Comparison of four nuclear isolation buffers for plant DNA flow cytometry

• Background and Aims DNA flow cytometry requires preparationof suspensions of intact nuclei, which are stained using a DNA-specificfluorochrome prior to analysis. Various buffer formulas weredeveloped to preserve nuclear integrity, protect DNA from degradationand facilitate its stoichiometric staining. Although nuclearisolation buffers differ considerably in chemical composition,no systematic comparison of their performance has been madeuntil now. This knowledge is required to select the appropriatebuffer for a given species and tissue. • Methods Four common lysis buffers (Galbraith's, LB01,Otto's and Tris.MgCl₂) were used to prepare samples from leaftissues of seven plant species (Sedum burrito, Oxalis pes-caprae,Lycopersicon esculentum, Celtis australis, Pisum sativum, Festucarothmaleri and Vicia faba). The species were selected to covera wide range of genome sizes (1·30–26·90pg per 2C DNA) and a variety of leaf tissue types. The followingparameters were assessed: forward (FS) and side (SS) light scatters,fluorescence of propidium iodide-stained nuclei, coefficientof variation of DNA peaks, presence of debris background andthe number of nuclei released from sample tissue. The experimentswere performed independently by two operators and repeated onthree different days. • Key Results Clear differences among buffers were observed.With the exception of O. pes-caprae, any buffer provided acceptableresults for all species. LB01 and Otto's were generally thebest buffers, with Otto's buffer providing better results inspecies with low DNA content. Galbraith's buffer led to satisfactoryresults and Tris.MgCl₂ was generally the worst, although ityielded the best histograms in C. australis. A combined analysisof FS and SS provided a ‘fingerprint’ for each buffer.The variation between days was more significant than the variationbetween operators. • Conclusions Each lysis buffer tested responded to a specificproblem differently and none of the buffers worked best withall species. These results expand our knowledge on nuclear isolationbuffers and will facilitate selection of the most appropriatebuffer depending on species, tissue type and the presence ofcytosolic compounds interfering with DNA staining.     

Simultaneous Measurement of DAPI-Sulforhodamine 101 Stained Nuclear DNA and Protein in Higher Plants by Flow Cytometry

A 2-step staining procedure is presented for simultaneous measurement of nuclear DNA and protein content in higher plants by flow cytometry. To release nuclei, plant tissues were chopped and stirred in the presence of the DNA specific fluorochrome 4', 6-diamidino-2-phenylindole (DAPI) and the nonionic detergent Triton X-100. Plant protoplasts were stirred in the DAPI dye solution with the detergent. After a short incubation period a second dye solution containing DAPI and the protein fluorochrome sulforhodamine 101 (SR 101) without detergent was added. Following another incubation, and after filtration through nylon gauze, the highly fluorescent nuclei were analyzed with an impulse cytophotometer. Accurate bivariate DNA-protein histograms were obtained with CV-values of about 2% or less for the 2C-peak of the univariate DNA parameter. The method presented here can be used for basic and applied cytogenetic studies of higher plants, for characterization of subcompartments of the cell cycle phases, or for examination of heterogeneity in plant tissues.     

适合流式细胞仪分析的小麦细胞核提取方法比较

为制备供流式细胞仪分析的高纯度小麦细胞核悬液,以冬小麦"临优2018"为材料,分别采用酶解法和直接剪切法对其幼苗的细胞核进行提取,对所得到的细胞核在形态结构和数量等方面进行了分析和比较,根据其优缺点优化出最适合流式细胞仪分析的小麦幼苗细胞核的提取方法。结果显示:在直接剪切法所用的3种细胞核提取缓冲液中,MgSO4缓冲液的提取效果最好,细胞核形态及内部结构完整,且得到的细胞核量多;OttoⅠ对细胞核的提取效果显著,但是在加入OttoⅡ后细胞核破裂明显;Tris.MgCl2缓冲液提取的细胞核数量较少;酶解法制备的细胞核悬浮液中杂质较多,且需时较长。结果表明采用MgSO4提取缓冲液的直接剪切法是适合流式细胞仪对小麦幼苗DNA含量的分析。     

High-resolution flow cytometry of nuclear DNA in higher plants

Summary High-resolution flow cytometry of nuclear DNA in higher plants has been performed from chopped plant tissues and plant protoplasts. A preparation and staining procedure with the DNA specific fluorochrome DAPI, successfully employed for precise flow cytometric DNA analysis of animal and human cells has been used in a slightly modified manner for the DNA analysis of plant cell material. High-resolution DNA histograms coefficients of variation about 1–1.5% have been obtained routinely from plant species with different DNA content. Staining of nuclei with DAPI in combination with the protein fluorochrome sulforhodamine 101 allows bi-parametric analysis of nuclear DNA and protein. The described simple and precise method might be very promising for the analysis of DNA in basic and applied cytogenetic investigations of plant cell research.Abbreviations CV coefficient of variation - DAPI 4,6-diamidino-2-phenylindole - SR 101 sulforhodamine 101     

应用流式细胞术测定绢毛苣属4种8居群基因组大小

以绢毛苣属（Soroseris）植物肉菊（S.umbrella（Franch.）Lipsch）、绢毛苣（S.glomerata（Decne.）Stebbins）、羽裂绢毛苣（S.hirsuta（Anth.）Shih）和金沙绢毛菊（S.gillii（S.Moore）Stebbins）4个种的8个居群为材料,采用WPB和Galbraith's buffer两种裂解液分别处理样品,运用流式细胞术测定DNA含量,并使用常规染色体压片法确定其细胞倍性。结果显示：两种裂解液中,最适于绢毛苣属植物DNA含量测定的裂解液为WPB;4个物种中,羽裂绢毛苣的基因组最小;细胞倍性水平对1Cx-值的影响较小。此外,本研究发现可采用种子萌发形成的幼苗代替难以获取的植物幼嫩叶片作为实验材料。     

Isolation of plant nuclei suitable for flow cytometry from recalcitrant tissue by use of a filtration column

Flow cytometry is widely applied in the determination of nuclear DNA content and ploidy level in many organisms. However, a difficulty with flow cytometry is the method's intrinsic inability to tolerate large particles that associate with the isolated nuclei. A suspension of plant nuclei can often contain a high level of crystalline calcium oxalate, which blocks the fluidics system of the flow cytometer. We designed a cotton column and added polyvinylpyrrolidone-40 to the buffer to remove phenolic impurities and cytoplasmic compounds from plant nuclei, making the suspension suitable for flow cytometry. This simple and highly efficient protocol enables isolation of intact nuclei from plant tissues containing high levels of polysaccharides, calcium oxalate crystals and other metabolites. Our protocol resulted in the isolation of intact nuclei from mature orchid leaves. This method can be used on recalcitrant tissues and is particularly effective on plants containing calcium oxalate crystals.     

Are seeds suitable for flow cytometric estimation of plant genome size?

BACKGROUND: Nuclear DNA content in plants is commonly estimated using flow cytometry (FCM). Plant material suitable for FCM measurement should contain the majority of its cells arrested in the G0/G1 phase of the cell cycle. Usually young, rapidly growing leaves are used for analysis. However, in some cases seeds would be more convenient because they can be easily transported and analyzed without the delays and additional costs required to raise seedlings. Using seeds would be particularly suitable for species that contain leaf cytosol compounds affecting fluorochrome accessibility to the DNA. Therefore, the usefulness of seeds or their specific tissues for FCM genome size estimation was investigated, and the results are presented here. METHODS: The genome size of six plant species was determined by FCM using intercalating fluorochrome propidium iodide for staining isolated nuclei. Young leaves and different seed tissues were used as experimental material. Pisum sativum cv. Set (2C = 9.11 pg) was used as an internal standard. For isolation of nuclei from species containing compounds that interfere with propidium iodide intercalation and/or fluorescence, buffers were used supplemented with reductants. RESULTS: For Anethum graveolens, Beta vulgaris, and Zea mays, cytometrically estimated genome size was the same in seeds and leaves. For Helianthus annuus, different values for DNA amounts in seeds and in leaves were obtained when using all but one of four nuclei isolation buffers. For Brassica napus var. oleifera, none of the applied nuclei isolation buffers eliminated differences in genome size determined in the seeds and leaves. CONCLUSIONS: The genome size of species that do not contain compounds that influence fluorochrome accessibility appears to be the same when estimated using specific seed tissues and young leaves. Seeds can be more suitable than leaves, especially for species containing staining inhibitors in the leaf cytosol. Thus, use of seeds for FCM nuclear DNA content estimation is recommended, although for some species a specific seed tissue (usually the radicle) should be used. Protocols for preparation of samples from endospermic and endospermless seeds have been developed.     

流式细胞仪检测高等植物细胞核DNA含量的方法

相对于动物和微生物而言,流式细胞术在植物科学上的应用会因植物组织与细胞(如细胞壁、中央液泡、特殊细胞器等)的特殊结构以及次生代谢产物等特殊成分,造成样品在前期处理、染色及测试等方面的困难,甚至导致检测失败或结果不准确。笔者在长期运用流式细胞仪测试工作中,积累了大量的植物样本检测经验,并参考国内外相关文献,总结出从植物取材、样品制备到植物细胞核DNA流式检测的方法和技巧,可为植物科学研究者及从事流式细胞检测的技术人员提供实验参考。     

Plant DNA flow cytometry and estimation of nuclear genome size

BACKGROUND: DNA flow cytometry describes the use of flow cytometry for estimation of DNA quantity in cell nuclei. The method involves preparation of aqueous suspensions of intact nuclei whose DNA is stained using a DNA fluorochrome. The nuclei are classified according to their relative fluorescence intensity or DNA content. Because the sample preparation and analysis is convenient and rapid, DNA flow cytometry has become a popular method for ploidy screening, detection of mixoploidy and aneuploidy, cell cycle analysis, assessment of the degree of polysomaty, determination of reproductive pathway, and estimation of absolute DNA amount or genome size. While the former applications are relatively straightforward, estimation of absolute DNA amount requires special attention to possible errors in sample preparation and analysis. SCOPE: The article reviews current procedures for estimation of absolute DNA amounts in plants using flow cytometry, with special emphasis on preparation of nuclei suspensions, stoichiometric DNA staining and the use of DNA reference standards. In addition, methodological pitfalls encountered in estimation of intraspecific variation in genome size are discussed as well as problems linked to the use of DNA flow cytometry for fieldwork. CONCLUSIONS: Reliable estimation of absolute DNA amounts in plants using flow cytometry is not a trivial task. Although several well-proven protocols are available and some factors controlling the precision and reproducibility have been identified, several problems persist: (1) the need for fresh tissues complicates the transfer of samples from field to the laboratory and/or their storage; (2) the role of cytosolic compounds interfering with quantitative DNA staining is not well understood; and (3) the use of a set of internationally agreed DNA reference standards still remains an unrealized goal.     

Flow cytometric and microscopic analysis of the effect of tannic acid on plant nuclei and estimation of DNA content

• Background and Aims Flow cytometry (FCM) is extensivelyused to estimate DNA ploidy and genome size in plants. In orderto determine nuclear DNA content, nuclei in suspension are stainedby a DNA-specific fluorochrome and fluorescence emission isquantified. Recent studies have shown that cytosolic compoundsmay interfere with binding of fluorochromes to DNA, leadingto flawed data. Tannic acid, a common phenolic compound, maybe responsible for some of the stoichiometric errors, especiallyin woody plants. In this study, the effect of tannic acid onestimation of nuclear DNA content was evaluated in Pisum sativumand Zea mays, which were chosen as model species. • Methods Nuclear suspensions were prepared from P. sativumleaf tissue using four different lysis buffers (Galbraith's,LB01, Otto's and Tris.MgCl₂). The suspensions were treated withtannic acid (TA) at 13 different initial concentrations rangingfrom 0·25 to 3·50 mg mL^–1. After propidiumiodide (PI) staining, samples were analysed using FCM. In additionto the measurement of nuclei fluorescence, light scatter propertieswere assessed. Subsequently, a single TA concentration was chosenfor each buffer and the effect of incubation time was assessed.Similar analyses were performed on liquid suspensions of P.sativum and Z. mays nuclei that were isolated, treated and analysedsimultaneously. FCM analyses were accompanied by microscopicobservations of nuclei suspensions. • Key Results TA affected PI fluorescence and light scatterproperties of plant nuclei, regardless of the isolation bufferused. The least pronounced effects of TA were observed in Tris.MgCl₂buffer. Samples obtained using Galbraith's and LB01 bufferswere the most affected by this compound. A newly described ‘tannicacid effect’ occurred immediately after the addition ofthe compound. With the exception of Otto's buffer, nuclei ofP. sativum and Z. mays were affected differently, with pea nucleiexhibiting a greater decrease in fluorescence intensity. • Conclusions A negative effect of a secondary metabolite,TA, on estimation of nuclear DNA content is described and recommendationsfor minimizing the effect of cytosolic compounds are presented.Alteration in light scattering properties of isolated nucleican be used as an indicator of the presence of TA, which maycause stoichiometric errors in nuclei staining using a DNA intercalator,PI.     

Anthocyanin inhibits propidium iodide DNA fluorescence in Euphorbia pulcherrima: implications for genome size variation and flow cytometry

BACKGROUND: Measuring genome size by flow cytometry assumes direct proportionality between nuclear DNA staining and DNA amount. By 1997 it was recognized that secondary metabolites may affect DNA staining, thereby causing inaccuracy. Here experiments are reported with poinsettia (Euphorbia pulcherrima) with green leaves and red bracts rich in phenolics. METHODS: DNA content was estimated as fluorescence of propidium iodide (PI)-stained nuclei of poinsettia and/or pea (Pisum sativum) using flow cytometry. Tissue was chopped, or two tissues co-chopped, in Galbraith buffer alone or with six concentrations of cyanidin-3-rutinoside (a cyanidin-3-rhamnoglucoside contributing to red coloration in poinsettia). KEY RESULTS: There were large differences in PI staining (35-70 %) between 2C nuclei from green leaf and red bract tissue in poinsettia. These largely disappeared when pea leaflets were co-chopped with poinsettia tissue as an internal standard. However, smaller (2.8-6.9 %) differences remained, and red bracts gave significantly lower 1C genome size estimates (1.69-1.76 pg) than green leaves (1.81 pg). Chopping pea or poinsettia tissue in buffer with 0-200 microm cyanidin-3-rutinoside showed that the effects of natural inhibitors in red bracts of poinsettia on PI staining were largely reproduced in a dose-dependent way by this anthocyanin. CONCLUSIONS: Given their near-ubiquitous distribution, many suspected roles and known affects on DNA staining, anthocyanins are a potent, potential cause of significant error variation in genome size estimations for many plant tissues and taxa. This has important implications of wide practical and theoretical significance. When choosing genome size calibration standards it seems prudent to select materials producing little or no anthocyanin. Reviewing the literature identifies clear examples in which claims of intraspecific variation in genome size are probably artefacts caused by natural variation in anthocyanin levels or correlated with environmental factors known to induce variation in pigmentation.     

Nuclear DNA content of Solanum species grown in vitro, as determined by flow cytometry

The DNA relative content in nuclei from several Solanum species, which were used as partners for somatic hybridization, were determined using a flow cytometry method. The nuclei were isolated mechanically or via protoplasts from leaves of in vitro grown plants. In the case of S. nigrum as well as S. tuberosum cv. Bzura and dihaploid clone H8105, the nuclei were also obtained from suspension cultured cells by lysis of protoplasts. The source and the method of nuclei isolation affected the pattern of nuclear DNA in the genotypes studied. The mesophyll nuclei showed two distinct peaks on the DNA histograms, whereas the DNA peaks produced by cell suspension nuclei were broad and less distinct. The DNA content in the nuclei, calculated from the DNA histograms of the samples and a DNA standard historgam (Trout Red Blood Cells, having DNA content of 5.05 pg per nucleus), were much lower in mesophyll nuclei than in those obtained from the cell suspension for the same genotypes. The results are discussed in respect of the genetic instability of Solanum genotypes. The usefulness of a flow cytometry approach in somatic hybridization research is also discussed.     

Flow cytometric analysis of polysomaty and in vitro genetic instability in potato

The analysis of nuclear DNA contents in various tissues of potato genotypes showed that flow cytometry is a rapid method to characterize large populations of cells for polysomaty, that is, the occurrence of cells with normal DNA levels together with cells containing endoreduplicated nuclei. The proportion of endoreduplicated nuclei varied in different tissues and genotypes of potato. The analysis of callus and cell cultures showed that the temporal changes in nuclear DNA contents during in vitro growth can be followed and the degree of polyploidization quantified. It is concluded that flow cytometry is a highly suitable method to detect ploidy changes in differentiated plant tissues and calli which are often not amenable for chromosome number determination.     

Estimation of nuclear DNA content of plants by flow cytometry

A rapid and simple protocol for estimation of nuclear DNA content of plants is described. Suspensions of intact nuclei are prepared either by chopping plant tissues or lysing protoplasts in a MgSO₄ buffer, mixed with DNA standards, and stained with propidium iodide in a solution containing DNAase-free RNAase. Fluorescence intensities of the stained nuclei are measured by a flow cytometer. Values for nuclear DNA content are estimated by comparing fluorescence intensities of the nuclei of the test population with those of appropriate internal DNA standards. The same procedure can also be used for rapid determination of ploidy in plant tissues.     

Plant Genome Size Measurement with DNA Image Cytometry

To test the reliability of DNA image cytometry for the measurement of nuclear DNA content in plant material, we conducted independent experiments in two laboratories using different image analysis instruments for densitometric measurement of nuclear DNA amount in Feulgen-stained squash preparations of root tips. The 2C nuclear DNA content of the nine species studied spanned a 100-fold range (approx. 0.3–33 pg). The estimates of nuclear DNA content measured with image cytometry methods were comparable to values obtained previously using both photometric cytometry and flow cytometry. Image cytometry methods showed little variation among repeated experiments within each laboratory or among different operators using the same instrument. Furthermore, the interphase-peak method (measurement of several hundred interphase nuclei per slide) was comparable to the classical prophase/telophase approach (measurement of ten early prophase and ten late telophase nuclei per slide). Hence, DNA image cytometry gives accurate and reproducible results and may be used as an alternative to photometric cytometry in plant nuclear DNA content measurements. In the present study, we propose that two standards for quality control of nuclear DNA content measurement are used in plant DNA image cytometry: (1) the coefficient of variation of the peak should be lower than 6%, and (2) the 4C/2C ratio should be between 1.9 and 2.1.     

COMPARISON OF PLANT DNA CONTENTS DETERMINED BY FEULGEN MICROSPECTROPHOTOMETRY AND LASER FLOW CYTOMETRY

An improved procedure is reported for determining DNA amounts of plant nuclei. Nuclei stained with propidium iodide, isolated from chopped plant leaves, were passed through an Ortho Cytofluorograph with a Lexel model 95 argon laser (514 nm) and the fluorescence measured, integrated, and recorded using an Ortho 2140 Data Acquisition computer. All nuclear samples were mixed with nuclei of Sultan barley (2C DNA content = 11.12 pg [picogram]) as an internal standard. DNA contents of ten plant species, ranging from 2C = 1.7 pg to 36.1 pg measured by flow cytometry, correlated strongly (r = 0.99, slope = + 1.00) with DNA contents determined from Feulgen-stained nuclei of the same species using microspectrophotometry. The flow cytometric procedures were sufficiently sensitive to detect differences in DNA content between inbred lines of corn and their F1 hybrids. Our results obtained with improved procedures, specifically using propidium iodide as a fluorochrome and plant nuclei instead of chicken erythrocytes as an internal standard, demonstrate that laser flow cytometry can be a precise, rapid, and reliable method for determining nuclear DNA content of plants.     

Estimation of the nuclear DNA content of gossypium species

BACKGROUND AND AIMS: Gossypium is an economically important, globally distributed taxon comprising more than 50 species. DNA content estimates from about half of the species indicate over a 3-fold variation exists. However, the nine DNA content estimates for G. hirsutum reveal over a 2-fold difference for this species alone. Recent reports have shown that several plant compounds can bias DNA content estimates obtained by commonly used methods. The purpose of this research was to examine the standardization procedures used for DNA content determinations with flow cytometry as applied to Gossypium, and generate revised DNA content estimates for all available Gossypium species using best-standard practices. METHODS: Flow cytometry was used to measure fluorescence of isolated Gossypium nuclei stained with propidium iodide. Fluorescence values were converted to DNA content estimates based on the nuclear fluorescence of standard genotypes of barley, corn and rice. Various combinations of nuclei preparations relative to the standards were evaluated for their influence on the estimates. KEY RESULTS: Both external standardization and internal standardization with Oryza sativa 'IR36' yielded statistically similar DNA content estimates for Gossypium. Internal standardization with Hordeum vulgare 'Sultan' resulted in a high estimate of DNA content. Nuclear DNA content estimates were generated for 37 Gossypium species using external standardization. Estimates of ancestral genome sizes reveal that both increases and decreases in nuclear DNA content have occurred. Variation in intraspecific and intragenomic DNA content was low, and the allopolyploid AD-genome size was nearly the additive of its progenitor genomes. CONCLUSIONS: Due to unknown factors, internal standardization with H. vulgare 'Sultan' may not be appropriate for DNA content determinations of Gossypium. The current DNA content estimates support accepted cytogenetic divisions of the genus. Gossypium is a genus that exhibits genome constancy both through speciation within genomic groups and allopolyploidization.     

Analysis of Nuclear DNA content in plant cells by Flow cytometry

Flow cytometry was used to analyse the DNA content of nuclei isolated from intact plant tissues and from callus and cell suspension cultures invitro. Cell nuclei were isolated either mechanically (chopping, syringing) or by a hypotonic lysis of isolated protoplasts. Although both methods gave similar results, a slight shift to lower ploidy levels was observed after protoplast isolation from intact tissues and calli. No differences were observed if the two methods were compared using cell suspension cultures. The results showed that flow cytometry is a rapid method of nuclear DNA content analysis in intact plant tissues and variousin vitro cultures.     

Preparation of HMW DNA from Plant Nuclei and Chromosomes Isolated from Root Tips

Simple, fast and cost-effective method for preparation of DNA with high molecular weight (HMW DNA) from plant nuclei and mitotic chromosomes has been developed. The technique involves mechanical homogenization of formaldehyde-fixed root tips, purification of nuclei and/or chromosomes on sucrose gradient, embedding in low-melting-point agarose, and DNA isolation in agarose plugs. Alternatively, nuclei and chromosomes may be purified using flow cytometry. Majority of DNA obtained is megabase-sized and well digestible by restriction endonucleases. The method is highly efficient as microgram amounts of DNA can be obtained from only several milligrams of plant tissue. Handling negligible amounts of plant material reduces the consumption of chemicals. Furthermore, the use of root tips makes it possible to obtain high-quality DNA even from plant species with leaves that are rigid or rich in secondary metabolites such as polyphenols. It is expected that preparation of HMW DNA from root tip nuclei will facilitate long-range mapping and construction of large-insert DNA libraries also in these species. Successful isolation of HMW DNA from flow-sorted chromosomes opens a way for construction of chromosome-specific large-insert libraries in plants. This revised version was published online in July 2006 with corrections to the Cover Date.     

Nuclear DNA content in species ofEleusine (Gramineae): A critical re-evaluation using laser flow cytometry

Interest in dinitroaniline herbicide resistant biotypes ofEleusine indica, and an as yet undetermined taxon ofEleusine, necessitated a revaluation of reported nuclear genome size estimates for available species in the genus. Laser flow cytometry showed that the nuclear DNA content of six of the seven species examined had 15 to 50% less DNA than reported previously. It was also determined that roots, as contrasted to leaves, possessed a large fraction of nuclei at the 4C or 8C DNA content level, in diploid or tetraploid species, respectively (i.e. the G2/M peak). Two major reasons for the previously reported overestimation may include sampling only of root tissues where endopolyploid and normal diploid nuclei both occur and the inappropriate choice of onion nuclei as an internal standard.