Laboratory of flow cytometry Průhonice

J. Suda1,2 & A. Krahulcová2 1Department of Botany, Faculty of Science, Charles University

2Institute of Botany, Academy of Sciences of the Czech Republic

E q u i p m e n tE q u i p m e n tE q u i p m e n tE q u i p m e n t

Partec PA II

HBO lamp (UV, DAPI staining)

air-cooled argon ion laser (488 nm, PI staining)

2 parameters - FL1, FL2

B a s i c d a t aB a s i c d a t aB a s i c d a t aB a s i c d a t a

Startup: September 2000

Total number of samples: 10 517

Botanical Institute: 5 509 (cca 52 %)

Department of Botany: 5 008 (cca 48 %)

Operating: ca 940 days

Out of order: ca 200 days (!)

(13.9. 02 - 23.1. 03)

(6.6. 03 - 14.8. 03)

954

2307

3841

3415

0

1000

2000

3000

4000

Nu

mb

er o

f sa

mp

les

2000 2001 2002 2003

M a t e r i a l a n a l y z e dM a t e r i a l a n a l y z e dM a t e r i a l a n a l y z e dM a t e r i a l a n a l y z e d

Other taxa

Agropyron, Agrostis, Allium, Asplenium, Aster, Betula, Butomus,

Carduus, Centaurea, Dryopteris, Echium, Lythrum, Orchis,

Phragmites, Poa, Ranunculus, Tragopogon, Vaccinium,

Valerianella

Most frequent taxa

Hieracium subg. Pilosella - 4 494

Taraxacum - 1 205

Macaronesian endemics - 1 082

Galeobdolon - 696

Oxycoccus - 467

Campanula - 418

Empetrum - 326

Reynoutria - 297

Anthoxanthum - 149

Hieracium subgen.PilosellaTaraxacum

Kanárské endemity

Galeobdolon

Oxycoccus

Campanula

Empetrum

Reynoutria

Anthoxanthum

A p p l i c a t i o n sA p p l i c a t i o n sA p p l i c a t i o n sA p p l i c a t i o n s

Nuclear DNA amount analysis

Ploidy estimationDetermination of closely related taxa

Determination of hybrid plants

Detection of rare cytotypes

Sympatric growth of various cytotypes

Breeding mode detection

Absolute nuclear DNA contentCorrelation with environmental conditions

and/or evolutionary history

Discrimination of plants with the same

chromosome number

Genome composition in allopolyploid taxa

M e t h o d sM e t h o d sM e t h o d sM e t h o d s

Nuclei isolation

(chopping in hypotonic buffer)

Staining (DAPI or PI)

Fluorescence analysis

Two step procedure Otto I + II (Otto 1990)

(with centrifugation)

Simplified Otto I + II (without centrifugation)

LB01 (Doležel et al. 1989)

Matzk (seeds) (Matzk et al. 2000)

Tris-MgCl2 (Pfosser et al. 1995)

0

80

160

240

320

400

0 250 500 750 1000

2 x 3 x 4 x

G 2G 2

G 2

R e la tiv e f lu o re sc e n c e

Num

ber

of n

ucle

i

G r a n t p r o j e c t s G r a n t p r o j e c t s G r a n t p r o j e c t s G r a n t p r o j e c t s

2002-6 (GAAV): The influence of environmental parameters on the genetic structure of young agamic complex: Hieracium subgen. Pilosella (project leader: F. Krahulec)

2002-4 (GAČR): Evolution within hybridogenous population of Tragopogon: does Czech population differ from those ones in US? (project leader: F. Krahulec)

2002 (FRVŠ): An employment of flow cytometry and isozyme analyses in the study of the genus Taraxacum (project leader: L. Záveský)

2003 (FRVŠ): Genome size in the genus Reynoutria – stable or variable? (project leader: J. Suda, researcher: Z. Hoták)

2002 (FRVŠ): A biosystematic study of the polyploid Campanula patula L. (project leader: J. Suda, researcher: H. Látalová)

2001 (FRVŠ): An employment of flow cytometry in the taxonomic study of the genus Galeodolon (project leader: J. Suda, researcher: R. Rosenbaumová)

2002-4 (GAUK): Genome size – a key to the knowledge of relationships among central European taxa from Hieracium subg. Pilosella (project leader: J. Suda, researchers: A. Krahulcová, T. Peckert, R. Rosenbaumová)

2001-3 (GAUK): A biosystematic study of the genus Empetrum (project leader: J. Suda)

M a s t e r T h e s e s M a s t e r T h e s e s M a s t e r T h e s e s M a s t e r T h e s e s

2000 - 2: R. Rosenbaumová: A taxonomic study of the genus Galeobdolon Hudson

(supervisor: J. Suda)

2000 - 2: S. Papoušková: Reproduction mode variability in the facultatively apomictic hawkweed

Hieracium rubrum (supervisor: A. Krahulcová)

2001 - ?: V. Košťálová: Hybridization between Hieracium bauhinii and H. pilosella in mixed

populations (supervisor: F. Krahulec)

2003 - ?: T. Urfus: Sources of variation in Hieracium pilosella (supervisor: F. Krahulec)

2003 - ?: T. Mandáková: Biosystematic and ecological study of various cytotypes in Aster amellus

(supervisor: Z. Münzbergová)

2003 - ?: K. Mozolová: Cytogeography and population structure in Pimpinella saxifraga

(supervisor: J. Suda)

2003 - ?: L. Grattová: Morphological and phytocoenological differentiation of the taxa in the genus

Empetrum (supervisor: J. Suda)

2003 - ?: R. Holubová: Morphological and karyological study of the Dryopetris dilatata alliance

(supervisor: J. Suda)

2003 - ?: P. Vít: Biosystematic study of the Czech endemic apomicts from the genus Sorbus

(supervisor: J. Suda)

P h. D. T h e s e s P h. D. T h e s e s P h. D. T h e s e s P h. D. T h e s e s

2000 - 3/4: J. Suda: An employment of flow cytometry in the biosystematics of vascular plants

(supervisor: J. Kirschner)

2001 - ?: T. Peckert: A taxonomic study of Hieracium sect. Echinina in Central Europe

(supervisor: J. Chrtek jun.)

2001 - ?: L. Záveský: Apomixis vs. sexuality: different approaches to the investigation in the genus

Taraxacum Wigg (supervisor: J. Štěpánek)

2001 - ?: V. Mahelka: An effect of potential hybridization between crops and weedy species: a risk of

genetic material transfer (with Elytrigia spp. as a model organism)

2002 - ?: R. Rosenbaumová: Relationships between breeding mode and population structure in

Hieracium subg. Pilosella (supervisor: F. Krahulec)

P r o s a n d C o n sP r o s a n d C o n sP r o s a n d C o n sP r o s a n d C o n s

high accuracy of measurements (when properly adjusted)

sub-optimal design of the instrument

non-professional Partec service

absolute incompetence of the Biotech (Czech representative)

P e r s p e c t i v e sP e r s p e c t i v e sP e r s p e c t i v e sP e r s p e c t i v e s

• new instrument (Cy Flow with green laser)

• utilization of forward scatter

• image cytometry

• volumetric measurements (Coulter counter)