or transmission T, it is not permissible to calculate the IOD based on averaged grey values multiplied by the area of a nucleus, because a nucleus is never homogeneous in dye distribution. The algorithm must calculate the E of every single pixel versus a background value.
Scanning machines such as the Vickers M86 or the much less widely distributed Zeiss or Leitz systems are no longer on the market, or even in use, and satisfactory DNA image analysis systems such as the CIRES (Cell Image Retrieval and Evaluation System, Kontron, Munich, distributed by Zeiss; also expired) are rarely found in botany laboratories. However, older non-scanning cytophotometers, such as the Amplival Photometrie (VEB Carl Zeiss Jena), the Leitz MPV or the Zeiss and the Nikon photometers or non-serial instruments, are still in use. Although publications using such instruments rarely give precise information about the mode of measurement, it is evident that the so-called ‘one-wavelength-method’ (e.g. Sharma and Sharma, 1980) is usually conducted, i.e. no correction is made for distributional error. The two-wavelength method is hardly in use nowadays, which is a pity as many data are obviously compromised by the use of the one-wavelength method, since a small financial investment in two interference filters, yellow and blue-green, or a continuous interference filter over the visible spectrum, could solve the problem. This is particularly true when small genomes are measured against a large genome such as Allium cepa for standardization. The two-wavelength method yields reasonable data. For instance, my C-values for the human, mouse, chicken and several other animals measured against Allium cepa (Greilhuber et al., 1983), are even today among the most accurate estimates for these taxa (compare Galbraith et al., 1983; Sohn et al., 2000). Because it may be of help to some workers, I will briefly explain the practice (not the theory) of the two-wavelength method here and give the calculation formula (based on Patau, 1952).
The two-wavelength method
There is proportionality of E and chromophore quantity in a given field of measurement only if the dye is distributed homogeneously. When the same chromophore amount is distributed in a smaller area while the measurement field remains the same, more light from a clear area reaches the photomultiplier than is additionally absorbed by a now denser area (Fig. 1). An irregular chromophore distribution has a comparable effect. This is the distributional error. Thus, if a nucleus of constant DNA amount is smaller at constant field area, the measured extinction of the field is less. The denser the nucleus becomes, the larger is the error. Density can be ‘manipulated’ by selecting a wavelength of monochromatic light off the maximum extinction, in which a nucleus then appears less densely stained and the error is also less. The ‘improvement’ of selecting a shorter wavelength varies with the inhomogeneity of the field. Patau (1952) and Ornstein (1952) used this for a correction formula which can be computed easily, so that calculation tables are no longer necessary (see below). With Schiff's reagent made from pararosanilin (basic fuchsin), the maximum extinction was found at 575 nm (yellow) and the half-maximum extinction at 505 nm (blue-green).
|
Usually a variable measurement diaphragm is available. A measurement field is selected which surrounds a nucleus not too closely. Its area B needs to be given only in relative terms (i.e. r2 or a x b in ocular scale units for circular or polygonal, or rectangular areas, respectively). A nucleus is measured first at the wavelength of maximum extinction
max (yellow) and then (for mathematical simplicity of the correction formula) at the wavelength of half-maximum extinction max1/2 (blue-green). Towards this at any time, a blank is first set to T = 1·0, the nucleus is moved into the field and the transmission is measured. The transmissions (T1 at max1/2 and T2 at max) are noted. The formula for calculating the relative amount of DNA, M, of a nucleus is this: |
|
Completely substituted the formula reads:
|
|
The formula is so simple because we work with one dye with a constant molar extinction coefficient and use a biological reference material for calculating ‘absolute’ DNA amounts. [See also the treatise of the two-wavelength method by Swift and Rasch (1956).]
In practice the extinction ratio at max x max1/2–1 can be slightly lower than 2·0, because the error introduced is largely the same in the test object and standard. Also max does not need to be precisely in the absorbance maximum, but can be shifted slightly towards the green (Van Oostveld and Boeken, 1977). The wavelengths are determined using a very small homogeneous area of a nucleus of average density (E < 0·7) to establish an extinction curve. This needs a continuous interference filter or a monochromator, althouth if wavelengths are known this step is not necessary. It is advisable to maintain a chosen pair of filters or continuous interference filter or monochromator positions throughout, i.e. it is not necessary to rearrange the two wavelengths for different nuclei or sets of measurements. If the ratios change, there is likely to be some staining interference from secondary compounds. Köhler illumination is important to reduce stray light (flare), i.e. light which is reflected from the microscope lenses back to the object, causing a brightening of the image and loss of contrast. Field illumination must be homogeneous. Although unnecessary light in the optical path should be avoided, flare influences sample and standard nuclei to a similar degree. Therefore, calibrated nuclear DNA contents are largely the same in scanning instruments with step-motor-driven stages and narrow field diaphragms (about 5 µm diameter at a spot size of 0·25 µm2 in the MPV2) with little flare and high local extinction, or in video-based systems or flying-spot scanning photometers with full-sized Köhler-illuminated fields and lower local extinction at a given chromophore concentration [compare Doleel et al. (1998) and Vilhar et al. (2001)]. Measurements are done using immersion optics.
The Feulgen reaction
Schiff's reagent is a colourless solution of pararosaniline or basic fuchsin, a red anilin compound, in sulfurous acid, in which the dye molecule reacts with SO2 and is then called leucobase. The reagent reacts with aldehyde groups and turns purple, because a quinoid structure is re-established in the molecule. With DNA, acid hydrolysis is used to remove purin bases from deoxyribose, whereby free aldehyde groups originate when the sugar ring opens. (NB In the presence of methanol the ring does not open and this can lead to erratic staining, if methanol is still present.) In ribose the ring opens much less frequently than in deoxyribose. This is the main reason why the Feulgen reaction is specific for DNA. Moreover, cellular RNA seems to be degraded and washed out more easily. Nucleoli are therefore Feulgen-negative, save occasional intranucleolar chromatin. Lillie (1977, p. 263) mentioned two different reactions of Schiff's reagent with deoxyribose. However, a definite clarification of which one occurs (i.e. the Wieland-Scheuing or the sulfonic acid formulation) has yet to be reached.
Although many papers have been published on methodological aspects of the Feulgen reaction, most are concerned with medical material (e.g. tumours, lymphocytes) and procedures (special fixations, embeddings, sections). In the botanical field, Fox (1969) contributed much to better methods thanks to his promotion of the ‘cold hydrolysis’ approach using 5 N HCl at 20 °C, and in his demonstration of the suitability of formaldehyde fixation. The advantage of cold hydrolysis for experimental handling, namely a much extended hydrolysis curve compared with the classical ‘hot’ hydrolysis (1 N HCl at 60 °C; Feulgen and Rossenbeck, 1924) is not always appreciated, even in recent work. The desirability of formaldehyde fixation was even explicitly denied by Berlyn et al. (1990), but with questionable reasons (Greilhuber, 1997). Greilhuber and Temsch (2001) revisited these basic questions and considered, perhaps for the first time, less obvious aspects, such as the effect of fixation storage at different temperatures, time and temperature of fixation with formaldehyde and the effect of washing after hydrolysis and after staining. Some rules for best practice in handling material for the quantitative Feulgen reaction can be derived from this.
Type of fixation
Two different fixatives, acetic methanol or ethanol (1 : 3, v/v) and phosphate-buffered formaldehyde (4 %, pH 7), are of prime importance for DNA content measurements. Formaldehyde has the advantage of promoting a better performance of the Feulgen reaction in the presence of endogenous polyphenols, especially condensed tannins. The latter are located in vacuoles and are polymerized in situ by formaldehyde. With acetic-alcohol fixation they ‘tan’ everything in their environment (Greilhuber, 1986). Formaldehyde must therefore not be applied in alcoholic mixtures when tannins are present, because a good preservation of the tonoplast is essential for polymerization to occur before the tannin molecules can leak out of the vacuoles. Figures 2A and B show Feulgen-stained root tip cells of Picea abies and Allium cepa side by side, fixed with 4 % formaldehyde and acetic methanol, respectively. Figure 2C shows the vanillin–sulfuric acid test for condensed tannins, demonstrating the presence of tannins in spruce cells and absence in onion cells.
|
Formaldehyde has the disadvantage of diminishing the staining intensity over time and at elevated temperature, so the standard material should be co-fixed, which is inconvenient in fieldwork. Nevertheless, buffered dilute formaldehyde is stable in contrast to stock solutions (Helander, 2000) and is easily washed out from fixed tissues by repeatedly changed 1 : 3 acetic methanol. This is significantly more effective than water (Greilhuber and Temsch, 2001).
Acetic alcohol 1 : 3 fixation is simple, and with material stored at –20 °C the staining result remains unimpaired for 7 years (Greilhuber and Temsch, 2001 and unpublished) and probably longer. However, it is advisable to replace the fixative with 96 % ethanol, because at higher temperatures a more rapid decay of 1 : 3-stored material has been noted. This means, the presence of water and protons should be reduced to a minimum.
The studies of Greilhuber and Baranyi (1999) and Greilhuber and Temsch (2001) further pointed to (a) the importance of a stringent hydrolysis regime with respect to temperature and time, especially with 1 : 3 fixation; (b) a lability of hydrolysed DNA in water; (c) restoration of relative stability in Schiff's reagent and afterwards in water; (d) the need to optimize the time for repeated washing of unbound Schiff's reagent with SO2-water (30–45 min) and softening in acetic acid before squashing (15 min); (e) a relative stability of stained and air-dried slides if stored in the dark. Air-drying of slides after coverslip removal upon freezing promotes flattening of nuclei, which is welcome. A short rinse in ethanol after coverslip removal promotes quicker drying of slides, but is not mandatory.
Hydrolysis curves with a range of 1 : 3-fixed material (oomycota, higher plants, and higher plants with purportedly different hydrolysis optima) point to a consistent optimum of 60 min when using 5 N HCl at 20·0 °C (Voglmayr and Greilhuber, 1998; Greilhuber and Baranyi, 1999). At present there is no reliable evidence that different organisms have different hydrolysis optima.
The slides do not need coverslips (mounting in a medium may even be a disadvantage) and measurements are taken under oil-immersion optics. A x63 objective is suitable for very small genomes (e.g. Arabidopsis thaliana) up to relatively large ones (e.g. Scilla siberica). Note, that the size of the standard genome is much less critical with Feulgen densitometry than with flow cytometry. There is no need to use a standard genome close in size to the unknown.
Type of tissue
The Feulgen reaction for genome size measurements is usually conducted on meristematic tissues suitable for squash preparations. Solid tissues with prominent cell walls are less amenable and may suffer from imperfect penetration of reagents. Price et al. (1980) used a leaf epidermis peeling technique, although this approach does not seem to have been adopted outside their laboratory. With this technique, epidermis was peeled off fresh leaves, fixed, stained, squashed onto glass slides, frozen and dried. Then cellulase was applied and the cuticle was peeled off. Epidermis nuclei free of cuticule and cell walls were obtained. Making squashes before hydrolysis, sometimes after the application of enzymatic maceration, makes sense if the material is suspended as very small particles, which are difficult to handle with forceps. Generally, staining of small pieces of soft tissue in toto gives regular staining and needs only millilitre quantities of reagents.
Use of dormant seeds
Instead of using active meristems, I have recently used a different approach, i.e. tissues from dormant seeds. It is now known that embryo and endosperm provide suitable nuclei for flow cytometric analysis (Matzk et al., 2000; Sliwinska et al., 2005). Fresh tissues of sundew, Drosera rotundifolia (Droseraceae), provide problems for Feulgen densitometry because of the presence of condensable tannins and probably other metabolites. To circumvent these, the testa was removed from mature dry seeds of Drosera rotundifolia in 4 % formaldehyde (pH 7) and the endosperms with embryos inside were crushed with forceps. At the same time, radicles from dormant mature seeds of Pisum sativum were minced in the fixative. After 1·5 h at room temperature the formaldehyde was replaced by several changes of acetic methanol and finally ethanol overnight. The post-fixation procedure removed not only formaldehyde but also abundant lipids. After thorough hydration the Feulgen reaction was conducted and squashes were made. In the sundew material, besides a brown membraneous inner integument and a starchy endosperm without nuclei, there were well-stained embryo nuclei in 2C and endosperm nuclei in 3C (Fig. 3). The vital endosperm in these mature seeds obviously consisted of a monolayer around the embryo. There was no evidence of staining interference by endogenous tannins and the peaks were symmetrical and narrow at coefficient of variation (CV) values mostly below 3 % (Fig. 4). A 2C-value of 2·73 pg was determined, which is considerably higher than a value of 1·76 pg published by Rothfels and Heimburger (1968), who noted staining irregularities but at that time had no information available about the effect of tannins. Pea nuclei (1C = 4·42 pg; Greilhuber and Ebert, 1994) were in 2C, 4C and 8C and their staining intensity was normal compared with fresh root tip nuclei. By using dormant seeds one avoids the need of germination, has tissue available with possibly less or no tannin, and has embryonic 2C nuclei in large numbers. Thus, notwithstanding the benefits of flow cytometry, it is possible also to use the ‘dormant seed approach’ with Feulgen densitometry. However, to date there is only limited experience with this type of material in other species.
|
|
Feulgen densitometry is still a useful methodology
Dole
el et al. (1998) demonstrated the excellent correspondence of genome size measurements done with flow cytometry (using PI as stain) and Feulgen scanning densitometry. In some respects Feulgen densitometry has advantages over flow cytometry: it is applicable to single cells; very small DNA amounts of single particles can be measured; there is visual control; long-term storage of material is possible; there is no debris in the histogram; having no fluidics the instruments are not sensitive to microbial growth when standing. For workers wanting to do measurements only occasionally densitometry is thus an alternative to flow cytometry, which should be practiced frequently (Doleel et al., 2007a). However, densitometry is time-consuming and thus in future will mainly play the role of a supplementary technique. It also seems that the Feulgen reaction is possibly more sensitive to inhibition by secondary metabolites than DNA staining with fluorochromes in flow cytometry. |
FLOW CYTOMETRY |
|---|
|
TOPABSTRACTA TRIBUTE TO THE...FEULGEN DENSITOMETRYFLOW CYTOMETRYSTAINING INHIBITORSCONCLUSIONSAPPENDIXACKNOWLEDGEMENTSLITERATURE CITED |
|---|
Flow cytometry may be characterized as a kind of dynamic fluorescence microphotometry, with a (hydrodynamically) focused stream of suspended particles (nuclei) instead of a microscope slide. Probably the first successful isolation of nuclei for plant flow cytometry was made from Vicia faba root tips by Heller (1973), but for several reasons the plant community hesitated to adopt the method for a long time. The instruments were expensive and purportedly capricious and the results of the pioneering paper may have not been convincing enough to justify a large financial investment. It was not until 10 years later that the paper by Galbraith et al. (1983) paved the way for the technology by reporting a simple technique for making nuclear suspensions. This initially unorthodox ‘chopping technique’ finally became the method of choice for everybody (Dole
el et al., 2007a, b). Fresh tissue, frequently almost completely expanded leaves, is chopped with a razor blade in isolation buffer, the slurry is sieved and fluorochrome is added (or is already a component of the isolation fluid). Within minutes the nuclear isolate is ready for running on the flow cytometer. Depending on the type of stain RNase is added. It is widely accepted that in work involving different species intercalating dyes (almost) without base preference should be used, principally propidium iodide (PI), (Doleel et al., 1992). Base-specific dyes, such as Hoechst dyes and 4'6-diamidino-2-phenylindole (DAPI), are suitable for ploidy analyses or cell cycle studies, but should be avoided if there are differences in base content (this applies also to heterochromatic or satellite DNA within a genome). Nevertheless, these dyes play a role in the determination of base content with FCM methods (Meister and Barow, 2007).
Isolation buffers
Loureiro et al. (2006a, 2007a) list ten frequently used buffers, which have been described for isolation of plant nuclei. Otto's buffer (Otto, 1990) differs from the rest in that it consists essentially of two buffer components identical to McIlvaine's buffer system (Rauen, 1964, pp. 92 and 95). Nuclei are isolated in citric acid plus detergent. The dye is added dissolved in the second, basic, component (Na2HPO4 solution) to achieve neutral proton concentration (pH 7). Loureiro et al. (2006a) studied four buffers in a number of species which were difficult to analyse by flow cytometry because of their phenolics, slime, xeromorphic anatomy or acidic cell sap. No buffer worked equally well in all species, but Otto's buffer and Doleel's lysis buffer LB01 (Doleel et al., 1989) were preferable in most cases in terms of CV and nuclear yield. Also, slight differences in the target/standard ratio were noted, which has bearings on the comparability of results between laboratories and between tests. It is obvious that the content of detergent, reductants, polyvinyl pyrrolidone (PVP) and chromatin stabilizers (such as spermine or magnesium ions) can influence the quality of measurements.
Choice of material
The usual material is fresh differentiated tissue, mostly leaves. The content (or better, the absence) of fluorescence inhibitors is crucial (see below). Therefore, coloured plant organs should be used cautiously, and only if absolutely necessary. Remarkably, it has been found that dried herbarium material can be used for FCM, opening up the possibility of making direct ploidy level studies. Previously, such data were obtainable only using indirect methods such as measurements of pollen diameter and stomata size (Suda and Trávní
ek, 2006; Suda et al., 2007). Another unorthodox approach has been to use seed material (Matzk et al., 2000; Sliwinska et al., 2005). FCM may be applied to a suspension prepared from dry seed chopped or gently ground with fine sand paper (Matzk, 2007). Peaks from embryo and endosperm nuclei are obtained and can be used for testing the reproductive mode in various plants (Matzk, 2007). Sliwinska et al. (2005) found that, among other plants, in Brassica napus the hypocotyl from dry seeds gave better results than leaves. This may be due to a lower level of inhibitors in dormant embryos compared with leaves. As already noted, this approach is also fruitful with static Feulgen densitometry. The use of unconventional materials, i.e. dried vegetative parts and seeds, considerably extends the time scale of applicability of FCM.
|
STAINING INHIBITORS |
|---|
|
TOPABSTRACTA TRIBUTE TO THE...FEULGEN DENSITOMETRYFLOW CYTOMETRYSTAINING INHIBITORSCONCLUSIONSAPPENDIXACKNOWLEDGEMENTSLITERATURE CITED |
|---|
In plants, endogenous metabolites capable of reducing the quality of DNA measurements occur very frequently. While both Feulgen densitometry and FCM are affected, the various compounds may have different effects, because the DNA staining mechanisms are very different. Research on this is still in its infancy and should be one of the urgent themes for the future.
Stoichiometric errors by staining inhibitors in Feulgen cytophotometry
The action of tannins as staining inhibitors in the Feulgen reaction was first reported by Milovidov (1936), who had previously noted the stabilizing effect of tannins on the morphology of nuclei following cell death (Milovidov, 1932). He reported a reduced intensity of the reaction in conifer root tips and subsequently a similar effect when tannic acid was applied to onion root tip sections (Milovidov, 1936). This was long before Feulgen cytophotometric measurements could be done. In the same year, Hurel-Py (1936), who had access to unpublished information from Milovidov, indicated tannins as the reason for a negative Feulgen reaction in Rosa (Rosaceae). Milovidov's results were, however, soon doubted by Hillary (1939) and later by Ishida (1961), and for a long time after these observations were scarcely considered in the literature, although Esteves de Sousa (1950) mentioned, without further analysis, that tannins were possible Feulgen staining inhibitors in Stapeliaceae. Hillary's 1939 paper seems to have been especially influential, although the approach was a doubtful one. Hillary worked with isolated DNA embedded in agar cubes of 5 mm side length, with tannic acid applied or not, with the Feulgen staining result evaluated visually. It is still unclear whether tannic acid could inhibit the Feulgen reaction with purified DNA, but it is completely clear that the approach did not reflect the situation in cells in situ and on microscope slides. However, pollen mother cells of Tradescantia were treated in a similar way and no effect of tannins was noted. It is possible that upon visual inspection (without immersion optics) an effect may remain undetected. However, Greilhuber (1986) found a strong inhibitory effect of tannic acid in aqueous solution if applied to fixed root tips of onion before Feulgen staining.
Ishida (1961) worked with purified DNA and his verdict on the results of Milovidov (1936) was questionable as well. He applied tannic acid to hydrolysed DNA in vitro. Later, Warden (1968, 1974) observed staining inhibition in Bryophyllum (Crassulaceae), but associated the effect with proteins rather than tannins, since cytochemical tests seemed to indicate this. My own experience with tannins occurred first in 1986 when I stained spruce and pine root tips (Greilhuber, 1986; J. Greilhuber, unpubl. res.) to investigate the question of large-scale genome size variation in conifers. The usual fixation with acetic methanol yielded a strikingly defective result, despite a good mitotic index. Teoh and Rees (1976), in a refutation of intraspecific genome size variation in Picea glauca, P. engelmannii and Pinus contorta reported by Miksche (1968, 1971), had used formaldehyde fixation for DNA content measurement, without giving reasons for choosing this fixative. Applying formaldehyde, I found that the condensed tannins were excellently preserved as solid bodies of various shapes in the vacuoles (Fig. 2A), and it was evident that they were the reason for the staining inhibition, being invisible after acetic-methanol (1 : 3) fixation (Fig. 2B). However, the chemical identity of these bodies as condensed tannins became known only after application of the vanillin test (Greilhuber, 1986). The colour tests with aldehydes such as vanillin aldehyde (Fig. 2C) or cinnamomum aldehyde (Feucht and Schmid, 1983) do not work well after formaldehyde fixation (the reactive groups of the tannins are largely bound) and are better when conducted without fixation or after 1 : 3 fixation.
Naturally occurring tannins are found in red wine, while white wine has almost none. Raspberries are rich in ellagitannins, a class of hydrolyzable tannins. Application of red wine (Fig. 5) or raspberry juice (Fig. 6) to 1 : 3-fixed or formaldehyde-fixed Pisum sativum seedling root tips before Feulgen staining exhibited a strong inhibitory effect, with only about one-third or even smaller amounts of staining expressed than was found in the control. White wine had hardly any effect (data not shown). These endogenous substances of supposedly phenolic nature also had an inhibitory effect with flow cytometry using propidium iodide as stain (Fig. 7), but as the applied concentrations were about 10-fold lower, the degree of inhibition under both methods is difficult to assess at present.
|
|
|
The mechanism causing the staining inhibition by tannins appears to be probably a steric blocking of the dye access by a tight binding of the tannin molecules to chromosomal proteins and DNA, but there is no proof of this, and detailed physico-chemical studies are necessary.
It is one of the paradoxes in the DNA photometric literature that the role of tannins as staining inhibitors was denied in print as late as 1990 by Berlyn et al. (1990), a group who had worked for a long time largely on DNA photometry in conifers, with challenged results (Murray, 1998). A detailed criticism is found in Greilhuber (1997), where the historical aspects are treated in more detail.
The good side of condensed tannins is that they are polymerized with formaldehyde, so that the error can be avoided with the appropriate fixation (Greilhuber, 1986, 1988a, b). Other metabolites such as gallotannins, flavonoids, chalcones and probably many others are not solidified and may cause stoichiometric errors, which can be controlled only with difficulty (e.g. in flower bud tissues of Ranunculus; Paun et al., 2006). Generally, formaldehyde fixation can provide better results than 1 : 3 fixation (Greilhuber, 1988a, b).
Stoichiometric errors in flow cytometry
Fluorochromes stain DNA in a way different from the Feulgen reaction. DAPI and Hoechst dyes (AT preference) and chromomycin (GC preference) bind to the minor groove of the double helix, while PI and ethidium bromide intercalate into the DNA with negligible base preference. The staining intensity depends, among others, on dye concentration, ions present, pH and temperature. Thus internal standardization should be conducted whenever possible. Figure 8 shows graphically the considerable fluctuation over time of peak positions of Pisum sativum and co-chopped Secale cereale at constant gain of the flow cytometer. However, the relative position of S. cereale versus P. sativum varies only very little (Greilhuber et al., 2007, see here also the corresponding table 4·4). Moreover, it seems highly probable that endogenous molecules, which bind by hydrogen bonds or covalently to DNA and chromosomal proteins or intercalate into DNA, can interfere with fluorochromes. The use of a co-chopped internal standard can minimize the error through these staining inhibitors.
|
Polyhydroxyphenols can crosslink proteins, possibly also DNA, and can act as steric barriers. Phenolics occur in the reduced state, in which they are often colourless, and in the oxidized state, in which they often assume a brown colour. Oxidized phenolics bind irreversibly to their target, while reduced phenolics bind by hydrogen bonds and can theoretically be stripped (Endres, 1961). It is a common experience, that browned isolates yield inferior results with FCM. Authors have added reductants such as mercaptoethanol, ascorbic acid or metabisulfite to the isolation buffer, and PVP as a binding competitor (Loureiro et al., 2006a, 2007a). PVP can even reactivate enzymes inactivated by tannins (Schneider and Hallier, 1970). So, a significant improvement of the CVs can be achieved (Bharathan et al., 1994; Yokoya et al., 2000).
Targeted FCM studies on fluorescence inhibition are rare. Noirot et al. (2000, 2002, 2003, 2005) studied the phenomenon in Coffea (Rubiaceae). Cytosol from Coffea leaves reduced the fluorescence of the standard nuclei, Petunia hybrida. The phenolic compound chlorogenic acid, a typical metabolite of the coffee tree, also showed this effect. Caffeine, which is known to react with tannins, could partly revert the inhibition (Noirot et al., 2003). Elevated temperature led to a stronger staining of Coffea nuclei with PI, probably because of improved access of the dye to the more widely dispersed chromatin (Noirot et al., 2005). Heat is known to relax hydrogen bonds and acts as a tannin stripper.
Helianthus annuus (Compositae) is an example of metabolite effects on fluorescence yield that can mimic biological DNA content variation. FCM data by Michaelson et al. (1991) were first indicative of a plastic genome, which apparently became smaller as a sunflower plant grew, and Price and Johnston (1996) and Price et al. (1998) presented evidence that far-red light was effective in reducing genome size down to about 50 % of the original DNA content. However, more recently Price et al. (2000) demonstrated that this variation was caused by endogenous inhibitors, which can be assumed to vary with age and environment, thereby inducing changes in fluorescence yield. This finding led to the designing of a test that could detect the presence of such inhibitors (Price et al., 2000). At the International Botanical Congress in Vienna (2005) M. D. Bennett and J. S. Johnston presented evidence for the inhibitory effect of the endogenous metabolite cyanidin-3-rutinoside in Poinsettia (Euphorbiaceae), a plant which contains this compound in red-coloured bracts but not in green leaves (see Bennett and Leitch, 2005: Report on the workshop on Genome Size: A Research Discipline in Development, and Bennett et al. (2008).
Loureiro et al. (2006b) investigated the effect of tannic acid (0·25–3·5 mg mL–1) on nuclear preparations of Pisum sativum and Zea mays using FCM and light microscopy. Four different isolation buffers were applied. Tannic acid is the glycoside of gallic acid and a molecule of considerable size, which makes it suitable for tanning leather. Its presence in nuclear isolates in lower concentrations led to an aggregation of anucleate particles of low fluorescence onto a fraction of the nuclei, which then showed even higher mean fluorescence (e.g. up to 7·7 % more fluorescence with LB01 buffer at 0·50 mg mL–1 tannic acid), visible as a right-hand shoulder of the peaks. Aggregates of anucleate debris caused increasing background noise, first seen at the left side of the histogram. At intermediate and higher tannic acid concentrations, a reduction in nuclear fluorescence also occurred. At high concentrations of tannic acid the histograms collapsed because of a general precipitation of the sample. According to the different buffers used for nuclear isolation, there were different tannic acid concentrations at which increased side scatter, increased nuclear fluorescence, reduced nuclear fluorescence and collapse of the sample occurred. The emergence of ‘coatings of debris’ (Greilhuber et al., 2007) in the presence of polyphenols explains, for the first time, such right-hand shoulders or peak asymmetries in histograms, which tend to occur in samples of species known to harbour tannins or similar substances and which are often encountered. Side scatter (if available as a parameter on the cytometer) allows a convenient gating out of coated nuclei on the fluorescence/side-scatter cytogram. There, such coated nuclei appear as a tail of higher side-scatter attached to the straight cloud of presumably integer nuclei. If these ‘integer’ nuclei are nevertheless quenched in fluorescence this can be evaluated only by a test for inhibitors as described below.
FCM test for inhibitors
Inhibitors are released into the isolation buffer upon chopping and react with all nuclei, both sample and standard. An inhibitory effect should become evident if standard alone and sample plus standard, prepared under identical conditions, are compared. Lower fluorescence yields or skewed peaks caused by debris coatings in the co-chopped standard indicate effects of metabolites. Also, addition of a nucleus-free sample homogenate to a standard isolate should indicate an inhibitory effect, if the fluorescence yield of the standard is diminished. Unfortunately, in present-day FCM work, tests like this are still rather the exception than the rule.
The simultaneous effect of inhibitors on sample and standard is also a strong argument for internal standardization in genome size studies, because inhibitor effects are more or less balanced out. However, standard nuclei (from cells without tannins) would always receive dilute inhibitor, while many nuclei from the sample would receive the full dose from their own cells at the moment of cell destruction. Therefore, J. Loureiro et al. (University of Aveiro, Portugal, unpubl. res.) suggested that standard and sample should be chopped in a sandwich-like fashion.
Internal standardization is now widely applied in FCM genome size studies (Loureiro et al., 2007a). This is not the case for inhibitor tests, which should nevertheless become an essential part of best-practice technique (Greilhuber et al., 2007).
Inhibitors – a major problem in cytophotometry
As is clear from the above, there are several phenomena which must be kept apart. (a) In FCM, coating of nuclei mediated by molecules sticking low-fluorescent particles externally onto nuclei (‘coatings of debris’) leads, at least primarily, to an increase in fluorescence. This has been demonstrated experimentally (Loureiro et al., 2006b). Whether molecules attached outside the nuclear membrane can act as a barrier for fluorochromes and so reduce nuclear fluorescence seems possible, but has not been demonstrated. (b) Molecules bind to DNA and chromosomal proteins, which compete with fluorochromes or present steric barriers to these or, in the case of Feulgen staining, to leucofuchsin. From differential chromosome staining it is known that minor-grove-binding GC and AT dyes such as chromomycin and DAPI compete with each other and lead to an enhanced banding pattern in chromosomes when applied synchronously. Actinomycin can dislodge fluorochromes, but with unclear base-preference. Distamycin A quenches DAPI fluorescence by energy transfer and in human material highlights a few segments by strongly quenching the rest (Schweizer, 1981). We may suppose that comparable competition phenomena could occur with intercalating endogenous metabolites and fluorochromes such as PI. (c) Binding endogenous substances may themselves be fluorescent. This could modify the fluorescence yield under certain circumstances. The significance of this remains unexplored. These effects do not exclude each other.
Substances which bind to proteins and DNA may also modify DNA staining. All natural substances to which mutagenic or anticancer activity is ascribed belong here. Many of these are phenolics. Cyanidin forms complexes with the sugar-phosphate backbone of DNA (Sarma and Sharma, 1999). Quercetin, after peroxidase-mediated oxidation, binds covalently to DNA and significantly stronger to protein (Walle et al., 2003). Ellagic acid is a widely distributed phenolic and a constituent of ellagitannin, a hydrolysable tannin. Macromolecules are cross-linked upon oxidation of ellagic acid (Whitley et al., 2003). Binding of ellagic acid by an irreversible intercalation mechanism was significantly stronger to DNA than to proteins (Whitley et al., 2003). These properties make ellagitannins and ellagic acid major candidates for inhibitory compounds in both FCM and Feulgen densitometry (Figs 6 and 7). Coumarins are phenolics which intercalate into DNA and, after UV irradiation, cause AT adducts and cross-links (Sastry et al., 1993). In the legume Bituminaria bituminosa, DNA amounts as measured with FCM co-varied with coumarin content, dependent on seasonal temperature (Walker et al., 2006), which was thus considered as a causative agent of methodologically mimicked genome size variation.
Plant slimes are another kind of ‘inhibitor’, which can make FCM and sometimes densitometric measurements difficult. In Ulmaceae, Loureiro et al. (2007b) investigated this problem in Ulmus species and found an improvement when a nuclear isolation buffer with higher detergent concentration was used. The best results were obtained with the fruits (samaras) of these species. The orchid Polystachia is another example of extreme slime-mediated recalcitrance, even with the Feulgen method applied to ovules (personal observation). The use of dry seeds could provide a solution to this problem.
|
CONCLUSIONS |
|---|
|
TOPABSTRACTA TRIBUTE TO THE...FEULGEN DENSITOMETRYFLOW CYTOMETRYSTAINING INHIBITORSCONCLUSIONSAPPENDIXACKNOWLEDGEMENTSLITERATURE CITED |
|---|
The routine methods now available to the botanist to measure nuclear DNA amounts are flow cytometry and Feulgen absorbance cytophotometry (densitometry). It is the high throughput of nuclei in a short time, and thus high precision, which makes flow cytometry the method of choice in many applications. Nevertheless, due to the fluidics system in a flow cytometer, such machines should be operated frequently. Flow cytometry is not a method for occasional measurements only. Thus, in certain situations static cytometry can be useful or even indispensible. With Feulgen densitometry single nuclei and very small DNA amounts can be measured, which would be impossible with common FCM instruments. However, the Feulgen densitometric method must be applied in a qualified way, which is often not the case. Endogenous secondary metabolites (‘inhibitors’), mostly of phenolic nature, are a major problem for plant flow and static cytometry. The clarification of their chemical identity and the mechanisms of their interference with the staining process is an important theme for the future. This can be achieved only by a co-operation of phytochemists, molecular biologists and cytologists. Here we still have a long way to go.
|
APPENDIX |
|---|
|
TOPABSTRACTA TRIBUTE TO THE...FEULGEN DENSITOMETRYFLOW CYTOMETRYSTAINING INHIBITORSCONCLUSIONSAPPENDIXACKNOWLEDGEMENTSLITERATURE CITED |
|---|
Original results presented here were obtained with Feulgen densitometry on the Cell Image Retrieval and Evaluation System (CIRES, Kontron, Munich) (compare Greilhuber and Temsch, 2001) and by flow cytometry using propidium iodide (PI) as stain (50 mg L–1) and Otto's buffer system for nuclei isolation and staining (compare Greilhuber et al., 2007). The flow cytometers were either a Partec PA II equipped with a mercury 50-W lamp or a Partec CyFlow ML equipped with a Cobolt Samba laser (output power 150 mW) working at 532 nm. Details are given under the corresponding figures.
|
ACKNOWLEDGEMENTS |
|---|
|
TOPABSTRACTA TRIBUTE TO THE...FEULGEN DENSITOMETRYFLOW CYTOMETRYSTAINING INHIBITORSCONCLUSIONSAPPENDIXACKNOWLEDGEMENTSLITERATURE CITED |
|---|
I thank Eva M. Temsch for her help with preparing the figures, Hermann Voglmayr for taking the photograph of Fig. 3, and Kornelija Pranji
and Ingeborg Lang (Faculty of Life Sciences, University of Vienna) for permission to quote from our unpublished work on Drosera rotundifolia. This is also a good occasion to thank Dieter Schweizer, whose support in building up a cytophotometry laboratory and whose introduction to cytophotometry in the seventies was of great help to me. I thank Ilia Leitch for useful comments and Peter Brandham (Kew) for linguistically improving this manuscript. |
LITERATURE CITED |
|---|
|
TOPABSTRACTA TRIBUTE TO THE...FEULGEN DENSITOMETRYFLOW CYTOMETRYSTAINING INHIBITORSCONCLUSIONSAPPENDIXACKNOWLEDGEMENTSLITERATURE CITED |
|---|
-
Baranyi M, Greilhuber J. Flow cytometric analysis of genome size variation in cultivated and wild Pisum sativum (Fabaceae). Plant Systematics and Evolution (1995) 194:231–239.[CrossRef][Web of Science]
Bennett MD. The duration of meiosis. Proceedings of the Royal Society of London, Series B (1971) 178:277–299.
Bennett MD. Nuclear DNA content and minimum generation time in herbaceous plants. Proceedings of the Royal Society of London, Series B (1972) 179:109–135.
Bennett MD. Variation in genomic form in plants and its ecological implications. New Phytologist (1987) 106:177–200.[Web of Science]
Bennett MD, Leitch IJ. Plant DNA C-values database (release 4·0, October 2005). (2005).
Bennett MD, Smith JB. Nuclear DNA amounts in angiosperms. Philosophical Transactions of the Royal Society of London, Series B (1976) 274:227–274.[Web of Science][Medline]
Bennett MD, Heslop-Harrison JS, Smith JB, Ward JP. DNA density in mitotic and meiotic metaphase chromosomes of plants and animals. Journal of Cell Science (1983) 63:173–179.[Abstract]
Bennett MD, Leitch IJ, Hanson L. DNA amounts in two samples of angiosperm weeds. Annals of Botany (1998) 82((Suppl. A)):121–134.
Bennett MD, Leitch IJ, Price HJ, Johnston JS. Comparisons with Caenorhabditis (
100 Mb) and Drosophila (175 Mb) using flow cytometry show genome size in Arabidopsis to be 157 Mb and thus 25 % larger than the Arabidopsis genome initiative estimate of 125 Mb. Annals of Botany (2003) 91:547–557.
Bennett MD, Price HJ, Johnston JS. Anthocyanin inhibits propidium iodide DNA fluorescence in Euphorbia pulcherrima: implications for genome size variation and flow cytometry. Annals of Botany (2008) 101.
Berlyn GP, Royte JL, Anoruo AO. Cytophotometric differentiation of high elevation spruces: physiological and ecological implications. Stain Technology (1990) 65:1–14.[Web of Science][Medline]
Bharathan G, Lambert G, Galbraith DW. Nuclear DNA content of monocotyledons and related taxa. American Journal of Botany (1994) 81:381–386.[CrossRef][Web of Science]
Cavallini A, Natali L. Nuclear DNA variability within Pisum sativum (Leguminosae): cytophotometric analyses. Plant Systematics and Evolution (1990) 173:179–185.[CrossRef][Web of Science]
Doleel J, Binarová P, Lucretti S. Analysis of nuclear DNA content in plant cells by flow cytometry. Biologia Plantarum (1989) 31:113–120.[Web of Science]
Doleel J, Sgorbati S, Lucretti S. Comparison of three DNA fluorochromes for flow cytometric estimation of nuclear DNA content in plants. Physiologia Plantarum (1992) 85:625–631.[CrossRef]
Doleel J, Greilhuber J, Lucretti S, Meister A, Lysák M, Nardi L, Obermayer R. Plant genome size estimation by flow cytometry: inter-laboratory comparison. Annals of Botany (1998) 82((Suppl. A)):17–26.
Doleel J, Greilhuber J, Suda J. Flow cytometry with plants: an overview. In: Flow cytometry with plant cells.—Doleel J, Greilhuber J, Suda J, eds. (2007) a. Weinheim: Wiley-VCH Verlag. 41–65.
Doleel J, Greilhuber J, Suda J. Flow cytometry with plant cells (2007) b. Weinheim: Wiley-VCH Verlag.
Endres H. Die Gerbwirkung niedermolekularer Polyhydroxyphenole. Leder (1961) 12:294–297.
Esteves de Sousa A. About the inhibition of the Feulgen nuclear reaction in plants of the tribe Stapelieae. Portugaliae Acta Biologica, Series A (1950) 3:147–149.
Evans GM. Induced chromosomal changes in flax. Heredity (1968) 23:301–310.[Web of Science]
Evans GM, Durrant A, Rees H. Associated nuclear changes in the induction of flax genotrophs. Nature (1966) 212:697–699.[CrossRef]
Feucht W, Schmid PPS. Selective histochemical staining of flavanols (catechins) with p-dimethylaminocinnamaldehyde in shoots from some fruit crops. Gartenbauwissenschaft (1983) 48:119–124.[Web of Science]
Feulgen R, Rossenbeck H. Mikroskopisch-chemischer Nachweis einer Nucleinsäure vom Typus der Thymonukleinsäure und die darauf beruhende elektive Färbung von Zellkernen in mikroskopischen Präparaten. Hoppe-Seyler's Zeitschrift für physiologische Chemie (1924) 135:203–248.[Web of Science]
Fox DP. Some characteristics of the cold hydrolysis technique for staining plant tissues by the Feulgen reaction. Journal of Histochemistry (1969) 17:206–272.
Galbraith DW, Harkins KR, Maddox JM, Ayres NM, Sharma DP, Firoozabady E. Rapid flow cytometric analysis of the cell cycle in intact plant tissues. Science (1983) 220:1049–1051.
Greilhuber J. Nuclear DNA and heterochromatin contents in the Scilla hohenackeri group, S. persica, and Puschkinia scilloides (Liliaceae). Plant Systematics and Evolution (1977) 128:243–257.[CrossRef][Web of Science]
Greilhuber J. Severely distorted Feulgen DNA amounts in Pinus (Coniferophytina) after nonadditive fixations as a result of meristematic self-tanning with vacuole contents. Canadian Journal of Genetics and Cytology (1986) 28:409–415.[Web of Science]
Greilhuber J. ‘Self-tanning’ – a new and important source of stoichiometric error in cytophotometric determination of nuclear DNA content in plants. Plant Systematics and Evolution (1988) 158a:87–96.[Web of Science]
Greilhuber J. Critical reassessment of DNA content variation in plants. In: Kew Chromosome Conference III—Brandham PE, ed. (1988) b. London: HMSO. 39–50.
Greilhuber J. The problem of variable genome size in plants (with special reference to woody plants). In: Cytogenetic studies of forest trees and shrub species—Borzan Z, Schlarbaum SE, eds. (1997) 8–11 September 1993. Brijuni National Park, Croatia, Zagreb: Croatian Forests, Inc. and Faculty of Forestry, University of Zagreb. 13–34. Proceedings of the First IUFRO Cytogenetics Working Party S2·04–08 Symposium.
Greilhuber J. Intraspecific variation in genome size: a critical reassessment. Annals of Botany (1998) 82((Suppl. A)):27–35.
Greilhuber J. Intraspecific variation in genome size in angiosperms: identifying its existence. Annals of Botany (2005) 95:91–98.
Greilhuber J, Baranyi M. Feulgen densitometry: importance of a stringent hydrolysis regime. Plant Biology (1999) 1:538–540.
Greilhuber J, Ebert I. Genome size variation in Pisum sativum. Genome. (1994) 37:646–655.
Greilhuber J, Temsch EM. Feulgen densitometry: some observations relevant to best practice in quantitative nuclear DNA content determination. Acta Botanica Croatica (2001) 60:285–298.
Greilhuber J, Volleth M, Loidl J. Genome size of man and animals relative to the plant Allium cepa. Canadian Journal of Genetics and Cytology (1983) 25:554–560.[Web of Science]
Greilhuber J, Doleel J, Lysák MA, Bennett MD. The origin, evolution and proposed stabilization of the terms ‘genome size’ and ‘C-value’ to describe nuclear DNA contents. Annals of Botany (2005) 95:255–260.
Greilhuber J, Temsch EM, Loureiro JCM. Nuclear DNA content measurement. In: Flow cytometry with plant cells.—Doleel J, Greilhuber J, Suda J, eds. (2007) Weinheim: Wiley-VCH Verlag. 67–101.
Hardie DC, Gregory TR, Hebert PDN. From pixels to picograms: a beginner's guide to genome quantification by Feulgen image analysis densitometry. Journal of Histochemistry and Cytochemistry (2002) 50:735–749.
Helander KG. Formaldehyde prepared from paraformaldehyde is stable. Biotechnic & Histochemistry (2000) 75:19–22.[Web of Science][Medline]
Heller FO. DNS-Bestimmung an Keimwurzeln von Vicia faba L. mit Hilfe der Impulscytophotometrie. Berichte der Deutschen Botanischen Gesellschaft (1973) 86:437–441.[Web of Science]
Hillary BB. Use of the Feulgen reaction in cytology. I. Effect of fixatives on the reaction. Botanical Gazette (1939) 101:276–300.
Hurel-Py G. Lés réactions de Feulgen sur la cellule végétale. Revue Cytologie e Cytophysiologie Végétale (1936) 2:67–76.
Ishida MR. A cytochemical study of nucleic acids in plant cells. VII. Causal analysis of negative Feulgen staining. Memoirs of the College of Science of the University of Kyoto, Series B (1961) 28:81–94.
König C, Ebert I, Greilhuber J. A DNA cytophotometric and chromosome banding study in Hedera helix (Araliaceae), with reference to differential DNA replication associated with juvenile-adult phase change. Genome (1987) 29:498–503.
Lillie RD, ed. H. J. Conn's biological stains – a handbook on the nature and uses of the dyes employed in the biological laboratory (1977) 9th edn. Baltimore, MD: The Williams and Wilkins Company.
Loureiro J, Rodriguez E, Doleel J, Santos C. Comparison of four nuclear isolation buffers for plant DNA flow cytometry. Annals of Botany (2006) 98a:679–689.
Loureiro J, Rodriguez E, Doleel J, Santos C. Flow cytometric and microscopic analysis of the effect of tannic acid on plant nuclei and estimation of DNA content. Annals of Botany (2006) 98b.
Loureiro J, Suda J, Doleel J, Santos C. FLOWER: a plant DNA flow cytometry database. In: Flow cytometry with plant cells.—Doleel J, Greilhuber J, Suda J, eds. (2007) a. Weinheim: Wiley-VCH Verlag. 423–438.
Loureiro J, Rodriguez E, Gomes A, Santos C. Genome size estimation in Ulmus minor Mill. Ulmus glabra Huds. and Celtis australis L. using flow cytometry. Plant Biology (2007) 9b:541–544.[CrossRef][Medline]
Matzk F. Reproduction mode screening. In: Flow cytometry with plant cells.—Doleel J, Greilhuber J, Suda J, eds. (2007) Weinheim: Wiley-VCH Verlag. 131–152.
Matzk F, Meister A, Schubert I. An efficient screen for reproductive pathways using mature seeds of monocots and dicots. The Plant Journal (2000) 21:97–108.[CrossRef][Web of Science][Medline]
Meister A, Barow M. DNA base composition of plant genomes. In: Flow cytometry with plant cells.—Doleel J, Greilhuber J, Suda J, eds. (2007) Weinheim: Wiley-VCH Verlag. 177–215.
Michaelson MJ, Price HJ, Johnston JS, Ellison JR. Variation of nuclear DNA content in Helianthus annuus (Asteraceae). American Journal of Botany (1991) 78:1238–1243.[CrossRef][Web of Science]
Miksche JP. Quantitative study of intraspecific variation of DNA per cell in Picea glauca and Pinus banksiana. Canadian Journal of Genetics and Cytology (1968) 10:590–600.[Web of Science]
Miksche JP. Intraspecific variation of DNA per cell between Picea sitchensis (Bong.) Carr. provenances. Chromosoma (1971) 32:343–352.[Web of Science][Medline]
Milovidov PF. Einfluß von Wasser hoher Temperatur auf den Kern der Pflanzenzellen im Lichte der Nuklealreaktion. Protoplasma (1932) 17:32–88.[CrossRef]
Milovidov PF. Zur Theorie und Technik der Nuklealfärbung. Protoplasma (1936) 25:570–597.[CrossRef]
Milovidov PF. Bibliographie der Nucleal- und Plasmalreaktion. Protoplasma (1938) 31:246–266.[CrossRef]
Muñoz M, Riegel R, Seemann P. Use of image cytometry for the early screening of induced autopolyploids. Plant Breeding (2006) 125:414–416.[CrossRef][Web of Science]
Murray BG. Nuclear DNA amounts in gymnosperms. Annals of Botany (1998) 82((Suppl. A)):3–15.
Noirot M, Barre P, Louarn J, Duperray C, Hamon S. Nucleus-cytosol interactions: a source of stoichiometric error in flow cytometric estimation of nuclear DNA content in plants. Annals of Botany (2000) 86:309–316.
Noirot M, Barre P, Louarn J, Duperray C, Hamon S. Consequences of stoichiometric error on nuclear DNA content evaluation in Coffea liberica var. dewevrei using DAPI and propidium iodide. Annals of Botany (2002) 89:385–389.
Noirot M, Barre P, Duperray C, Louarn J, Hamon S. Effects of caffeine and chlorogenic acid on propidium iodide accessibility to DNA: consequences on genome size evaluation in coffee tree. Annals of Botany (2003) 92:259–264.
Noirot M, Barre P, Duperray C, Hamon S, De Kochko A. Investigation on the causes of stoichiometric error in genome size estimation using heat experiments: consequences on data interpretation. Annals of Botany (2005) 95:111–118.
Ornstein L. The distributional error in microspectrophotometry. Laboratory Investigations (1952) 1:250–262.
Otto FJ. DAPI staining of fixed cells for high-resolution flow cytometry of nuclear DNA. In: Methods in cell biology—Crissman HA, Darzynskiewicz Z, eds. (1990) Vol. 33. New York, NY: Academic Press. 105–110.[Medline]
Patau K. Absorption microphotometry of irregular-shaped objects. Chromosoma (1952) 5:341–362.[CrossRef][Web of Science][Medline]
Paun O, Greilhuber J, Temsch EM, Hörandl E. Patterns, sources and ecological implications of clonal diversity in apomictic Ranunculus carpaticola (Ranunculus auricomus complex, Ranunculaceae). Molecular Ecology (2006) 15:897–910.[Medline]
Price HJ, Johnston JS. Influence of light on DNA content of Helianthus annuus Linnaeus. Proceedings of the National Academy of Sciences of the USA (1996) 93:11264–11267.
Price HJ, Bachmann K, Chambers KL, Riggs J. Detection of intraspecific variation in nuclear DNA content in Microseris douglasii. Botanical Gazette (1980) 141:195–198.
Price HJ, Morgan PW, Johnston JS. Environmentally correlated variation in 2C nuclear DNA content measurements in Helianthus annuus L. Annals of Botany (1998) 82((Suppl. A)):95–98.
Price HJ, Hodnett G, Johnston JS. Sunflower (Helianthus annuus) leaves contain compounds that reduce nuclear propidium iodide fluorescence. Annals of Botany (2000) 86:929–934.
Rauen HM. Biochemisches Taschenbuch, Zweiter Teil (1964) 2nd edn. Berlin: Springer Verlag.
Rothfels K, Heimburger M. Chromosome size and DNA values in sundews. Chromosoma (1968) 25:96–103.[CrossRef][Web of Science]
Sarma AD, Sharma R. Anthocyanin-DNA copigmentation complex: mutual protection against oxidative damage. Phytochemistry (1999) 52:1313–1318.[CrossRef][Web of Science]
Sastry SS, Spielmann HP, Hearst JE. Psoralens and their application to the study of molecular biological processes. Advances in Enzymology (1993) 66:85–148.[CrossRef]
Schäffner K-H, Nagl W. Differential DNA replication involved in transition from juvenile to adult phase in Hedera helix (Araliaceae). Plant Systematics and Evolution. Suppl. 2. Genome and chromatin: organization, evolution, function (1979) 105–110.
Schneider V, Hallier UW. Polyvinylpyrrolidone als Schutzstoff bei der Untersuchung gerbstoffgehemmter Enzymreaktionen. Planta (1970) 94:134–139.[CrossRef][Web of Science]
Schweizer D. Counterstain-enhanced chromosome banding. Human Genetics (1981) 57:1–14.[CrossRef][Web of Science][Medline]
Sharma AK, Sharma A. Chromosome techniques: theory and practice (1980) 3rd edn. London: Butterworths.
Sliwinska E, Zielinska E, Jedrzejczyk I. Are seeds suitable for flow cytometric estimation of plant genome size? Cytometry A (2005) 64:72–79.[Medline]
Sohn LL, Saleh OA, Facer GR, Beavis AJ, Allan RS, Notterman DA. Capacitance cytometry: measuring biological cells one by one. Proceedings of the National Academy of Sciences of the USA (2000) 97:10687–10690.
Suda J, Trávníek P. Reliable DNA ploidy determination in dehydrated tissues of vascular plants by DAPI flow cytometry – new prospects for plant research. Cytometry A (2006) 69:273–280.[Medline]
Suda J, Kron P, Husband BC, Trávníek P. Flow cytometry and ploidy: applications in plant systematics, ecology and evolutionary biology. In: Flow cytometry with plant cells.—Doleel J, Greilhuber J, Suda J, eds. (2007) Weinheim: Wiley-VCH Verlag. 103–130.
Swift H. The constancy of desoxyribose nucleic acid in plant nuclei. Proceedings of the National Academy of Sciences of the USA (1950) 36:643–654.
Swift H, Rasch E. Microphotometry with visible light. In: Physical techniques in biological research—Oster J, Pollister AW, eds. (1956) New York, NY: Academic Press. 353–400.
Teoh SB, Rees H. Nuclear DNA amounts in populations of Picea and Pinus species. Heredity (1976) 36:123–137.[Web of Science]
Van Oostveldt P, Boeken G. Two-wavelength cytophotometry: the choice of the wavelengths from a practical point of view. Journal of Histochemistry and Cytochemistry (1977) 25:1337–1344.[Abstract]
Vilhar B, Dermastia M. Standardization of instrumentation in plant DNA image cytometry. Acta Botanica Croatica (2002) 61:11–26.
Vilhar B, Greilhuber J, Dolenc Koce J, Temsch EM, Dermastia M. Plant genome size measurement with DNA image cytometry. Annals of Botany (2001) 87:719–728.
Voglmayr H, Greilhuber J. Genome size determination in Peronosporales (Oomycota) by Feulgen image analysis. Fungal Genetics and Biology (1998) 25:181–195.[CrossRef][Web of Science][Medline]
Walker DJ, Monino I, Correal E. Genome size in Bituminaria bituminosa (L.) C.H. Stirton (Fabaceae) populations: separation of ‘true’ differences from environmental effects on DNA determination. Environmental and Experimental Botany (2006) 55:258–265.[CrossRef][Web of Science]
Walle T, Vincent TS, Walle UK. Evidence of covalent binding of the dietary flavonoid quercetin to DNA and protein in human intestinal and hepatic cells. Biochemical Pharmacology (2003) 65:1603–1610.[CrossRef][Web of Science][Medline]
Warden J. Protein interference in the localization of nucleic acids in Bryophyllum: staining with methyl-green and pyronin. Portugaliae Acta Biologica, Series A (1968) 10:55–74.
Warden J. Urea and pyridine in Feulgen staining of Bryophyllum. Acta Histochemica (1974) 50:98–104.[Web of Science][Medline]
Whitley AC, Stoner GD, Darby MV, Walle T. Intestinal epithelial cell accumulation of the cancer preventive polyphenol ellagic acid—extensive binding to protein and DNA. Biochemical Pharmacology (2003) 66:907–915.[CrossRef][Web of Science][Medline]
Yokoya K, Roberts AV, Mottley J, Lewis R, Brandham PE. Nuclear DNA amounts in the roses. Annals of Botany (2000) 85:557–562.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  E. Wolny and R. Hasterok Comparative cytogenetic analysis of the genomes of the model grass Brachypodium distachyon and its close relatives Ann. Bot., October 1, 2009; 104(5): 873 - 881. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. J. Leitch, I. Kahandawala, J. Suda, L. Hanson, M. J. Ingrouille, M. W. Chase, and M. F. Fay Genome size diversity in orchids: consequences and evolution Ann. Bot., August 1, 2009; 104(3): 469 - 481. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. J. Leitch and M. F. Fay Plant Genome Horizons: Michael Bennett's Contribution to Genome Research Ann. Bot., April 1, 2008; 101(6): 737 - 746. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||