Plant Flow Cytometry22Far Beyond the Stone Age

Jo~ao Loureiro,1,2* Jaroslav Dolezel,3 Johann Greilhuber,4 Conceic~ao Santos,5 Jan Suda1,2

� Key termsflow cytometry; plant sciences; DNA ploidy; genome size; chro-mosome sorting

WHEN using flow cytometry (by that time called Impuls-

cytophotometrie, i.e., impulse cytophotometry in Germany)

to quantify nuclear DNA content in faba bean (Vicia faba),

the German botanist Friedrich Otto Heller could have hardly

imagined that in 1973, he laid the foundation stone of a new

scientific discipline, plant flow cytometry (1). In fact, his pio-

neering work received little attention, with just a dozen of cita-

tions on the Web of Science�, and only a few of his contem-

poraries recognized the potential of flow cytometry (FCM) for

plant sciences. The turning point was the work of David Gal-

braith, 10 years later, who came with an ingenious method for

rapid isolation of cell nuclei by mechanical homogenization of

plant tissues using a razor blade (2). This method replaced

lengthy enzymatic treatments, provided the conceptual basis

for routine analysis of nuclear DNA content and set the stage

for the spread of FCM into plant laboratories (3). However,

the dominance of biomedical research in the design of com-

mercially available instruments, coupled with their relatively

high cost, as well as a rather low interest of both the scientific

and the industrial community slowed down the progress. It

was not until the early 1990s that the number of publications

on plant FCM started to rise markedly (4). The last decade has

witnessed an ever-increasing number of applications in both

basic and applied research, as well as in industry and plant

breeding (5), making plant FCM a vital and dynamic research

discipline with a great potential.

Plant material has several unique features not paralleled

in animals and humans, which makes its analyses by flow

cytometry challenging. First, plant cells have rigid walls and

are held together by extra cellular matrix to form complex

three-dimensional tissues. It is not a trivial task to produce

liquid suspensions of single and regularly shaped cells and

subcellular particles such as nuclei, mitochondria, chloro-

plasts, and chromosomes. Other problems are due to the

chemical composition of the cytosol. Plant cells produce a vast

array of secondary metabolites that may interfere with a parti-

cular assay, for example with the staining of nuclear DNA (6).

In addition, autofluorescence of some cellular components

like the chloroplasts and cell walls can override the weak fluo-

rescence of stained targets. Concerning the methods and pro-

tocols, plant FCM suffers from the fact that the majority of

them were adopted from other fields (e.g., biomedical

research) and only a few attempts have been made to deal

with the peculiarities of plant samples.

A rather old-fashioned feature of plant FCM is the pre-

dominance of single-parameter analyses. This is clearly a

consequence of the enormous success of the method for

estimation of nuclear DNA content, which can be done

quite reliably with just one fluorescence parameter. The use

of FCM for addressing other issues (e.g., particle structure

and/or volume based on scatter properties) and for multi-

parameter analyses in particular (e.g., cell cycle studies) has

been scarce. Despite this, FCM contributed significantly to a

large number of remarkable and exciting results with far-

reaching implications. Plant breeding (5) and plant population

1Department of Botany, Faculty of Science, Charles University inPrague, Prague, Czech Republic2Institute of Botany, Academy of Sciences of the Czech Republic,Pruhonice, Czech Republic3Laboratory of Molecular Cytogenetics and Cytometry, Institute ofExperimental Botany, Olomouc, Czech Republic4Department of Systematic and Evolutionary Botany, University ofVienna, Vienna, Austria5CESAM & Department of Biology, University of Aveiro, Aveiro,Portugal

Received 2 April 2008; Accepted 6 April 2008

*Correspondence to: Jo~ao Loureiro, Department of Botany, Faculty ofScience, Charles University in Prague, Ben�atsk�a 2, Prague 128 01,Czech Republic.

E-mail: jloureiro@natur.cuni.cz

Published online in Wiley InterScience(www.interscience.wiley.com)

DOI: 10.1002/cyto.a.20578

© 2008 International Society for Advancement of Cytometry

Commentary

Cytometry Part A � 73A: 579�580, 2008

biology (7) are perhaps the two most profoundly affected

areas.

Modern, small, and relatively cheap multiparameter

instruments that measure several fluorescence parameters and

light scatter allow convenient and rapid DNA ploidy screening

using fresh and desiccated (8) plant tissues, high-precision ge-

nome size estimation (9), cell cycle studies (10), quantification

of the extent of endopolyploidy (11), as well as reproductive

mode screening using mature seeds (12). All these applications

may be successfully integrated in plant breeding programs, for

instance to characterize the available germplasm (13), control

the ploidy stability at various steps of breeding programs

(including in vitro cultures) (14), screen for desired cytotypes

after ploidy manipulation and/or hybridization (15), and

identify reproductive pathways (16).

Any method to be used by industry must be cost-effec-

tive, possibly high-throughput, saving time and replacing ex-

pensive workforce. There is no doubt that DNA flow cytome-

try has the characteristics of such a technique. However,

although FCM offers much more, other areas have not been

exploited sufficiently. Promising avenues include the analysis

of gene expression (17), the study of subcellular processes,

which comprise photosynthesis in plastids, respiration in mi-

tochondria, membrane studies, and apoptosis-associated

events (18) and the analysis and sorting of mitotic chromo-

somes (19). The latter application, which requires more so-

phisticated and expensive instruments, facilitates the analysis

of complex genomes and gene cloning in crops with massive

genomes, as is the case of barley and wheat.

Although FCM has significant impacts in various fields of

plant research, there are still some challenges that must be

overcome before its potential is fully realized. Identification of

secondary metabolites that may compromise the quality of

FCM assays (6) and optimization of protocols to ameliorate

their negative effect are badly needed. Alternative methods for

the preparation of nuclear suspensions for FCM such as the

bead beating protocol (20) and the use of tissues with reduced

amounts of interfering compounds [e.g., seeds (21)] are

potential solutions that need further research. In addition, the

possibilities for storing plant samples prior to analysis (i.e.,

fixation, freezing, and/or dehydration) are limited and more

work needs to be done in this area. Some recent studies

demonstrated the feasibility of sample storage (8), but much

more work is still ahead. Finally, we would like to emphasize

the increasing importance of educational aspects, mainly for

effective knowledge dissemination concerning best practices

(e.g., type of standardization, selection of fluorochromes and

standards, presentation of data and measures of result quality,

use of appropriate terminology).

Many of the topics covered in this commentary are thor-

oughly discussed in the first book dedicated to plant flow

cytometry, which was published last year (22). In fact, the year

2007 marked a new era in plant FCM, as in addition to the

book, the first database (http://flower.web.ua.pt/) (23), first

forum (http://flowerdatabase.20.forumer.com/), and the first

blog (http://flowerdatabase.blogspot.com/) were launched on-

line, all devoted to the applications of FCM in plant sciences.

Complementing the recent comprehensive review on applica-

tions of FCM to plant evolutionary and population biology

(7), the review of Ochatt in the current issue of Cytometry (5)

maps the applications of FCM in the field of plant breeding

and fills some of the remaining gaps. Along with the tradi-

tional topics (e.g., ploidy screening), the review provides a sum-

mary of some less-known uses of FCM in plants, including the

analysis of cell wall components and in vitro regeneration com-

petence, and may thus serve as a handy source of information

for plant breeders and biotechnologists. It is evident that plants

became a strong player on the FCM stage in recent years; a

‘‘tangible’’ evidence for this may come from the recent ISAC

2008 Congress in Budapest, where—for the first time—a tuto-

rial and two workshops fully devoted to plant systems was held.

LITERATURE CITED

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3. Galbraith DW. Cytometry and plant sciences: A personal retrospective. CytometryPart A 2004;58A:37–44.

4. Loureiro J, Suda J, Dolezel J, Santos C. FLOWER: A plant DNA flow cytometry data-base. In: Dolezel J, Greilhuber J, Suda J, editors. Flow Cytometry with PlantCells: Analysis of Genes, Chromosomes and Genomes. Weinheim: Wiley-VCH; 2007.pp 423–438.

5. Ochatt SJ. Flow cytometry in plant breeding. Cytometry Part A 2008;73A: in press.doi: 10.1002/cyto.a. 20562 (this issue).

6. Loureiro J, Rodriguez E, Dolezel J, Santos C. Flow cytometric and microscopic analy-sis of the effect of tannic acid on plant nuclei and estimation of DNA content. AnnBot 2006;98:515–527.

7. Kron P, Suda J, Husband BC. Applications of flow cytometry to evolutionary andpopulation biology. Annu Rev Ecol Evol Syst 2007;38:847–876.

8. Suda J, Tr�avn�ıcek P. Reliable DNA ploidy determination in dehydrated tissues of vas-cular plants by DAPI flow cytometry—New prospects for plant research. CytometryPart A 2006;69A:273–280.

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11. Barow M. Endopolyploidy in seed plants. Bioessays 2006;28:271–281.

12. Matzk F. Reproduction mode screening. In: Dolezel J, Greilhuber J, Suda J, editors.Flow Cytometry with Plant Cells: Analysis of Genes, Chromosomes and Genomes.Weinheim: Wiley-VCH; 2007. pp 131–152.

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15. Eeckhaut TGR, Werbrouck SPO, Leus LWH, Van Bockstaele EJ, Debergh PC. Chemi-cally induced polyploidization in Spathiphyllum wallisii Regel through somaticembryogenesis. Plant Cell Tissue Organ Cult 2004;78:241–246.

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19. Dolezel J, Kubal�akov�a M, Such�ankov�a P, Kov�arova P, Bartos J, Simkov�a H. Chromo-some analysis and sorting. In: Dolezel J, Greilhuber J, Suda J, editors. Flow Cytometrywith Plant Cells: Analysis of Genes, Chromosomes and Genomes. Weinheim: Wiley-VCH; 2007. pp 373–403.

20. Roberts AV. The use of bead beating to prepare suspensions of nuclei for flow cyto-metry from fresh leaves, herbarium leaves, petals and pollen. Cytometry Part A2007;71A:1039–1044.

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23. Loureiro J, Rodriguez E, Santos C, Dolezel J, Suda J. FLOWER: A plant DNA flowcytometry database (release 1, January 2008). Available at: http://flower.web.ua.pt/.

COMMENTARY

580 Plant Flow Cytometry