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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)