comparison of four nuclear isolation buffers for plant dna flow cytometry
TRANSCRIPT
TECHNICAL ARTICLE
Comparison of Four Nuclear Isolation Buffers for Plant DNA Flow Cytometry
JOAO LOUREIRO 1 ELEAZAR RODRIGUEZ 1 JAROSLAV DOLEZEL2 and
CONCEICAO SANTOS1
1Laboratory of Biotechnology and Cytomics Department of Biology University of Aveiro Campus Universitario
de Santiago 3810-193 Aveiro Portugal and 2Laboratory of Molecular Cytogenetics and Cytometry Institute of
Experimental Botany Sokolovska Olomouc CZ-77200 Czech Republic
Received 15 March 2006 Returned for revision 24 April 2006 Accepted 22 May 2006 Published electronically 4 July 2006
Background and Aims DNA flow cytometry requires preparation of suspensions of intact nuclei which are stainedusing a DNA-specific fluorochrome prior to analysis Various buffer formulas were developed to preserve nuclearintegrity protect DNA from degradation and facilitate its stoichiometric staining Although nuclear isolation buffersdiffer considerably in chemical composition no systematic comparison of their performance has been made untilnow This knowledge is required to select the appropriate buffer for a given species and tissue Methods Four common lysis buffers (Galbraithrsquos LB01 Ottorsquos and TrisMgCl2) were used to prepare samplesfrom leaf tissues of seven plant species (Sedum burrito Oxalis pes-caprae Lycopersicon esculentum Celtisaustralis Pisum sativum Festuca rothmaleri and Vicia faba) The species were selected to cover a wide rangeof genome sizes (130ndash2690 pg per 2C DNA) and a variety of leaf tissue types The following parameters wereassessed forward (FS) and side (SS) light scatters fluorescence of propidium iodide-stained nuclei coefficientof variation of DNA peaks presence of debris background and the number of nuclei released from sample tissue Theexperiments were performed independently by two operators and repeated on three different days Key Results Clear differences among buffers were observed With the exception of O pes-caprae any bufferprovided acceptable results for all species LB01 and Ottorsquos were generally the best buffers with Ottorsquos bufferproviding better results in species with low DNA content Galbraithrsquos buffer led to satisfactory results andTrisMgCl2 was generally the worst although it yielded the best histograms in C australis A combined analysisof FS and SS provided a lsquofingerprintrsquo for each buffer The variation between days was more significant than thevariation between operators Conclusions Each lysis buffer tested responded to a specific problem differently and none of the buffers workedbest with all species These results expand our knowledge on nuclear isolation buffers and will facilitate selection ofthe most appropriate buffer depending on species tissue type and the presence of cytosolic compounds interferingwith DNA staining
Key words Angiosperms flow cytometry genome size lysis buffers nuclear DNA content nuclear isolation bufferspropidium iodide stoichiometric error
INTRODUCTION
Flow cytometry (FCM) was developed in the 1950s but itsapplication to plant sciences was delayed until the late1980s when it became an important technique for estima-tion of nuclear DNA content determination of DNAploidy level and cell cycle analysis (Galbraith 2004Shapiro 2004 Bennett and Leitch 2005) This delay wasmainly due to problems with the preparation ofsuspensions of intact nuclei from thick tissues consistingof cells with a rigid cell wall In his pioneering workHeller (1973) used hydrolytic enzymes to digest cell wallsand release nuclei from fixed tissues The method wastime consuming and rarely followed by others Amonglater investigators Ulrich and Ulrich (1986) Ulrich et al(1988) and Bergounioux et al (1988 1992) employed amodified approach in which cell nuclei were released afterhypotonic lysis of intact protoplasts In addition to beingtime consuming the need for intact protoplasts limited theapplication of this protocol to some species and certaintypes of tissues As an alternative Galbraith et al (1983)developed a rapid and convenient method for isolation of
plant nuclei by chopping plant tissues in a lysis bufferSince then this has been the main and most reliablemethod for nuclear isolation in plant FCM
In addition to releasing nuclei from intact cells lysisbuffers must ensure the stability of nuclei throughout theexperiment protect DNA from degradation and facilitatestoichiometric staining Since the late 1980s some labora-tories have developed their own buffer formulas Asa result about 25 different lysis buffers are knownalthough only eight are commonly used Their chemicalcomposition varies but it usually includes chromatinstabilizers (eg MgCl2 MgSO4 spermine) chelatoragents [eg ethylenediaminetetraacetic acid (EDTA)sodium citrate] to bind divalent cations which serveas nuclease cofactors inorganic salts (eg KCl NaCl)to achieve proper ionic strength organic buffers [eg3-(N-morpholino) propanesulfonic acid (MOPS) Tris-(hydroxymethyl)-aminomethane (Tris) and 4-(hydroxy-methyl)piperazine-1-ethanesulfonic acid (HEPES)] tostabilize the pH of the solution (usually set between 70and 80) and non-ionic detergents (eg Triton X-100Tween 20) to release nuclei disrupt chloroplasts asfluorescent entities remove and hinder cytoplasmatic
For correspondence E-mail jloureirobiouapt
Annals of Botany 98 679ndash689 2006
doi101093aobmcl141 available online at wwwaoboxfordjournalsorg
The Author 2006 Published by Oxford University Press on behalf of the Annals of Botany Company All rights reserved
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remnants from nuclei surface and decrease the aggrega-tion affinity of nuclei and debris (Coba de la Pena andBrown 2001 Dolezel and Bartos 2005)
Given the different chemical composition and diversityof plant tissues it may be expected that each buffer wouldperform differently This problem was exacerbated by therecent observation of the presence of cytosolic compoundsthat are released during nuclei isolation These compoundsinteract with nuclear DNA andor the fluorochromeaffecting sample quality and causing stoichiometric errorsin DNA staining (Noirot et al 2000 2003 Pinto et al2004 Loureiro et al 2006 Walker et al 2006) Loureiroet al (2006) observed that nuclei of Pisum sativum andZea mays responded differently to tannic acid a commonphenolic compound in plants when isolated in differentnuclear isolation buffers However no systematic com-parison of lysis buffers has been made until now
We set out to compare four of the most commonbuffers that differ in chemical composition Galbraithrsquosbuffer (Galbraith et al 1983) LB01 (Dolezel et al1989) Ottorsquos buffer (Otto 1992 Dolezel and Gohde1995) and TrisMgCl2 (Pfosser et al 1995) We evaluatedlight scatter and fluorescence properties of nuclei insuspension the presence of debris background and thenumber of nuclei released from sample tissue Sampleswere prepared from leaf tissue of seven plant species thatcover a wide range of genome sizes (130ndash2690 pg per 2CDNA) and whose tissues differ in structure and chemicalcomposition (Table 1) Pisum sativum Lycopersiconesculentum and Vicia faba are common DNA referencestandards for FCM Sedum burrito a species fromCrassulaceae has fleshy leaves with many reserve sub-stances Oxalis pes-caprae has an acidic cell sap (Castroet al 2005) Celtis australis releases mucilaginouscompounds after tissue homogenization (Rodriguez et al2005a) and Festuca rothmaleri has rigid leaves that aredifficult to chop The effect of instrumental drift andoperator were also evaluated The main goal of the studywas to provide data that facilitate selection of the
appropriate buffer and to propose strategies to minimizecommon problems in plant DNA flow cytometry
MATERIALS AND METHODS
Plant material
Plants of Lycopersicon esculentum lsquoStupickersquo(Solanaceae) Pisum sativum lsquoCtiradrsquo (Fabaceae) andVicia faba lsquoInovecrsquo (Fabaceae) were grown from seedsPlants of Festuca rothmaleri (Poaceae) and Oxalis pes-caprae (Oxalidaceae) were kindly provided by Prof PauloSilveira and Dr Sılvia Castro (Department of BiologyUniversity of Aveiro Portugal) respectively Plants ofSedum burrito (Crassulaceae) were obtained from Flor doCentro Horticultural Centre (Mira Portugal) All plantswere maintained in a greenhouse at 22 6 2 C with aphotoperiod of 16 h and a light intensity of 530 6 2 mmolm2 s1 Leaves of Celtis australis (Ulmaceae) werecollected directly from field-growing trees in AveiroPortugal
Sample preparation
Approximately 40ndash50 mg of young leaf tissue was usedfor sample preparation The amount of material requiredto release a sufficient number of nuclei in S burrito had tobe increased to approximately 500 mg due to the fleshynature of the leaves Nuclei suspensions were preparedaccording to Galbraith et al (1983) Four common nuclearisolation buffers (Dolezel and Bartos 2005) were used toprepare samples (Table 2) One millilitre of nucleisuspension was recovered and filtered through a 50-mmnylon filter to remove cell fragments and large debrisNuclei were stained with 50mg mL1 propidium iodide(PI) (Fluka Buchs Switzerland) and 50mg mL1 RNase(Sigma St Louis MO USA) was added to the nuclearsuspension to prevent staining of double-stranded RNASamples were incubated on ice and analysed within10 min
Flow cytometric analyses
Samples were analysed in a Coulter EPICS XL (CoulterElectronics Hialeah FL USA) flow cytometer equippedwith an air-cooled argon-ion laser tuned at 15 mW andoperating at 488 nm Fluorescence was collected through a645-nm dichroic long-pass filter and a 620-nm band-passfilter The results were acquired using the SYSTEM IIsoftware version 30 (Coulter Electronics) Prior toanalysis the instrument was checked for linearity withfluorescent beads (Coulter Electronics) and the amplifi-cation settings were kept constant throughout theexperiment
The following parameters were evaluated in eachsample forward light scatter (FS to estimate relativesize of particles) side light scatter (SS to estimate relativeoptical complexity of particles) relative fluorescenceintensity of PI-stained nuclei (FL) half peak coefficientof variation (CV) of the G0G1 peak (to estimatenuclei integrity and variation in DNA staining) a debris
T A B L E 1 Nuclear DNA content of the seven plant speciesused in this study
Species Family
NuclearDNA content(pg per 2C) Reference
Sedum burrito Crassulaceae 130 This workOxalis pes-caprae Oxalidaceae 137 Castro
et al (2005)LycopersiconesculentumlsquoStupickersquo
Solanaceae 196 Dolezelet al (1992)
Celtis australis Ulmaceae 246 Rodriguezet al (2005a)
Pisum sativum lsquoCtiradrsquo Fabaceae 909 Dolezelet al (1989)
Festuca rothmaleri Poaceae 1367 J Loureiro(unpubl data)
Vicia faba lsquoInovecrsquo Fabaceae 2690 Dolezelet al (1992)
680 Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM
background factor (DF to assess sample quality) and anuclear yield factor (YF to compare the amount of nucleiin suspension independently of the amount of leaftissue used)
CV () was calculated using the following formula
Half peak CV eth THORN
frac14 4246 middot width of peak at half the peak height
peak positioneth1THORN
DF () was calculated as follows
DF eth THORN frac14total number of particles total number of intact nuclei
total number of particlesmiddot 100
eth2THORN
DF increases as debris increasesYF (nuclei sndash1 mgndash1) was calculated using the formula
YF nucleis1mg1
frac14 total number of intact nuclei=number of seconds of run seth THORNweight of tissue mgeth THORN
eth3THORN
The flow rate was defined as low and was kept constantthroughout the experiment
The analysis was performed on three different days andby two operators (labelled here as A and B) Fivereplicates were performed per operator for each buffer andin each replicate at least 5000 nuclei were analysedHistograms of FL obtained with the best and worstperforming buffers were overlaid using WinMDI software(Trotter 2000) (Fig 1)
For S burrito nuclear genome size was estimated usingL esculentum lsquoStupickersquo (2C = 196 Dolezel et al 1992)as internal reference standard according to the followingformula
S burrito 2C nuclear DNA content pgeth THORN
frac14 S burrito G0=G1 peak mean
L esculentum G0=G1 peak meanmiddot 196 eth4THORN
Conversion of mass values into base-pair numbers wasachieved according to the factor 1 pg = 978 Mbp (Dolezelet al 2003)
Statistical analyses
Statistical analyses were performed using a three-wayANOVA (SigmaStat for Windows version 311) to assessfor differences among buffers and dates and betweenoperators When treatments were significantly different aHolmndashSidak multiple comparison test was used for pair-wise comparison Hierarchical cluster analyses wereperformed using NCSS 2004 (Hintze 2004) Dendrogramshighlighting dissimilarities among buffers and betweenoperators were obtained using FS SS FL CV BF andYF The Unweighted Pair Group Method with Arithmeticmean (UPGMA) was followed in each species as ityielded the highest co-phenetic correlation coefficient
RESULTS
With the exception of O pes-caprae for which meas-urable samples were only obtained with Ottorsquos andGalbraithrsquos buffers all buffers yielded acceptable histo-grams with all species tested In any analysis it waspossible to isolate a reasonable number of nuclei(approximately 20ndash60 nuclei per second in a low-speedconfiguration) and obtain well-defined histograms withDNA peaks with acceptable CV values (lt50 Galbraithet al 2002 Fig 1 Table 3) Table 3 indicates the bestperforming buffer(s) for each species The selectioncriteria were the highest FL and YF values and thelowest CV and DF values
Sedum burrito
This species was investigated because of its expectedsmall genome size and fleshy leaves FCM analysisrevealed the occurrence of polysomaty as demonstratedby the presence of discrete populations of nuclei withDNA contents of 2C 4C 8C 16C and higher In orderto observe a higher number of endopolyploidy
T A B L E 2 Four nuclear isolation buffers most frequently used in plant DNA flow cytometry
Buffer Composition Reference
Galbraith 45 mM MgCl2 30 mM sodium citrate 20 mM MOPS01 (vv) Triton X-100 pH 70
Galbraith et al (1983)
LB01 15 mM Tris 2 mM Na2EDTA 05 mM spermine4HCl80 mM KCl 20 mM NaCl 01 (vv) TritonX-100 pH 80dagger
Dolezel et al (1989)
Ottoz Otto I 100 mM citric acid 05 (vv) Tween 20 (pH 2ndash3)Otto II 400 mM Na2PO4
12H2O (pH 8ndash9)Otto (1992) Dolezel and Gohde (1995)
TrisMgCl2 200 mM Tris 4 mM MgCl26H2O 05 (vv)Triton X-100 pH 75
Pfosser et al (1995)
Final concentrations are givendagger The buffer formula contains 15 mM mercaptoethanol However as the other buffers were used without additives that suppress the negative effect of
phenols and other cytosolic compounds LB01 was used without mercaptoethanolz pH of the buffers is not adjusted The nuclei are isolated in Otto I buffer DNA staining is done in a mixture of Otto I and Otto II (1 2) with a final volume
of 1 mL
Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM 681
levels instrument gain was set such that the 2C peak wasapproximately on channel 100 The 2C nuclear DNAcontent was estimated as 130 6 009 pg (2C = 1271 Mbp)this is the first estimate for this species (Table 1) Com-parative analysis of the four buffers revealed that Ottorsquoswas the best in this species In general YF values were low
(the lowest values from all the test species) whereas DFvalues were high (approx 650 Table 3 Fig 1B)
Oxalis pes-caprae
Only Ottorsquos and Galbraithrsquos buffers provided acceptableresults with this species Ottorsquos was clearly and
0 75 1500
25
1023
43
3
2
2
2
2
2
2
1
1
1
1
1
PI
Peak FL CV() 1 108middot7 5middot87 2 118middot7 3middot00 3 206middot8 4middot10 4 231middot0 2middot55
TrismiddotMgCl2 bufferGreenOttorsquos bufferBlue
Peak FL CV() 1 97middot6 9middot74 2 187middot0 3middot50 3 201middot1 2middot69
TrismiddotMgCl2 bufferGreenGalbraithrsquos bufferOrangeOttorsquos bufferBlue
Peak FL CV() 1 226middot1 4middot09 2 252middot1 1middot96
TrismiddotMgCl2 bufferGreenLB01 bufferRed
Peak FL CV() 1 190middot3 2middot21 2 205middot8 3middot90
TrismiddotMgCl2 bufferGreenOttorsquos bufferBlue
Peak FL CV() 1 184middot1 2middot78 2 213middot8 3middot48
LB01 bufferRedOttorsquos bufferBlue
Peak FL CV() 1 168middot4 3middot32 2 202middot6 2middot10
Ottorsquos bufferBlueLB01 bufferRed
Peak FL CV() 1 180middot5 1middot52 2 188middot1 3middot07
Ottorsquos bufferBlueTrismiddotMgCl2 bufferGreen
0
50
75
100
125
Time (s)
Ottorsquos buffer
PI
225
0
450
0
512
128
00 PI 1023
300
Cou
nt
Cou
ntC
ount
Cou
nt
0
1024
Cou
nt
0
400
1
0
512
Cou
nt
2
1
1023PI0
512
0
Cou
nt
A B
C D
E F
G H
F I G 1 Cytogram of fluorescence intensity (PI relative channel numbers) vs time of Pisum sativum nuclei isolated with Ottorsquos buffer (A) and histograms ofrelative fluorescence intensities (BndashH) which show overlays of distributions obtained with the best and worst performing buffer for each species (B) Sedumburrito (C) Oxalis pes-caprae (D) Lycopersicon esculentum (E) Celtis australis (F) Pisum sativum (G) Festuca rothmaleri and (H) Vicia faba Relativemean channel numbers and coefficients of variation (CV ) of G0G1 peaks are given Four lysis buffers were compared LB01 (red) Galbraithrsquos (orange)
TrisMgCl2 (green) and Ottorsquos (blue)
682 Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM
TA
BL
E3
F
low
cyto
met
ric
pa
ram
eter
sa
sses
sed
inea
chsp
ecie
s
FS
(ch
ann
elu
nit
s)S
S(c
han
nel
un
its)
FL
(ch
ann
elu
nit
s)C
V(
)B
F(
)Y
F(n
ucl
eis
1m
g
1)
Sp
ecie
sB
uff
erM
ean
SD
Mea
nS
DM
ean
SD
Mea
nS
DM
ean
SD
Mea
nS
D
Sed
um
bu
rrit
oL
B0
15
76
6a
8 7
24
11 3
7a
2 4
48
11
0 9
a5
93
4 4
8a
0 4
07
69 1
5a
24 9
03
0 0
6a
0 0
04
Gal
bra
ith
41 0
6b
5 1
71
5 3
6b
2 2
99
11
2 1
a2
57
3 5
9ab
0 3
96
60 0
2a
16 6
31
0 0
5b
0 0
04
Tri
sM
gC
l 25
75
0a
8 5
56
13 3
4a
2 6
70
10
3 0
b6
19
5 6
1c
1 0
00
68 8
5a
13 8
74
0 0
5ab
0 0
04
Ott
o8 0
1c
2 1
79
2 5
8c
0 7
87
12
1 4
c3
93
3 2
7b
0 2
88
65 9
6a
6 3
30
05
ab
0 0
06
Oxa
lis
pes
-ca
pra
eL
B0
1ndash
ndashndash
ndashndash
ndashndash
ndashndash
ndashndash
ndashG
alb
rait
h1
71 3
3a
36 4
04
19 7
5a
6 9
20
17
8 5
a5
21
4 1
4a
0 7
78
7 0
5a
4 9
77
1 0
1a
0 3
64
Tri
sM
gC
l 2ndash
ndashndash
ndashndash
ndashndash
ndashndash
ndashndash
ndashO
tto
26 3
2b
4 9
33
3 9
0b
0 6
41
20
0 7
b6
24
3 0
3b
0 4
15
8 9
6a
5 9
08
0 6
2b
0 1
25
Lyc
oper
sico
nes
cule
ntu
mL
B0
11
5 4
2a
2 1
25
2 9
7a
0 4
94
25
7 9
a1
1 5
02 8
8a
0 8
80
21 2
1a
14 1
37
2 4
7a
1 5
12
Gal
bra
ith
21 5
3b
3 7
52
3 4
0a
0 7
13
20
8 5
b4
9 6
12 8
7a
0 7
12
16 8
7a
10 0
36
1 6
9ab
0 8
14
Tri
sM
gC
l 21
7 4
8a
4 9
70
4 7
6b
1 5
62
21
6 7
b2
7 2
73 7
0b
0 8
36
20 5
2a
12 9
18
1 7
2ab
1 7
45
Ott
o3 6
8c
1 5
37
1 9
5c
0 7
66
26
9 9
a1
1 1
72 1
8c
0 2
66
28 4
9b
13 2
54
0 9
1b
0 6
17
Cel
tis
au
stra
lis
LB
01
28 4
7a
4 8
47
11 4
5a
3 4
81
18
2 1
a1
3 1
33 0
2a
0 3
15
7 6
6a
3 5
65
0 5
0a
0 2
24
Gal
bra
ith
25 5
7a
1 8
42
4 5
4b
0 6
37
17
1 3
b7
76
2 8
7a
0 2
59
8 2
7a
3 4
45
0 3
5a
0 1
62
Tri
sM
gC
l 24
1 7
1b
6 9
44
16 0
8c
2 1
57
18
0 3
a1
0 4
42 8
5a
0 4
19
9 1
0b
5 0
85
0 4
9a
0 2
91
Ott
o8 2
0c
1 4
04
7 2
5b
1 7
87
19
2 8
c8
23
3 4
6b
0 4
05
23 9
6c
4 6
15
0 4
9a
0 2
00
Pis
um
sati
vum
LB
01
47 5
4a
1 5
50
10 8
7a
3 0
78
18
3 5
a5
06
2 8
1a
0 4
61
7 1
9a
2 8
23
2 3
3a
0 4
90
Gal
bra
ith
51 9
7a
3 6
24
5 2
9b
1 1
55
17
8 6
a3
36
3 0
2ab
0 4
65
6 3
4a
1 7
65
2 3
0a
0 8
76
Tri
sM
gC
l 25
9 3
7b
4 8
26
16 7
4c
2 9
14
17
7 7
a1
1 8
63 2
9b
0 5
15
6 3
9a
2 7
81
2 5
9a
0 8
94
Ott
o5 7
2c
0 9
93
4 7
9b
1 0
83
19
0 1
a6
06
1 9
4c
0 1
80
9 8
5b
2 6
68
1 1
5b
0 3
67
Fes
tuca
roth
ma
leri
LB
01
57 3
5a
1 8
25
8 5
7a
0 7
99
19
6 1
a6
07
3 2
4a
0 4
52
14 2
3ab
3 2
83
0 4
2a
0 1
82
Gal
bra
ith
61 0
3a
2 2
50
6 8
4b
0 6
49
18
2 7
b7
15
3 3
3ab
0 4
59
15 0
7a
1 9
74
0 2
7b
0 1
31
Tri
sM
gC
l 26
9 8
5a
1 6
03
17 7
2c
0 8
15
18
5 1
b8
06
3 6
6ab
0 3
12
17 3
0b
5 7
01
0 5
4a
0 1
95
Ott
o1
3 8
4b
2 8
46
8 5
5a
1 6
03
21
0 9
c6
77
3 7
6b
0 6
13
23 8
6c
12 9
92
0 1
1c
0 0
66
Vic
iafa
ba
LB
01
10
4 8
2a
6 0
21
11 6
1a
1 7
42
20
1 5
a4
45
2 4
0a
0 1
78
6 3
6a
2 3
26
0 8
7a
0 2
90
Ga
lbra
ith
11
4 5
5b
2 7
87
8 5
8b
1 3
06
19
6 9
a4
88
2 4
1a
0 1
48
5 8
0a
0 8
06
0 8
2a
0 2
35
Tri
sM
gC
l 21
15 4
5b
5 1
94
20 3
6c
1 4
43
19
1 5
a9
10
2 9
1b
0 2
72
6 8
5a
4 6
09
1 3
0b
0 3
45
Ott
o2
1 5
6c
8 7
21
14 0
5a
7 5
89
18
4 2
a1
6 9
22 2
2a
0 5
32
4 3
5a
1 7
51
0 4
5c
0 1
8
Val
ues
are
giv
enas
mea
nan
dst
and
ard
dev
iati
on
of
the
mea
n(S
D)
of
forw
ard
scat
ter
(FS
ch
ann
elu
nit
s)s
ide
scat
ter
(SS
chan
nel
un
its)
flu
ore
scen
ce(F
Lc
han
nel
un
its)
coef
fici
ento
fv
aria
tio
no
fG
0G
1D
NA
pea
k(C
V
)b
ack
gro
un
dfa
cto
r(B
F
)an
dn
ucl
eary
ield
fact
or(Y
Fn
ucl
eisndash
1m
gndash1)
Mea
ns
foll
ow
edb
yth
esa
me
lett
er(a
bo
rc)
are
no
tsta
tist
ical
lyd
iffe
ren
tacc
ord
ing
toth
em
ult
iple
com
par
ison
Ho
lmndashS
idak
test
atPlt
00
5
Buff
er(s
)th
atp
erfo
rmed
bes
tin
each
spec
ies
are
sho
wn
inb
old
typ
e
Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM 683
significantly better than Galbraithrsquos (Fig 1C) with highermean FL intensities and lower CV values (Table 3)Figure 1C also shows the histogram obtained after nuclearisolation with TrisMgCl2 buffer In this case a G0G1 peakwith an unacceptable CV value (974 ) and considerableloss of fluorescence was obtained A similar result wasobtained for nuclei isolated with LB01
Lycopersicon esculentum
Acceptable results were obtained with all four buffersTwo buffer groups with statistically significant differencesin FL were obtained Samples prepared with Galbraithrsquosand TrisMgCl2 buffers yielded lower mean FL valuesthan their counterparts prepared with LB01 and Ottorsquosbuffers (Fig 1D) The FL values were highly heterogen-eous among buffers Ottorsquos and LB01 were the bestbuffers with Ottorsquos providing lower CV values and higherFL but lower YF and higher BF than LB01 (Table 3)
Celtis australis
Low CV values (lt30 ) and low DF (lt100 ) wereobserved for this species Nevertheless it was not easy toobtain suffiecient nuclei and the second lowest YF valueswere observed in this species With regard to FL onlyLB01 and TrisMgCl2 buffers were not statistically dif-ferent with nuclei isolated from Galbraithrsquos buffer pre-senting the lowest mean FL and Ottorsquos the highest meanFL All parameters combined TrisMgCl2 and LB01 werethe best buffers as nuclei in TrisMgCl2 presented thelowest CV values and similar FL intensity and YF valuesas in LB01 (Table 3 Fig 1E)
Pisum sativum
In this species all buffers performed reasonably wellThe lowest FL intensities were obtained for nucleiisolated with TrisMgCl2 although no statistically signi-ficant differences were observed among the tested buffersThe best buffer for this species was Ottorsquos (Table 3Fig 1F) Among the investigated species P sativum wasthe one with the highest YF
Festuca rothmaleri
With the exception of CV values and YF overall resultsfor this species were satisfying with all four buffers testedNo statistically significant differences were found regard-ing the FL of nuclei isolated in TrisMgCl2 or Galbraithrsquosbuffers as nuclei from both buffers presented low FLvalues The best buffer for this species was LB01 (Table 3Fig 1G)
Vicia faba
This species gave the lowest CV and DF values amongthose tested Generally the results were very similar tothose obtained for P sativum FL was similar for all thebuffers and no statistically significant differences wereobserved Interestingly Vicia faba was the only species
for which Ottorsquos was not the buffer with the highest FL(Fig 1H) Despite low CV values of DNA peaks thisbuffer gave the worst results with the G0G1 peak shiftedtowards the lower channels This was due to fluorescenceinstability which decreased over time In all other speciesand with the remaining buffers FL was stable after 10 minof incubation with PI (Fig 1A) Results obtained withLB01 and Galbraithrsquos buffers were similar and the best forthis species (Table 3)
Analysis of FS and SS
Generally FS and SS values differed considerablyamong the test buffers Nevertheless in most of thespecies analysis of scatter parameters revealed that two ofthe four buffers were more similar than the others and nostatistically significant differences were observed betweenthem Interestingly and with the exception of S burritobuffers that had similar FS mean values were not thosethat had similar SS values This can be seen on cytogramsof FS vs SS obtained in P sativum (Fig 2) In thisspecies and for FS no statistically significant differenceswere observed between LB01 and Galbraithrsquos buffers forSS no difference was observed for Galbraithrsquos and Ottorsquosbuffers Interestingly simultaneous analysis of FS and SSresulted in a species-specific pattern that could be used asa fingerprint of each buffer
Effect of operator and date of analysis
In most cases no statistically significant differenceswere observed between operators and dates of analysisOperators provided more homogeneous results than datesin the former statistically significant differences wereobserved only for BF and YF in more than one speciesSignificant differences between the dates of analysis weredetected in some species for FS SS CV and BF Withregard to FL one of the most important parameters inFCM analyses significant among-day differences weredetected only in C australis and differences betweenoperators occurred only in L esculentum The two speciesmore susceptible to differences were L esculentum andV faba (Table 4)
Hierarchical cluster analysis
With the exception of the results for S burrito the fourbuffers fell into two highly dissimilar and consistentclusters one with Ottorsquos buffer and the other with theremaining buffers In S burrito (Fig 3A) one cluster wasformed with Ottorsquos and Galbraithrsquos buffers while theother comprised TrisMgCl2 and LB01 buffers InC australis (Fig 3C) Galbraithrsquos buffer was more similarto LB01 and TrisMgCl2 buffers than to Ottorsquos buffer inaddition lsquoTrisMgCl2 Arsquo was more similar to LB01 thanto lsquoTrisMgCl2 Brsquo In P sativum lsquoGalbraithrsquos Arsquo wasmore related to LB01 than to lsquoGalbraithrsquos Brsquo (Fig 3D)whereas for F rothmaleri lsquoLB01 Arsquo was more similar toGalbraithrsquos than to lsquoLB01 Brsquo (Fig 3E) In L esculentumtwo groups were formed from the second clusterTrisMgCl2 and lsquoGalbraith Arsquo formed one group and
684 Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM
FS log
C
SS lo
g
1 10 100 10000middot1
0middot1
1
10
100
1000
Meanchannel
FSSS 18middot2
TrisMgCl2
37middot8735middot6960middot7()
FPCV
Meanchannel
FSSS
LB01ASS
log
0middot1
1
10
100
1000
10middot045middot4
30middot0226middot09()
FPCV
FSSS 3middot1 37middot24
29middot936middot0
Ottorsquos
FS log
D
1 10 100 10000middot1
Meanchannel ()
FPCV
FS 52middot1 28middot42SS 5middot2 39middot04
GalbraithsB
Meanchannel ()
FPCV
F I G 2 Cytograms of forward scatter (logarithmic scale FS log) vs side scatter (logarithmic scale SS log) obtained after the analysis of Pisum sativum nucleiisolated with four lysis buffers (A) LB01 (B) Galbraithrsquos (C) TrisMgCl2 and (D) Ottorsquos The mean channel number and full peak coefficient of variation
(FPCV ) are given for both parameters Note that the patterns of distributions are characteristic for each buffer
T A B L E 4 Three-way ANOVA analysis of the dates (D) and operators (O) for the parameters evaluated on each species
FS SS FL CV BF YF
Species D O D O D O D O D O D O
Sedum burrito ns ns ns ns ns ns ns ns s ns ns nsOxalis pes-caprae s ns s ns ns ns s ns ns ns ns nsLycopersicon esculentum s ns ns ns ns s s ns s s s sCeltis australis ns ns ns ns s ns ns ns s ns ns nsPisum sativum ns ns s ns ns ns s ns s ns ns nsFestuca rothmaleri s ns ns ns ns ns ns ns ns s ns nsVicia faba ns s s s ns ns ns ns ns ns s s
Forward scatter FS side scatter SS fluorescence FL coefficient of variation of the G0G1 DNA peak CV background factor BF and nuclearyield factor YF
ns not significantly different s significantly different at P lt 005
Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM 685
LB01 and lsquoGalbraith Brsquo formed the other (Fig 3B) InV faba (Fig 3F) LB01 and Galbraithrsquos formed one groupowing to greater similarities between operators thanwithin each buffer As previously stated in O pes-capraeonly two buffers (Ottorsquos and Galbraithrsquos) providedacceptable results with considerable dissimilaritiesbetween them (data not shown)
DISCUSSION
Four nuclear isolation buffers were used with a setof species that were chosen to represent different types
of leaf tissues and different nuclear DNA content(130ndash2690 pg per 2C DNA) As expected popularDNA reference standards (P sativum V faba andL esculentum) were easy to work with Neverthelessnot all buffers worked well with L esculentumpossibly owing to the presence of cytosolic compoundsHowever as the aim of the study was to compare theperformance of basic buffer formulas the use of additivesthat could counteract the negative effects of cytosol wasavoided
Overall the best results were obtained with P sativumAs its 2C nuclear DNA content is in the middle of theknown range of genome sizes in plants this observation
A
C
E
B
D
F
Dissimilarity Dissimilarity
2middot0 1middot5 1middot0 0middot5 0middot02middot0 1middot5 1middot0 0middot5 0middot0
2middot0 1middot5 1middot0 0middot5 0middot0
2middot0 1middot5 1middot0 0middot5 0middot0 2middot0 1middot5 1middot0 0middot5 0middot0
2middot0 1middot5 1middot0 0middot5 0middot0
LB01A
LB01B
LB01A
LB01B
Ottos B
Ottos A
Galbraiths B
Galbraiths A
TrisMgCl2 B
TrisMgCl2 A
Ottos B
Ottos A
Galbraiths B
Galbraiths A
Galbraiths B
Galbraiths A
TrisMgCl2 B
TrisMgCl2 A
LB01A
LB01B
Ottos B
Ottos A
TrisMgCl2 B
TrisMgCl2 A
LB01A
LB01B
LB01A
LB01B
Ottos B
Ottos A
Galbraiths A
Galbraiths B
TrisMgCl2 A
TrisMgCl2 B
Ottos B
Ottos A
Galbraiths B
Galbraiths A
Galbraiths B
Galbraiths A
TrisMgCl2 B
TrisMgCl2 A
LB01A
LB01B
Ottos B
Ottos A
TrisMgCl2 B
TrisMgCl2 A
F I G 3 Dendrograms obtained after hierarchical cluster analysis of the following species (A) Sedum burrito (B) Lycopersicon esculentum (C) Celtisaustralis (D) Pisum sativum (E) Festuca rothmaleri (F) Vicia faba according to the parameters FS SS FL CV BF and YF With the exception of S burrito
the four buffers fell into two highly dissimilar clusters of the same buffers
686 Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM
underlines its position as one of the best standards forplant DNA FCM By contrast Sedum burrito was themost difficult species to analyse due to low DNA contentoccurrence of polysomaty and high leaf water contentwhich hampered sample preparation and analysis More-over its tissues may contain tannins (J Greilhuber perscomm 2006)
In O pes-caprae cytosol of which is highly acidic(pH lt 30) measurable samples could be prepared usingonly Ottorsquos and Galbraithrsquos buffers with Ottorsquos beinghighly superior This is in accordance with the results ofEmshwiller (2002) who analysed ploidy levels in OxalisAfter testing LB01 MgSO4 and Ottorsquos buffers sheobtained measurable samples only with Ottorsquos The formertwo buffers failed presumably as a result of the acidic cellsap which may have exceeded the buffering capacity ofLB01 and MgSO4
Celtis australis was the only woody plant speciesincluded in the present study and was chosen because ofthe presence of mucilaginous compounds (Rodriguez et al2005a) which increase sample viscosity restrain nucleirelease and cause their clumping Interestingly this wasthe only species for which TrisMgCl2 was the bestperforming buffer This was probably because of a higherconcentration of the non-ionic detergent which sup-pressed the effect of mucilaginous compounds Leaftissues of F rothmaleri were particularly hard anddifficult to chop In addition preliminary experimentswith this species revealed the presence of cytosoliccompounds which would be expected to interfere withDNA staining However given the pattern of FS and SSobtained the so-called lsquotannic acid effectrsquo (Loureiro et al2006) was absent indicating that these compounds werereleased at low concentration or not at all
In order to compare the performance of nuclear isolationbuffers a set of parameters was carefully selected toevaluate sample quality Furthermore stability of fluores-cence and light scatter properties of isolated nuclei overtime were confirmed Among the parameters chosen thecoefficient of variation of DNA peaks (CV) is of majorimportance Galbraith et al (2002) considered 50 asthe maximum acceptable CV value in plant DNA flowcytometry With the exception of TrisMgCl2 buffer whenused with S burrito all mean CV values obtained hereinwere below this limit Ideally a nuclei sample should befree of cell and tissue debris Emshwiller (2002) noted acorrelation between CV and background noise In thepresent study this correlation was found only in somespecies (eg F rothmaleri) Rather the results heresuggest that the extent of background debris is determinedby the buffer itself For example a higher detergentconcentration in a buffer could lead to chloroplast lysisand consequently decrease the number of fluorescentparticles contributing to debris signals (Coba de la Penaand Brown 2001)
LB01 buffer provided very good results with theexception of O pes-caprae and S burrito low CV highFL and YF values were obtained with this buffer Highnuclei FL intensities obtained with this buffer weredefinitely an advantage as compared with TrisMgCl2 and
Galbraithrsquos buffers Galbraithrsquos buffer seems well bal-anced as acceptable results were achieved in all speciesSurprisingly the buffer gave reasonable results also withO pes-caprae which is characterized by highly acidiccytosol The presence of MOPS in the buffer may shedlight on these findings as it has a pKa of 72 and a betterbuffering capacity than TRIS with a pKa of 81 Adisadvantage of this buffer was the low fluorescenceintensity of nuclei Collectively TrisMgCl2 was the worstperforming buffer Nevertheless it provided the bestresults for C australis This may have been due to thehigher concentration of non-ionic detergent (Table 1)which counteracted the agglutinating effect of mucilagin-ous compounds and decreased sample viscosity As withGalbraithrsquos buffer low FL values were obtained withTrisMgCl2
Ottorsquos buffer is unique in that sample preparationinvolves two steps The results obtained with Ottorsquos bufferwere excellent in many species especially those withlower nuclear DNA content Dolezel and Bartos (2005)highlighted the quality of this buffer which is known toprovide DNA content histograms with unequalled resolu-tion One explanation for this may be that citric acidimproves chromatin accessibility and lsquohomogenizesrsquo chro-matin structure eliminating differences in staining intens-ity among nuclei with the same DNA content but differentchromatin state This could explain the highest FL andlowest CV values as observed here Another characteristicof this buffer was that light scatter values (especially FS)were significantly lower than those of other buffers Thismay be explained by the action of citric acid whichcauses nuclei fixation (Dolezel and Bartos 2005) It isnoteworthy that the pattern of FS vs SS distribution wassimilar to that obtained when analysing nuclei fixed withformaldehyde (Rodriguez et al 2005b) Althoughproviding excellent results this buffer gave the highestBF and lowest YF values The former can be explained bynuclei instability after the addition of Otto II After thisstep nuclei deteriorate rapidly in some species (J Dolezelunpubl res) The lowest YF values could be due to thedetergent (Tween 20) which is weaker than Triton X-100thus having a lower capacity to release nuclei
An important consequence of the observation ofdifferent fluorescence values obtained with the differentbuffers is that different fluorescence ratios may beexpected with the same species pair if the samples areprepared in different buffers In fact Dolezel et al (1998)observed differences in peak ratios obtained in differentlaboratories Further studies are needed to confirm thisobservation as it may have important consequences forestimation of genome size
The present results showed that well-defined popula-tions of nuclei could be observed on cytograms of FS vsSS With the exception of S burrito for which LB01 andTrisMgCl2 buffers did not present statistically significantdifferences for both parameters the analysis of FS and SSprovided a fingerprint pattern for each buffer Loureiroet al (2006) showed that these parameters were sensitiveto the presence of tannic acid a cytosolic compoundcommon in plants and recommended the analysis of light
Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM 687
scatter to verify suitability of particular samples for plantDNA FCM
As nuclei samples are prepared manually it wasimportant to assess the effect of date of analysis andoperator on sample quality variation Date was found to bemore significant than the effect of different operatorsespecially for FS SS CV and DF YF which depends onthe way the sample is chopped and hence on the operatordid not vary It was expected that FL which is a primarysource of data in FCM analysis would not depend on theoperator or the day of chopping This was confirmed in allspecies except L esculentum and C australis In theformer species a significant variation was obtainedbetween operators These differences could be explainedby variable results obtained with Galbraithrsquos buffer InC australis significant differences were obtained amongdates In this case significant differences were due toresults obtained on one single day Emshwiller (2002)found significant differences when multiple preparationsfrom the same plant were run on different days Thesedifferences and those found in the present study wereprobably due to instrument drift (Kudo and Kimura2001) To avoid this type of error several authors haverecommended that each measurement be repeated at leastthree times on three different days (Suda 2004 Dolezeland Bartos 2005)
This is the first study that has systematically comparednuclear isolation buffers for DNA FCM The results showthat none of the buffers works best with all species andstatistically significant differences in sample quality wereobserved among the four buffers The results obtainedwith different species and contrasting types of leaf tissuescan serve as guidelines in buffer selection Nevertheless itis recommend that a range of buffers be tested whenworking with a new species and tissue type Once the bestbuffer has been identified additives should be tested ifrequired to suppress negative effects of phenols and othercytosolic compounds
ACKNOWLEDGEMENTS
We are grateful to Prof Johann Greilhuber and Dr JanSuda for critical reading of the manuscript This work wassupported by FCT project ref POCTIAGR606722004JL was supported by the Fellowship FCTBD90032002
LITERATURE CITEDBennett MD Leitch I 2005 Genome size evolution in plants In
Gregory T ed The evolution of the genome London ElsevierAcademic Press 89ndash162
Bergounioux C Perennes C Brown SC Gadal P 1988 Cytometricanalysis of growth regulator-dependent transcription and cell cycleprogression in Petunia protoplast cultures Planta 175 500ndash505
Bergounioux C Brown SC Petit PX 1992 Flow cytometry and plantprotoplast cell biology Physiologia Plantarum 85 374ndash386
Castro S Loureiro J Santos C Ayensa G Navarro L 2005 Differentploidy levels could explain reproductive matter of invasive Oxalispes-caprae L in Mediterranean regions In Brunel S ed Invasiveplants in Mediterranean type regions of the world StrasbourgCedex France Council of Europe Publishing 235
Coba de la Pena T Brown SC 2001 Cytometry and fluorimetryIn Hawes C Satiat-Jeunemaıtre B eds Plant cell biology
a practical approach New York Oxford University Press85ndash106
Dolezel J Bartos J 2005 Plant DNA flow cytometry and estimation ofnuclear genome size Annals of Botany 95 99ndash110
Dolezel J Gohde W 1995 Sex determination in dioecious plantsMelandrium album and M rubrum using high-resolution flowcytometry Cytometry 19 103ndash106
Dolezel J Binarova P Lucretti S 1989 Analysis of nuclear DNAcontent in plant cells by flow cytometry Biologia Plantarum 31113ndash120
Dolezel J Sgorbati S Lucretti S 1992 Comparison of three DNAfluorochromes for flow cytometric estimation of nuclear DNAcontent in plants Physiologia Plantarum 85 625ndash631
Dolezel J Greilhuber J Lucretti S Meister A Lysak M Nardi LObermayer R 1998 Plant genome size estimation byflow cytometry inter-laboratory comparison Annals of Botany 8217ndash26
Dolezel J Bartos J Voglmayr H Greilhuber J 2003 Nuclear DNAcontent and genome size of trout and human Cytometry 51A127ndash128
Emshwiller E 2002 Ploidy levels among species in the lsquoOxalis tuberosaAlliancersquo as inferred by flow cytometry Annals of Botany 89741ndash753
Galbraith DW 2004 Cytometry and plant sciences a personalretrospective Cytometry 58A 37ndash44
Galbraith DW Harkins KR Maddox JM Ayres NM Sharma DPFiroozabady E 1983 Rapid flow cytometric analysis of the cell-cycle in intact plant-tissues Science 220 1049ndash1051
Galbraith DW Lambert GM Macas J Dolezel J 2002 Analysis ofnuclear DNA content and ploidy in higher plants In Robinson JPDarzynkiewicz Z Dean PN Dressler LG Rabinovitch PS StewartCV Tanke HJ Wheeless LL eds Current protocols in cytometryNew York John Wiley amp Sons 761ndash7622
Heller FO 1973 DNS-Bestimmung an Keimwurzeln von Vicia faba Lmit Hilfe der Impulscytophotometrie Bericht der DeutschenBotanischen Gesellschaft 86 437ndash441
Hintze J 2004 NCSS and PASS Kaysville Utah Number CruncherStatistical Systems
Kudo N Kimura Y 2001 Flow cytometric evidence for endopolyploidyin seedlings of some Brassica species Theoretical and AppliedGenetics 102 104ndash110
Loureiro J Rodriguez E Dolezel J Santos C 2006 Flow cytometricand microscopic analysis of the effect of tannic acid on plantnuclei and estimation of DNA content Annals of Botany 98515ndash527
Noirot M Barre P Louarn J Duperray C Hamon S 2000 Nucleusndashcytosol interactionsmdashA source of stoichiometric error in flowcytometric estimation of nuclear DNA content in plants Annals ofBotany 86 309ndash316
Noirot M Barre P Duperray C Louarn J Hamon S 2003 Effects ofcaffeine and chlorogenic acid on propidium iodide accessibility toDNA consequences on genome size evaluation in coffee treeAnnals of Botany 92 259ndash264
Otto F 1992 Preparation and staining of cells for high-resolution DNAanalysis In Radbruch A ed Flow cytometry and cell sortingBerlin Springer-Verlag 101ndash104
Pfosser M Amon A Lelley T Heberle-Bors E 1995 Evaluation ofsensitivity of flow cytometry in detecting aneuploidy in wheat usingdisomic and ditelosomic wheat-rye addition lines Cytometry 21387ndash393
Pinto G Loureiro J Lopes T Santos C 2004 Analysis of the geneticstability of Eucalyptus globulus Labill somatic embryos by flowcytometry Theoretical and Applied Genetics 109 580ndash587
Rodriguez E Gomes A Loureiro J Dolezel J Santos C 2005aEstimation of the genome size of the Iberian Peninsula Ulmaceae In9th Iberian Congress of Cytometry Book of Abstracts PortoPortugal 37
Rodriguez E Loureiro J Dolezel J Santos C 2005b The adequacy ofusing formaldehyde fixation for nuclear DNA content analyses ofplant material In 9th Iberian Congress of Cytometry Book ofAbstracts Porto Portugal PO120-PB
Shapiro H 2004 Practical flow cytometry 4th edn New YorkWiley-Liss
688 Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM
Suda J 2004 An employment of flow cytometry into plant biosystematicsPhD thesis Charles University Czech Republic Available at httpwwwibotcasczfcmsudapresentationdisertationpdf
Trotter J 2000 WinMDI version 28 Available at httpfacsscrippsedusoftwarehtml
Ulrich I Ulrich W 1986 Flow cytometric DNA analysis of plantprotoplasts with DAPI Zeitschrift fur Naturforschung 411052ndash1056
Ulrich I Fritz B Ulrich W 1988 Application of DNA fluorochromes forflow cytometric DNA analysis of plant protoplasts Plant Science 55151ndash158
Walker D Monino I Correal E 2006 Genome size inBituminaria bituminosa (L) CH Stirton (Fabaceae) populationsseparation of lsquotruersquo differences from environmental effects on DNAdetermination Environmental and Experimental Botany 55258ndash265
Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM 689
remnants from nuclei surface and decrease the aggrega-tion affinity of nuclei and debris (Coba de la Pena andBrown 2001 Dolezel and Bartos 2005)
Given the different chemical composition and diversityof plant tissues it may be expected that each buffer wouldperform differently This problem was exacerbated by therecent observation of the presence of cytosolic compoundsthat are released during nuclei isolation These compoundsinteract with nuclear DNA andor the fluorochromeaffecting sample quality and causing stoichiometric errorsin DNA staining (Noirot et al 2000 2003 Pinto et al2004 Loureiro et al 2006 Walker et al 2006) Loureiroet al (2006) observed that nuclei of Pisum sativum andZea mays responded differently to tannic acid a commonphenolic compound in plants when isolated in differentnuclear isolation buffers However no systematic com-parison of lysis buffers has been made until now
We set out to compare four of the most commonbuffers that differ in chemical composition Galbraithrsquosbuffer (Galbraith et al 1983) LB01 (Dolezel et al1989) Ottorsquos buffer (Otto 1992 Dolezel and Gohde1995) and TrisMgCl2 (Pfosser et al 1995) We evaluatedlight scatter and fluorescence properties of nuclei insuspension the presence of debris background and thenumber of nuclei released from sample tissue Sampleswere prepared from leaf tissue of seven plant species thatcover a wide range of genome sizes (130ndash2690 pg per 2CDNA) and whose tissues differ in structure and chemicalcomposition (Table 1) Pisum sativum Lycopersiconesculentum and Vicia faba are common DNA referencestandards for FCM Sedum burrito a species fromCrassulaceae has fleshy leaves with many reserve sub-stances Oxalis pes-caprae has an acidic cell sap (Castroet al 2005) Celtis australis releases mucilaginouscompounds after tissue homogenization (Rodriguez et al2005a) and Festuca rothmaleri has rigid leaves that aredifficult to chop The effect of instrumental drift andoperator were also evaluated The main goal of the studywas to provide data that facilitate selection of the
appropriate buffer and to propose strategies to minimizecommon problems in plant DNA flow cytometry
MATERIALS AND METHODS
Plant material
Plants of Lycopersicon esculentum lsquoStupickersquo(Solanaceae) Pisum sativum lsquoCtiradrsquo (Fabaceae) andVicia faba lsquoInovecrsquo (Fabaceae) were grown from seedsPlants of Festuca rothmaleri (Poaceae) and Oxalis pes-caprae (Oxalidaceae) were kindly provided by Prof PauloSilveira and Dr Sılvia Castro (Department of BiologyUniversity of Aveiro Portugal) respectively Plants ofSedum burrito (Crassulaceae) were obtained from Flor doCentro Horticultural Centre (Mira Portugal) All plantswere maintained in a greenhouse at 22 6 2 C with aphotoperiod of 16 h and a light intensity of 530 6 2 mmolm2 s1 Leaves of Celtis australis (Ulmaceae) werecollected directly from field-growing trees in AveiroPortugal
Sample preparation
Approximately 40ndash50 mg of young leaf tissue was usedfor sample preparation The amount of material requiredto release a sufficient number of nuclei in S burrito had tobe increased to approximately 500 mg due to the fleshynature of the leaves Nuclei suspensions were preparedaccording to Galbraith et al (1983) Four common nuclearisolation buffers (Dolezel and Bartos 2005) were used toprepare samples (Table 2) One millilitre of nucleisuspension was recovered and filtered through a 50-mmnylon filter to remove cell fragments and large debrisNuclei were stained with 50mg mL1 propidium iodide(PI) (Fluka Buchs Switzerland) and 50mg mL1 RNase(Sigma St Louis MO USA) was added to the nuclearsuspension to prevent staining of double-stranded RNASamples were incubated on ice and analysed within10 min
Flow cytometric analyses
Samples were analysed in a Coulter EPICS XL (CoulterElectronics Hialeah FL USA) flow cytometer equippedwith an air-cooled argon-ion laser tuned at 15 mW andoperating at 488 nm Fluorescence was collected through a645-nm dichroic long-pass filter and a 620-nm band-passfilter The results were acquired using the SYSTEM IIsoftware version 30 (Coulter Electronics) Prior toanalysis the instrument was checked for linearity withfluorescent beads (Coulter Electronics) and the amplifi-cation settings were kept constant throughout theexperiment
The following parameters were evaluated in eachsample forward light scatter (FS to estimate relativesize of particles) side light scatter (SS to estimate relativeoptical complexity of particles) relative fluorescenceintensity of PI-stained nuclei (FL) half peak coefficientof variation (CV) of the G0G1 peak (to estimatenuclei integrity and variation in DNA staining) a debris
T A B L E 1 Nuclear DNA content of the seven plant speciesused in this study
Species Family
NuclearDNA content(pg per 2C) Reference
Sedum burrito Crassulaceae 130 This workOxalis pes-caprae Oxalidaceae 137 Castro
et al (2005)LycopersiconesculentumlsquoStupickersquo
Solanaceae 196 Dolezelet al (1992)
Celtis australis Ulmaceae 246 Rodriguezet al (2005a)
Pisum sativum lsquoCtiradrsquo Fabaceae 909 Dolezelet al (1989)
Festuca rothmaleri Poaceae 1367 J Loureiro(unpubl data)
Vicia faba lsquoInovecrsquo Fabaceae 2690 Dolezelet al (1992)
680 Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM
background factor (DF to assess sample quality) and anuclear yield factor (YF to compare the amount of nucleiin suspension independently of the amount of leaftissue used)
CV () was calculated using the following formula
Half peak CV eth THORN
frac14 4246 middot width of peak at half the peak height
peak positioneth1THORN
DF () was calculated as follows
DF eth THORN frac14total number of particles total number of intact nuclei
total number of particlesmiddot 100
eth2THORN
DF increases as debris increasesYF (nuclei sndash1 mgndash1) was calculated using the formula
YF nucleis1mg1
frac14 total number of intact nuclei=number of seconds of run seth THORNweight of tissue mgeth THORN
eth3THORN
The flow rate was defined as low and was kept constantthroughout the experiment
The analysis was performed on three different days andby two operators (labelled here as A and B) Fivereplicates were performed per operator for each buffer andin each replicate at least 5000 nuclei were analysedHistograms of FL obtained with the best and worstperforming buffers were overlaid using WinMDI software(Trotter 2000) (Fig 1)
For S burrito nuclear genome size was estimated usingL esculentum lsquoStupickersquo (2C = 196 Dolezel et al 1992)as internal reference standard according to the followingformula
S burrito 2C nuclear DNA content pgeth THORN
frac14 S burrito G0=G1 peak mean
L esculentum G0=G1 peak meanmiddot 196 eth4THORN
Conversion of mass values into base-pair numbers wasachieved according to the factor 1 pg = 978 Mbp (Dolezelet al 2003)
Statistical analyses
Statistical analyses were performed using a three-wayANOVA (SigmaStat for Windows version 311) to assessfor differences among buffers and dates and betweenoperators When treatments were significantly different aHolmndashSidak multiple comparison test was used for pair-wise comparison Hierarchical cluster analyses wereperformed using NCSS 2004 (Hintze 2004) Dendrogramshighlighting dissimilarities among buffers and betweenoperators were obtained using FS SS FL CV BF andYF The Unweighted Pair Group Method with Arithmeticmean (UPGMA) was followed in each species as ityielded the highest co-phenetic correlation coefficient
RESULTS
With the exception of O pes-caprae for which meas-urable samples were only obtained with Ottorsquos andGalbraithrsquos buffers all buffers yielded acceptable histo-grams with all species tested In any analysis it waspossible to isolate a reasonable number of nuclei(approximately 20ndash60 nuclei per second in a low-speedconfiguration) and obtain well-defined histograms withDNA peaks with acceptable CV values (lt50 Galbraithet al 2002 Fig 1 Table 3) Table 3 indicates the bestperforming buffer(s) for each species The selectioncriteria were the highest FL and YF values and thelowest CV and DF values
Sedum burrito
This species was investigated because of its expectedsmall genome size and fleshy leaves FCM analysisrevealed the occurrence of polysomaty as demonstratedby the presence of discrete populations of nuclei withDNA contents of 2C 4C 8C 16C and higher In orderto observe a higher number of endopolyploidy
T A B L E 2 Four nuclear isolation buffers most frequently used in plant DNA flow cytometry
Buffer Composition Reference
Galbraith 45 mM MgCl2 30 mM sodium citrate 20 mM MOPS01 (vv) Triton X-100 pH 70
Galbraith et al (1983)
LB01 15 mM Tris 2 mM Na2EDTA 05 mM spermine4HCl80 mM KCl 20 mM NaCl 01 (vv) TritonX-100 pH 80dagger
Dolezel et al (1989)
Ottoz Otto I 100 mM citric acid 05 (vv) Tween 20 (pH 2ndash3)Otto II 400 mM Na2PO4
12H2O (pH 8ndash9)Otto (1992) Dolezel and Gohde (1995)
TrisMgCl2 200 mM Tris 4 mM MgCl26H2O 05 (vv)Triton X-100 pH 75
Pfosser et al (1995)
Final concentrations are givendagger The buffer formula contains 15 mM mercaptoethanol However as the other buffers were used without additives that suppress the negative effect of
phenols and other cytosolic compounds LB01 was used without mercaptoethanolz pH of the buffers is not adjusted The nuclei are isolated in Otto I buffer DNA staining is done in a mixture of Otto I and Otto II (1 2) with a final volume
of 1 mL
Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM 681
levels instrument gain was set such that the 2C peak wasapproximately on channel 100 The 2C nuclear DNAcontent was estimated as 130 6 009 pg (2C = 1271 Mbp)this is the first estimate for this species (Table 1) Com-parative analysis of the four buffers revealed that Ottorsquoswas the best in this species In general YF values were low
(the lowest values from all the test species) whereas DFvalues were high (approx 650 Table 3 Fig 1B)
Oxalis pes-caprae
Only Ottorsquos and Galbraithrsquos buffers provided acceptableresults with this species Ottorsquos was clearly and
0 75 1500
25
1023
43
3
2
2
2
2
2
2
1
1
1
1
1
PI
Peak FL CV() 1 108middot7 5middot87 2 118middot7 3middot00 3 206middot8 4middot10 4 231middot0 2middot55
TrismiddotMgCl2 bufferGreenOttorsquos bufferBlue
Peak FL CV() 1 97middot6 9middot74 2 187middot0 3middot50 3 201middot1 2middot69
TrismiddotMgCl2 bufferGreenGalbraithrsquos bufferOrangeOttorsquos bufferBlue
Peak FL CV() 1 226middot1 4middot09 2 252middot1 1middot96
TrismiddotMgCl2 bufferGreenLB01 bufferRed
Peak FL CV() 1 190middot3 2middot21 2 205middot8 3middot90
TrismiddotMgCl2 bufferGreenOttorsquos bufferBlue
Peak FL CV() 1 184middot1 2middot78 2 213middot8 3middot48
LB01 bufferRedOttorsquos bufferBlue
Peak FL CV() 1 168middot4 3middot32 2 202middot6 2middot10
Ottorsquos bufferBlueLB01 bufferRed
Peak FL CV() 1 180middot5 1middot52 2 188middot1 3middot07
Ottorsquos bufferBlueTrismiddotMgCl2 bufferGreen
0
50
75
100
125
Time (s)
Ottorsquos buffer
PI
225
0
450
0
512
128
00 PI 1023
300
Cou
nt
Cou
ntC
ount
Cou
nt
0
1024
Cou
nt
0
400
1
0
512
Cou
nt
2
1
1023PI0
512
0
Cou
nt
A B
C D
E F
G H
F I G 1 Cytogram of fluorescence intensity (PI relative channel numbers) vs time of Pisum sativum nuclei isolated with Ottorsquos buffer (A) and histograms ofrelative fluorescence intensities (BndashH) which show overlays of distributions obtained with the best and worst performing buffer for each species (B) Sedumburrito (C) Oxalis pes-caprae (D) Lycopersicon esculentum (E) Celtis australis (F) Pisum sativum (G) Festuca rothmaleri and (H) Vicia faba Relativemean channel numbers and coefficients of variation (CV ) of G0G1 peaks are given Four lysis buffers were compared LB01 (red) Galbraithrsquos (orange)
TrisMgCl2 (green) and Ottorsquos (blue)
682 Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM
TA
BL
E3
F
low
cyto
met
ric
pa
ram
eter
sa
sses
sed
inea
chsp
ecie
s
FS
(ch
ann
elu
nit
s)S
S(c
han
nel
un
its)
FL
(ch
ann
elu
nit
s)C
V(
)B
F(
)Y
F(n
ucl
eis
1m
g
1)
Sp
ecie
sB
uff
erM
ean
SD
Mea
nS
DM
ean
SD
Mea
nS
DM
ean
SD
Mea
nS
D
Sed
um
bu
rrit
oL
B0
15
76
6a
8 7
24
11 3
7a
2 4
48
11
0 9
a5
93
4 4
8a
0 4
07
69 1
5a
24 9
03
0 0
6a
0 0
04
Gal
bra
ith
41 0
6b
5 1
71
5 3
6b
2 2
99
11
2 1
a2
57
3 5
9ab
0 3
96
60 0
2a
16 6
31
0 0
5b
0 0
04
Tri
sM
gC
l 25
75
0a
8 5
56
13 3
4a
2 6
70
10
3 0
b6
19
5 6
1c
1 0
00
68 8
5a
13 8
74
0 0
5ab
0 0
04
Ott
o8 0
1c
2 1
79
2 5
8c
0 7
87
12
1 4
c3
93
3 2
7b
0 2
88
65 9
6a
6 3
30
05
ab
0 0
06
Oxa
lis
pes
-ca
pra
eL
B0
1ndash
ndashndash
ndashndash
ndashndash
ndashndash
ndashndash
ndashG
alb
rait
h1
71 3
3a
36 4
04
19 7
5a
6 9
20
17
8 5
a5
21
4 1
4a
0 7
78
7 0
5a
4 9
77
1 0
1a
0 3
64
Tri
sM
gC
l 2ndash
ndashndash
ndashndash
ndashndash
ndashndash
ndashndash
ndashO
tto
26 3
2b
4 9
33
3 9
0b
0 6
41
20
0 7
b6
24
3 0
3b
0 4
15
8 9
6a
5 9
08
0 6
2b
0 1
25
Lyc
oper
sico
nes
cule
ntu
mL
B0
11
5 4
2a
2 1
25
2 9
7a
0 4
94
25
7 9
a1
1 5
02 8
8a
0 8
80
21 2
1a
14 1
37
2 4
7a
1 5
12
Gal
bra
ith
21 5
3b
3 7
52
3 4
0a
0 7
13
20
8 5
b4
9 6
12 8
7a
0 7
12
16 8
7a
10 0
36
1 6
9ab
0 8
14
Tri
sM
gC
l 21
7 4
8a
4 9
70
4 7
6b
1 5
62
21
6 7
b2
7 2
73 7
0b
0 8
36
20 5
2a
12 9
18
1 7
2ab
1 7
45
Ott
o3 6
8c
1 5
37
1 9
5c
0 7
66
26
9 9
a1
1 1
72 1
8c
0 2
66
28 4
9b
13 2
54
0 9
1b
0 6
17
Cel
tis
au
stra
lis
LB
01
28 4
7a
4 8
47
11 4
5a
3 4
81
18
2 1
a1
3 1
33 0
2a
0 3
15
7 6
6a
3 5
65
0 5
0a
0 2
24
Gal
bra
ith
25 5
7a
1 8
42
4 5
4b
0 6
37
17
1 3
b7
76
2 8
7a
0 2
59
8 2
7a
3 4
45
0 3
5a
0 1
62
Tri
sM
gC
l 24
1 7
1b
6 9
44
16 0
8c
2 1
57
18
0 3
a1
0 4
42 8
5a
0 4
19
9 1
0b
5 0
85
0 4
9a
0 2
91
Ott
o8 2
0c
1 4
04
7 2
5b
1 7
87
19
2 8
c8
23
3 4
6b
0 4
05
23 9
6c
4 6
15
0 4
9a
0 2
00
Pis
um
sati
vum
LB
01
47 5
4a
1 5
50
10 8
7a
3 0
78
18
3 5
a5
06
2 8
1a
0 4
61
7 1
9a
2 8
23
2 3
3a
0 4
90
Gal
bra
ith
51 9
7a
3 6
24
5 2
9b
1 1
55
17
8 6
a3
36
3 0
2ab
0 4
65
6 3
4a
1 7
65
2 3
0a
0 8
76
Tri
sM
gC
l 25
9 3
7b
4 8
26
16 7
4c
2 9
14
17
7 7
a1
1 8
63 2
9b
0 5
15
6 3
9a
2 7
81
2 5
9a
0 8
94
Ott
o5 7
2c
0 9
93
4 7
9b
1 0
83
19
0 1
a6
06
1 9
4c
0 1
80
9 8
5b
2 6
68
1 1
5b
0 3
67
Fes
tuca
roth
ma
leri
LB
01
57 3
5a
1 8
25
8 5
7a
0 7
99
19
6 1
a6
07
3 2
4a
0 4
52
14 2
3ab
3 2
83
0 4
2a
0 1
82
Gal
bra
ith
61 0
3a
2 2
50
6 8
4b
0 6
49
18
2 7
b7
15
3 3
3ab
0 4
59
15 0
7a
1 9
74
0 2
7b
0 1
31
Tri
sM
gC
l 26
9 8
5a
1 6
03
17 7
2c
0 8
15
18
5 1
b8
06
3 6
6ab
0 3
12
17 3
0b
5 7
01
0 5
4a
0 1
95
Ott
o1
3 8
4b
2 8
46
8 5
5a
1 6
03
21
0 9
c6
77
3 7
6b
0 6
13
23 8
6c
12 9
92
0 1
1c
0 0
66
Vic
iafa
ba
LB
01
10
4 8
2a
6 0
21
11 6
1a
1 7
42
20
1 5
a4
45
2 4
0a
0 1
78
6 3
6a
2 3
26
0 8
7a
0 2
90
Ga
lbra
ith
11
4 5
5b
2 7
87
8 5
8b
1 3
06
19
6 9
a4
88
2 4
1a
0 1
48
5 8
0a
0 8
06
0 8
2a
0 2
35
Tri
sM
gC
l 21
15 4
5b
5 1
94
20 3
6c
1 4
43
19
1 5
a9
10
2 9
1b
0 2
72
6 8
5a
4 6
09
1 3
0b
0 3
45
Ott
o2
1 5
6c
8 7
21
14 0
5a
7 5
89
18
4 2
a1
6 9
22 2
2a
0 5
32
4 3
5a
1 7
51
0 4
5c
0 1
8
Val
ues
are
giv
enas
mea
nan
dst
and
ard
dev
iati
on
of
the
mea
n(S
D)
of
forw
ard
scat
ter
(FS
ch
ann
elu
nit
s)s
ide
scat
ter
(SS
chan
nel
un
its)
flu
ore
scen
ce(F
Lc
han
nel
un
its)
coef
fici
ento
fv
aria
tio
no
fG
0G
1D
NA
pea
k(C
V
)b
ack
gro
un
dfa
cto
r(B
F
)an
dn
ucl
eary
ield
fact
or(Y
Fn
ucl
eisndash
1m
gndash1)
Mea
ns
foll
ow
edb
yth
esa
me
lett
er(a
bo
rc)
are
no
tsta
tist
ical
lyd
iffe
ren
tacc
ord
ing
toth
em
ult
iple
com
par
ison
Ho
lmndashS
idak
test
atPlt
00
5
Buff
er(s
)th
atp
erfo
rmed
bes
tin
each
spec
ies
are
sho
wn
inb
old
typ
e
Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM 683
significantly better than Galbraithrsquos (Fig 1C) with highermean FL intensities and lower CV values (Table 3)Figure 1C also shows the histogram obtained after nuclearisolation with TrisMgCl2 buffer In this case a G0G1 peakwith an unacceptable CV value (974 ) and considerableloss of fluorescence was obtained A similar result wasobtained for nuclei isolated with LB01
Lycopersicon esculentum
Acceptable results were obtained with all four buffersTwo buffer groups with statistically significant differencesin FL were obtained Samples prepared with Galbraithrsquosand TrisMgCl2 buffers yielded lower mean FL valuesthan their counterparts prepared with LB01 and Ottorsquosbuffers (Fig 1D) The FL values were highly heterogen-eous among buffers Ottorsquos and LB01 were the bestbuffers with Ottorsquos providing lower CV values and higherFL but lower YF and higher BF than LB01 (Table 3)
Celtis australis
Low CV values (lt30 ) and low DF (lt100 ) wereobserved for this species Nevertheless it was not easy toobtain suffiecient nuclei and the second lowest YF valueswere observed in this species With regard to FL onlyLB01 and TrisMgCl2 buffers were not statistically dif-ferent with nuclei isolated from Galbraithrsquos buffer pre-senting the lowest mean FL and Ottorsquos the highest meanFL All parameters combined TrisMgCl2 and LB01 werethe best buffers as nuclei in TrisMgCl2 presented thelowest CV values and similar FL intensity and YF valuesas in LB01 (Table 3 Fig 1E)
Pisum sativum
In this species all buffers performed reasonably wellThe lowest FL intensities were obtained for nucleiisolated with TrisMgCl2 although no statistically signi-ficant differences were observed among the tested buffersThe best buffer for this species was Ottorsquos (Table 3Fig 1F) Among the investigated species P sativum wasthe one with the highest YF
Festuca rothmaleri
With the exception of CV values and YF overall resultsfor this species were satisfying with all four buffers testedNo statistically significant differences were found regard-ing the FL of nuclei isolated in TrisMgCl2 or Galbraithrsquosbuffers as nuclei from both buffers presented low FLvalues The best buffer for this species was LB01 (Table 3Fig 1G)
Vicia faba
This species gave the lowest CV and DF values amongthose tested Generally the results were very similar tothose obtained for P sativum FL was similar for all thebuffers and no statistically significant differences wereobserved Interestingly Vicia faba was the only species
for which Ottorsquos was not the buffer with the highest FL(Fig 1H) Despite low CV values of DNA peaks thisbuffer gave the worst results with the G0G1 peak shiftedtowards the lower channels This was due to fluorescenceinstability which decreased over time In all other speciesand with the remaining buffers FL was stable after 10 minof incubation with PI (Fig 1A) Results obtained withLB01 and Galbraithrsquos buffers were similar and the best forthis species (Table 3)
Analysis of FS and SS
Generally FS and SS values differed considerablyamong the test buffers Nevertheless in most of thespecies analysis of scatter parameters revealed that two ofthe four buffers were more similar than the others and nostatistically significant differences were observed betweenthem Interestingly and with the exception of S burritobuffers that had similar FS mean values were not thosethat had similar SS values This can be seen on cytogramsof FS vs SS obtained in P sativum (Fig 2) In thisspecies and for FS no statistically significant differenceswere observed between LB01 and Galbraithrsquos buffers forSS no difference was observed for Galbraithrsquos and Ottorsquosbuffers Interestingly simultaneous analysis of FS and SSresulted in a species-specific pattern that could be used asa fingerprint of each buffer
Effect of operator and date of analysis
In most cases no statistically significant differenceswere observed between operators and dates of analysisOperators provided more homogeneous results than datesin the former statistically significant differences wereobserved only for BF and YF in more than one speciesSignificant differences between the dates of analysis weredetected in some species for FS SS CV and BF Withregard to FL one of the most important parameters inFCM analyses significant among-day differences weredetected only in C australis and differences betweenoperators occurred only in L esculentum The two speciesmore susceptible to differences were L esculentum andV faba (Table 4)
Hierarchical cluster analysis
With the exception of the results for S burrito the fourbuffers fell into two highly dissimilar and consistentclusters one with Ottorsquos buffer and the other with theremaining buffers In S burrito (Fig 3A) one cluster wasformed with Ottorsquos and Galbraithrsquos buffers while theother comprised TrisMgCl2 and LB01 buffers InC australis (Fig 3C) Galbraithrsquos buffer was more similarto LB01 and TrisMgCl2 buffers than to Ottorsquos buffer inaddition lsquoTrisMgCl2 Arsquo was more similar to LB01 thanto lsquoTrisMgCl2 Brsquo In P sativum lsquoGalbraithrsquos Arsquo wasmore related to LB01 than to lsquoGalbraithrsquos Brsquo (Fig 3D)whereas for F rothmaleri lsquoLB01 Arsquo was more similar toGalbraithrsquos than to lsquoLB01 Brsquo (Fig 3E) In L esculentumtwo groups were formed from the second clusterTrisMgCl2 and lsquoGalbraith Arsquo formed one group and
684 Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM
FS log
C
SS lo
g
1 10 100 10000middot1
0middot1
1
10
100
1000
Meanchannel
FSSS 18middot2
TrisMgCl2
37middot8735middot6960middot7()
FPCV
Meanchannel
FSSS
LB01ASS
log
0middot1
1
10
100
1000
10middot045middot4
30middot0226middot09()
FPCV
FSSS 3middot1 37middot24
29middot936middot0
Ottorsquos
FS log
D
1 10 100 10000middot1
Meanchannel ()
FPCV
FS 52middot1 28middot42SS 5middot2 39middot04
GalbraithsB
Meanchannel ()
FPCV
F I G 2 Cytograms of forward scatter (logarithmic scale FS log) vs side scatter (logarithmic scale SS log) obtained after the analysis of Pisum sativum nucleiisolated with four lysis buffers (A) LB01 (B) Galbraithrsquos (C) TrisMgCl2 and (D) Ottorsquos The mean channel number and full peak coefficient of variation
(FPCV ) are given for both parameters Note that the patterns of distributions are characteristic for each buffer
T A B L E 4 Three-way ANOVA analysis of the dates (D) and operators (O) for the parameters evaluated on each species
FS SS FL CV BF YF
Species D O D O D O D O D O D O
Sedum burrito ns ns ns ns ns ns ns ns s ns ns nsOxalis pes-caprae s ns s ns ns ns s ns ns ns ns nsLycopersicon esculentum s ns ns ns ns s s ns s s s sCeltis australis ns ns ns ns s ns ns ns s ns ns nsPisum sativum ns ns s ns ns ns s ns s ns ns nsFestuca rothmaleri s ns ns ns ns ns ns ns ns s ns nsVicia faba ns s s s ns ns ns ns ns ns s s
Forward scatter FS side scatter SS fluorescence FL coefficient of variation of the G0G1 DNA peak CV background factor BF and nuclearyield factor YF
ns not significantly different s significantly different at P lt 005
Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM 685
LB01 and lsquoGalbraith Brsquo formed the other (Fig 3B) InV faba (Fig 3F) LB01 and Galbraithrsquos formed one groupowing to greater similarities between operators thanwithin each buffer As previously stated in O pes-capraeonly two buffers (Ottorsquos and Galbraithrsquos) providedacceptable results with considerable dissimilaritiesbetween them (data not shown)
DISCUSSION
Four nuclear isolation buffers were used with a setof species that were chosen to represent different types
of leaf tissues and different nuclear DNA content(130ndash2690 pg per 2C DNA) As expected popularDNA reference standards (P sativum V faba andL esculentum) were easy to work with Neverthelessnot all buffers worked well with L esculentumpossibly owing to the presence of cytosolic compoundsHowever as the aim of the study was to compare theperformance of basic buffer formulas the use of additivesthat could counteract the negative effects of cytosol wasavoided
Overall the best results were obtained with P sativumAs its 2C nuclear DNA content is in the middle of theknown range of genome sizes in plants this observation
A
C
E
B
D
F
Dissimilarity Dissimilarity
2middot0 1middot5 1middot0 0middot5 0middot02middot0 1middot5 1middot0 0middot5 0middot0
2middot0 1middot5 1middot0 0middot5 0middot0
2middot0 1middot5 1middot0 0middot5 0middot0 2middot0 1middot5 1middot0 0middot5 0middot0
2middot0 1middot5 1middot0 0middot5 0middot0
LB01A
LB01B
LB01A
LB01B
Ottos B
Ottos A
Galbraiths B
Galbraiths A
TrisMgCl2 B
TrisMgCl2 A
Ottos B
Ottos A
Galbraiths B
Galbraiths A
Galbraiths B
Galbraiths A
TrisMgCl2 B
TrisMgCl2 A
LB01A
LB01B
Ottos B
Ottos A
TrisMgCl2 B
TrisMgCl2 A
LB01A
LB01B
LB01A
LB01B
Ottos B
Ottos A
Galbraiths A
Galbraiths B
TrisMgCl2 A
TrisMgCl2 B
Ottos B
Ottos A
Galbraiths B
Galbraiths A
Galbraiths B
Galbraiths A
TrisMgCl2 B
TrisMgCl2 A
LB01A
LB01B
Ottos B
Ottos A
TrisMgCl2 B
TrisMgCl2 A
F I G 3 Dendrograms obtained after hierarchical cluster analysis of the following species (A) Sedum burrito (B) Lycopersicon esculentum (C) Celtisaustralis (D) Pisum sativum (E) Festuca rothmaleri (F) Vicia faba according to the parameters FS SS FL CV BF and YF With the exception of S burrito
the four buffers fell into two highly dissimilar clusters of the same buffers
686 Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM
underlines its position as one of the best standards forplant DNA FCM By contrast Sedum burrito was themost difficult species to analyse due to low DNA contentoccurrence of polysomaty and high leaf water contentwhich hampered sample preparation and analysis More-over its tissues may contain tannins (J Greilhuber perscomm 2006)
In O pes-caprae cytosol of which is highly acidic(pH lt 30) measurable samples could be prepared usingonly Ottorsquos and Galbraithrsquos buffers with Ottorsquos beinghighly superior This is in accordance with the results ofEmshwiller (2002) who analysed ploidy levels in OxalisAfter testing LB01 MgSO4 and Ottorsquos buffers sheobtained measurable samples only with Ottorsquos The formertwo buffers failed presumably as a result of the acidic cellsap which may have exceeded the buffering capacity ofLB01 and MgSO4
Celtis australis was the only woody plant speciesincluded in the present study and was chosen because ofthe presence of mucilaginous compounds (Rodriguez et al2005a) which increase sample viscosity restrain nucleirelease and cause their clumping Interestingly this wasthe only species for which TrisMgCl2 was the bestperforming buffer This was probably because of a higherconcentration of the non-ionic detergent which sup-pressed the effect of mucilaginous compounds Leaftissues of F rothmaleri were particularly hard anddifficult to chop In addition preliminary experimentswith this species revealed the presence of cytosoliccompounds which would be expected to interfere withDNA staining However given the pattern of FS and SSobtained the so-called lsquotannic acid effectrsquo (Loureiro et al2006) was absent indicating that these compounds werereleased at low concentration or not at all
In order to compare the performance of nuclear isolationbuffers a set of parameters was carefully selected toevaluate sample quality Furthermore stability of fluores-cence and light scatter properties of isolated nuclei overtime were confirmed Among the parameters chosen thecoefficient of variation of DNA peaks (CV) is of majorimportance Galbraith et al (2002) considered 50 asthe maximum acceptable CV value in plant DNA flowcytometry With the exception of TrisMgCl2 buffer whenused with S burrito all mean CV values obtained hereinwere below this limit Ideally a nuclei sample should befree of cell and tissue debris Emshwiller (2002) noted acorrelation between CV and background noise In thepresent study this correlation was found only in somespecies (eg F rothmaleri) Rather the results heresuggest that the extent of background debris is determinedby the buffer itself For example a higher detergentconcentration in a buffer could lead to chloroplast lysisand consequently decrease the number of fluorescentparticles contributing to debris signals (Coba de la Penaand Brown 2001)
LB01 buffer provided very good results with theexception of O pes-caprae and S burrito low CV highFL and YF values were obtained with this buffer Highnuclei FL intensities obtained with this buffer weredefinitely an advantage as compared with TrisMgCl2 and
Galbraithrsquos buffers Galbraithrsquos buffer seems well bal-anced as acceptable results were achieved in all speciesSurprisingly the buffer gave reasonable results also withO pes-caprae which is characterized by highly acidiccytosol The presence of MOPS in the buffer may shedlight on these findings as it has a pKa of 72 and a betterbuffering capacity than TRIS with a pKa of 81 Adisadvantage of this buffer was the low fluorescenceintensity of nuclei Collectively TrisMgCl2 was the worstperforming buffer Nevertheless it provided the bestresults for C australis This may have been due to thehigher concentration of non-ionic detergent (Table 1)which counteracted the agglutinating effect of mucilagin-ous compounds and decreased sample viscosity As withGalbraithrsquos buffer low FL values were obtained withTrisMgCl2
Ottorsquos buffer is unique in that sample preparationinvolves two steps The results obtained with Ottorsquos bufferwere excellent in many species especially those withlower nuclear DNA content Dolezel and Bartos (2005)highlighted the quality of this buffer which is known toprovide DNA content histograms with unequalled resolu-tion One explanation for this may be that citric acidimproves chromatin accessibility and lsquohomogenizesrsquo chro-matin structure eliminating differences in staining intens-ity among nuclei with the same DNA content but differentchromatin state This could explain the highest FL andlowest CV values as observed here Another characteristicof this buffer was that light scatter values (especially FS)were significantly lower than those of other buffers Thismay be explained by the action of citric acid whichcauses nuclei fixation (Dolezel and Bartos 2005) It isnoteworthy that the pattern of FS vs SS distribution wassimilar to that obtained when analysing nuclei fixed withformaldehyde (Rodriguez et al 2005b) Althoughproviding excellent results this buffer gave the highestBF and lowest YF values The former can be explained bynuclei instability after the addition of Otto II After thisstep nuclei deteriorate rapidly in some species (J Dolezelunpubl res) The lowest YF values could be due to thedetergent (Tween 20) which is weaker than Triton X-100thus having a lower capacity to release nuclei
An important consequence of the observation ofdifferent fluorescence values obtained with the differentbuffers is that different fluorescence ratios may beexpected with the same species pair if the samples areprepared in different buffers In fact Dolezel et al (1998)observed differences in peak ratios obtained in differentlaboratories Further studies are needed to confirm thisobservation as it may have important consequences forestimation of genome size
The present results showed that well-defined popula-tions of nuclei could be observed on cytograms of FS vsSS With the exception of S burrito for which LB01 andTrisMgCl2 buffers did not present statistically significantdifferences for both parameters the analysis of FS and SSprovided a fingerprint pattern for each buffer Loureiroet al (2006) showed that these parameters were sensitiveto the presence of tannic acid a cytosolic compoundcommon in plants and recommended the analysis of light
Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM 687
scatter to verify suitability of particular samples for plantDNA FCM
As nuclei samples are prepared manually it wasimportant to assess the effect of date of analysis andoperator on sample quality variation Date was found to bemore significant than the effect of different operatorsespecially for FS SS CV and DF YF which depends onthe way the sample is chopped and hence on the operatordid not vary It was expected that FL which is a primarysource of data in FCM analysis would not depend on theoperator or the day of chopping This was confirmed in allspecies except L esculentum and C australis In theformer species a significant variation was obtainedbetween operators These differences could be explainedby variable results obtained with Galbraithrsquos buffer InC australis significant differences were obtained amongdates In this case significant differences were due toresults obtained on one single day Emshwiller (2002)found significant differences when multiple preparationsfrom the same plant were run on different days Thesedifferences and those found in the present study wereprobably due to instrument drift (Kudo and Kimura2001) To avoid this type of error several authors haverecommended that each measurement be repeated at leastthree times on three different days (Suda 2004 Dolezeland Bartos 2005)
This is the first study that has systematically comparednuclear isolation buffers for DNA FCM The results showthat none of the buffers works best with all species andstatistically significant differences in sample quality wereobserved among the four buffers The results obtainedwith different species and contrasting types of leaf tissuescan serve as guidelines in buffer selection Nevertheless itis recommend that a range of buffers be tested whenworking with a new species and tissue type Once the bestbuffer has been identified additives should be tested ifrequired to suppress negative effects of phenols and othercytosolic compounds
ACKNOWLEDGEMENTS
We are grateful to Prof Johann Greilhuber and Dr JanSuda for critical reading of the manuscript This work wassupported by FCT project ref POCTIAGR606722004JL was supported by the Fellowship FCTBD90032002
LITERATURE CITEDBennett MD Leitch I 2005 Genome size evolution in plants In
Gregory T ed The evolution of the genome London ElsevierAcademic Press 89ndash162
Bergounioux C Perennes C Brown SC Gadal P 1988 Cytometricanalysis of growth regulator-dependent transcription and cell cycleprogression in Petunia protoplast cultures Planta 175 500ndash505
Bergounioux C Brown SC Petit PX 1992 Flow cytometry and plantprotoplast cell biology Physiologia Plantarum 85 374ndash386
Castro S Loureiro J Santos C Ayensa G Navarro L 2005 Differentploidy levels could explain reproductive matter of invasive Oxalispes-caprae L in Mediterranean regions In Brunel S ed Invasiveplants in Mediterranean type regions of the world StrasbourgCedex France Council of Europe Publishing 235
Coba de la Pena T Brown SC 2001 Cytometry and fluorimetryIn Hawes C Satiat-Jeunemaıtre B eds Plant cell biology
a practical approach New York Oxford University Press85ndash106
Dolezel J Bartos J 2005 Plant DNA flow cytometry and estimation ofnuclear genome size Annals of Botany 95 99ndash110
Dolezel J Gohde W 1995 Sex determination in dioecious plantsMelandrium album and M rubrum using high-resolution flowcytometry Cytometry 19 103ndash106
Dolezel J Binarova P Lucretti S 1989 Analysis of nuclear DNAcontent in plant cells by flow cytometry Biologia Plantarum 31113ndash120
Dolezel J Sgorbati S Lucretti S 1992 Comparison of three DNAfluorochromes for flow cytometric estimation of nuclear DNAcontent in plants Physiologia Plantarum 85 625ndash631
Dolezel J Greilhuber J Lucretti S Meister A Lysak M Nardi LObermayer R 1998 Plant genome size estimation byflow cytometry inter-laboratory comparison Annals of Botany 8217ndash26
Dolezel J Bartos J Voglmayr H Greilhuber J 2003 Nuclear DNAcontent and genome size of trout and human Cytometry 51A127ndash128
Emshwiller E 2002 Ploidy levels among species in the lsquoOxalis tuberosaAlliancersquo as inferred by flow cytometry Annals of Botany 89741ndash753
Galbraith DW 2004 Cytometry and plant sciences a personalretrospective Cytometry 58A 37ndash44
Galbraith DW Harkins KR Maddox JM Ayres NM Sharma DPFiroozabady E 1983 Rapid flow cytometric analysis of the cell-cycle in intact plant-tissues Science 220 1049ndash1051
Galbraith DW Lambert GM Macas J Dolezel J 2002 Analysis ofnuclear DNA content and ploidy in higher plants In Robinson JPDarzynkiewicz Z Dean PN Dressler LG Rabinovitch PS StewartCV Tanke HJ Wheeless LL eds Current protocols in cytometryNew York John Wiley amp Sons 761ndash7622
Heller FO 1973 DNS-Bestimmung an Keimwurzeln von Vicia faba Lmit Hilfe der Impulscytophotometrie Bericht der DeutschenBotanischen Gesellschaft 86 437ndash441
Hintze J 2004 NCSS and PASS Kaysville Utah Number CruncherStatistical Systems
Kudo N Kimura Y 2001 Flow cytometric evidence for endopolyploidyin seedlings of some Brassica species Theoretical and AppliedGenetics 102 104ndash110
Loureiro J Rodriguez E Dolezel J Santos C 2006 Flow cytometricand microscopic analysis of the effect of tannic acid on plantnuclei and estimation of DNA content Annals of Botany 98515ndash527
Noirot M Barre P Louarn J Duperray C Hamon S 2000 Nucleusndashcytosol interactionsmdashA source of stoichiometric error in flowcytometric estimation of nuclear DNA content in plants Annals ofBotany 86 309ndash316
Noirot M Barre P Duperray C Louarn J Hamon S 2003 Effects ofcaffeine and chlorogenic acid on propidium iodide accessibility toDNA consequences on genome size evaluation in coffee treeAnnals of Botany 92 259ndash264
Otto F 1992 Preparation and staining of cells for high-resolution DNAanalysis In Radbruch A ed Flow cytometry and cell sortingBerlin Springer-Verlag 101ndash104
Pfosser M Amon A Lelley T Heberle-Bors E 1995 Evaluation ofsensitivity of flow cytometry in detecting aneuploidy in wheat usingdisomic and ditelosomic wheat-rye addition lines Cytometry 21387ndash393
Pinto G Loureiro J Lopes T Santos C 2004 Analysis of the geneticstability of Eucalyptus globulus Labill somatic embryos by flowcytometry Theoretical and Applied Genetics 109 580ndash587
Rodriguez E Gomes A Loureiro J Dolezel J Santos C 2005aEstimation of the genome size of the Iberian Peninsula Ulmaceae In9th Iberian Congress of Cytometry Book of Abstracts PortoPortugal 37
Rodriguez E Loureiro J Dolezel J Santos C 2005b The adequacy ofusing formaldehyde fixation for nuclear DNA content analyses ofplant material In 9th Iberian Congress of Cytometry Book ofAbstracts Porto Portugal PO120-PB
Shapiro H 2004 Practical flow cytometry 4th edn New YorkWiley-Liss
688 Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM
Suda J 2004 An employment of flow cytometry into plant biosystematicsPhD thesis Charles University Czech Republic Available at httpwwwibotcasczfcmsudapresentationdisertationpdf
Trotter J 2000 WinMDI version 28 Available at httpfacsscrippsedusoftwarehtml
Ulrich I Ulrich W 1986 Flow cytometric DNA analysis of plantprotoplasts with DAPI Zeitschrift fur Naturforschung 411052ndash1056
Ulrich I Fritz B Ulrich W 1988 Application of DNA fluorochromes forflow cytometric DNA analysis of plant protoplasts Plant Science 55151ndash158
Walker D Monino I Correal E 2006 Genome size inBituminaria bituminosa (L) CH Stirton (Fabaceae) populationsseparation of lsquotruersquo differences from environmental effects on DNAdetermination Environmental and Experimental Botany 55258ndash265
Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM 689
background factor (DF to assess sample quality) and anuclear yield factor (YF to compare the amount of nucleiin suspension independently of the amount of leaftissue used)
CV () was calculated using the following formula
Half peak CV eth THORN
frac14 4246 middot width of peak at half the peak height
peak positioneth1THORN
DF () was calculated as follows
DF eth THORN frac14total number of particles total number of intact nuclei
total number of particlesmiddot 100
eth2THORN
DF increases as debris increasesYF (nuclei sndash1 mgndash1) was calculated using the formula
YF nucleis1mg1
frac14 total number of intact nuclei=number of seconds of run seth THORNweight of tissue mgeth THORN
eth3THORN
The flow rate was defined as low and was kept constantthroughout the experiment
The analysis was performed on three different days andby two operators (labelled here as A and B) Fivereplicates were performed per operator for each buffer andin each replicate at least 5000 nuclei were analysedHistograms of FL obtained with the best and worstperforming buffers were overlaid using WinMDI software(Trotter 2000) (Fig 1)
For S burrito nuclear genome size was estimated usingL esculentum lsquoStupickersquo (2C = 196 Dolezel et al 1992)as internal reference standard according to the followingformula
S burrito 2C nuclear DNA content pgeth THORN
frac14 S burrito G0=G1 peak mean
L esculentum G0=G1 peak meanmiddot 196 eth4THORN
Conversion of mass values into base-pair numbers wasachieved according to the factor 1 pg = 978 Mbp (Dolezelet al 2003)
Statistical analyses
Statistical analyses were performed using a three-wayANOVA (SigmaStat for Windows version 311) to assessfor differences among buffers and dates and betweenoperators When treatments were significantly different aHolmndashSidak multiple comparison test was used for pair-wise comparison Hierarchical cluster analyses wereperformed using NCSS 2004 (Hintze 2004) Dendrogramshighlighting dissimilarities among buffers and betweenoperators were obtained using FS SS FL CV BF andYF The Unweighted Pair Group Method with Arithmeticmean (UPGMA) was followed in each species as ityielded the highest co-phenetic correlation coefficient
RESULTS
With the exception of O pes-caprae for which meas-urable samples were only obtained with Ottorsquos andGalbraithrsquos buffers all buffers yielded acceptable histo-grams with all species tested In any analysis it waspossible to isolate a reasonable number of nuclei(approximately 20ndash60 nuclei per second in a low-speedconfiguration) and obtain well-defined histograms withDNA peaks with acceptable CV values (lt50 Galbraithet al 2002 Fig 1 Table 3) Table 3 indicates the bestperforming buffer(s) for each species The selectioncriteria were the highest FL and YF values and thelowest CV and DF values
Sedum burrito
This species was investigated because of its expectedsmall genome size and fleshy leaves FCM analysisrevealed the occurrence of polysomaty as demonstratedby the presence of discrete populations of nuclei withDNA contents of 2C 4C 8C 16C and higher In orderto observe a higher number of endopolyploidy
T A B L E 2 Four nuclear isolation buffers most frequently used in plant DNA flow cytometry
Buffer Composition Reference
Galbraith 45 mM MgCl2 30 mM sodium citrate 20 mM MOPS01 (vv) Triton X-100 pH 70
Galbraith et al (1983)
LB01 15 mM Tris 2 mM Na2EDTA 05 mM spermine4HCl80 mM KCl 20 mM NaCl 01 (vv) TritonX-100 pH 80dagger
Dolezel et al (1989)
Ottoz Otto I 100 mM citric acid 05 (vv) Tween 20 (pH 2ndash3)Otto II 400 mM Na2PO4
12H2O (pH 8ndash9)Otto (1992) Dolezel and Gohde (1995)
TrisMgCl2 200 mM Tris 4 mM MgCl26H2O 05 (vv)Triton X-100 pH 75
Pfosser et al (1995)
Final concentrations are givendagger The buffer formula contains 15 mM mercaptoethanol However as the other buffers were used without additives that suppress the negative effect of
phenols and other cytosolic compounds LB01 was used without mercaptoethanolz pH of the buffers is not adjusted The nuclei are isolated in Otto I buffer DNA staining is done in a mixture of Otto I and Otto II (1 2) with a final volume
of 1 mL
Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM 681
levels instrument gain was set such that the 2C peak wasapproximately on channel 100 The 2C nuclear DNAcontent was estimated as 130 6 009 pg (2C = 1271 Mbp)this is the first estimate for this species (Table 1) Com-parative analysis of the four buffers revealed that Ottorsquoswas the best in this species In general YF values were low
(the lowest values from all the test species) whereas DFvalues were high (approx 650 Table 3 Fig 1B)
Oxalis pes-caprae
Only Ottorsquos and Galbraithrsquos buffers provided acceptableresults with this species Ottorsquos was clearly and
0 75 1500
25
1023
43
3
2
2
2
2
2
2
1
1
1
1
1
PI
Peak FL CV() 1 108middot7 5middot87 2 118middot7 3middot00 3 206middot8 4middot10 4 231middot0 2middot55
TrismiddotMgCl2 bufferGreenOttorsquos bufferBlue
Peak FL CV() 1 97middot6 9middot74 2 187middot0 3middot50 3 201middot1 2middot69
TrismiddotMgCl2 bufferGreenGalbraithrsquos bufferOrangeOttorsquos bufferBlue
Peak FL CV() 1 226middot1 4middot09 2 252middot1 1middot96
TrismiddotMgCl2 bufferGreenLB01 bufferRed
Peak FL CV() 1 190middot3 2middot21 2 205middot8 3middot90
TrismiddotMgCl2 bufferGreenOttorsquos bufferBlue
Peak FL CV() 1 184middot1 2middot78 2 213middot8 3middot48
LB01 bufferRedOttorsquos bufferBlue
Peak FL CV() 1 168middot4 3middot32 2 202middot6 2middot10
Ottorsquos bufferBlueLB01 bufferRed
Peak FL CV() 1 180middot5 1middot52 2 188middot1 3middot07
Ottorsquos bufferBlueTrismiddotMgCl2 bufferGreen
0
50
75
100
125
Time (s)
Ottorsquos buffer
PI
225
0
450
0
512
128
00 PI 1023
300
Cou
nt
Cou
ntC
ount
Cou
nt
0
1024
Cou
nt
0
400
1
0
512
Cou
nt
2
1
1023PI0
512
0
Cou
nt
A B
C D
E F
G H
F I G 1 Cytogram of fluorescence intensity (PI relative channel numbers) vs time of Pisum sativum nuclei isolated with Ottorsquos buffer (A) and histograms ofrelative fluorescence intensities (BndashH) which show overlays of distributions obtained with the best and worst performing buffer for each species (B) Sedumburrito (C) Oxalis pes-caprae (D) Lycopersicon esculentum (E) Celtis australis (F) Pisum sativum (G) Festuca rothmaleri and (H) Vicia faba Relativemean channel numbers and coefficients of variation (CV ) of G0G1 peaks are given Four lysis buffers were compared LB01 (red) Galbraithrsquos (orange)
TrisMgCl2 (green) and Ottorsquos (blue)
682 Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM
TA
BL
E3
F
low
cyto
met
ric
pa
ram
eter
sa
sses
sed
inea
chsp
ecie
s
FS
(ch
ann
elu
nit
s)S
S(c
han
nel
un
its)
FL
(ch
ann
elu
nit
s)C
V(
)B
F(
)Y
F(n
ucl
eis
1m
g
1)
Sp
ecie
sB
uff
erM
ean
SD
Mea
nS
DM
ean
SD
Mea
nS
DM
ean
SD
Mea
nS
D
Sed
um
bu
rrit
oL
B0
15
76
6a
8 7
24
11 3
7a
2 4
48
11
0 9
a5
93
4 4
8a
0 4
07
69 1
5a
24 9
03
0 0
6a
0 0
04
Gal
bra
ith
41 0
6b
5 1
71
5 3
6b
2 2
99
11
2 1
a2
57
3 5
9ab
0 3
96
60 0
2a
16 6
31
0 0
5b
0 0
04
Tri
sM
gC
l 25
75
0a
8 5
56
13 3
4a
2 6
70
10
3 0
b6
19
5 6
1c
1 0
00
68 8
5a
13 8
74
0 0
5ab
0 0
04
Ott
o8 0
1c
2 1
79
2 5
8c
0 7
87
12
1 4
c3
93
3 2
7b
0 2
88
65 9
6a
6 3
30
05
ab
0 0
06
Oxa
lis
pes
-ca
pra
eL
B0
1ndash
ndashndash
ndashndash
ndashndash
ndashndash
ndashndash
ndashG
alb
rait
h1
71 3
3a
36 4
04
19 7
5a
6 9
20
17
8 5
a5
21
4 1
4a
0 7
78
7 0
5a
4 9
77
1 0
1a
0 3
64
Tri
sM
gC
l 2ndash
ndashndash
ndashndash
ndashndash
ndashndash
ndashndash
ndashO
tto
26 3
2b
4 9
33
3 9
0b
0 6
41
20
0 7
b6
24
3 0
3b
0 4
15
8 9
6a
5 9
08
0 6
2b
0 1
25
Lyc
oper
sico
nes
cule
ntu
mL
B0
11
5 4
2a
2 1
25
2 9
7a
0 4
94
25
7 9
a1
1 5
02 8
8a
0 8
80
21 2
1a
14 1
37
2 4
7a
1 5
12
Gal
bra
ith
21 5
3b
3 7
52
3 4
0a
0 7
13
20
8 5
b4
9 6
12 8
7a
0 7
12
16 8
7a
10 0
36
1 6
9ab
0 8
14
Tri
sM
gC
l 21
7 4
8a
4 9
70
4 7
6b
1 5
62
21
6 7
b2
7 2
73 7
0b
0 8
36
20 5
2a
12 9
18
1 7
2ab
1 7
45
Ott
o3 6
8c
1 5
37
1 9
5c
0 7
66
26
9 9
a1
1 1
72 1
8c
0 2
66
28 4
9b
13 2
54
0 9
1b
0 6
17
Cel
tis
au
stra
lis
LB
01
28 4
7a
4 8
47
11 4
5a
3 4
81
18
2 1
a1
3 1
33 0
2a
0 3
15
7 6
6a
3 5
65
0 5
0a
0 2
24
Gal
bra
ith
25 5
7a
1 8
42
4 5
4b
0 6
37
17
1 3
b7
76
2 8
7a
0 2
59
8 2
7a
3 4
45
0 3
5a
0 1
62
Tri
sM
gC
l 24
1 7
1b
6 9
44
16 0
8c
2 1
57
18
0 3
a1
0 4
42 8
5a
0 4
19
9 1
0b
5 0
85
0 4
9a
0 2
91
Ott
o8 2
0c
1 4
04
7 2
5b
1 7
87
19
2 8
c8
23
3 4
6b
0 4
05
23 9
6c
4 6
15
0 4
9a
0 2
00
Pis
um
sati
vum
LB
01
47 5
4a
1 5
50
10 8
7a
3 0
78
18
3 5
a5
06
2 8
1a
0 4
61
7 1
9a
2 8
23
2 3
3a
0 4
90
Gal
bra
ith
51 9
7a
3 6
24
5 2
9b
1 1
55
17
8 6
a3
36
3 0
2ab
0 4
65
6 3
4a
1 7
65
2 3
0a
0 8
76
Tri
sM
gC
l 25
9 3
7b
4 8
26
16 7
4c
2 9
14
17
7 7
a1
1 8
63 2
9b
0 5
15
6 3
9a
2 7
81
2 5
9a
0 8
94
Ott
o5 7
2c
0 9
93
4 7
9b
1 0
83
19
0 1
a6
06
1 9
4c
0 1
80
9 8
5b
2 6
68
1 1
5b
0 3
67
Fes
tuca
roth
ma
leri
LB
01
57 3
5a
1 8
25
8 5
7a
0 7
99
19
6 1
a6
07
3 2
4a
0 4
52
14 2
3ab
3 2
83
0 4
2a
0 1
82
Gal
bra
ith
61 0
3a
2 2
50
6 8
4b
0 6
49
18
2 7
b7
15
3 3
3ab
0 4
59
15 0
7a
1 9
74
0 2
7b
0 1
31
Tri
sM
gC
l 26
9 8
5a
1 6
03
17 7
2c
0 8
15
18
5 1
b8
06
3 6
6ab
0 3
12
17 3
0b
5 7
01
0 5
4a
0 1
95
Ott
o1
3 8
4b
2 8
46
8 5
5a
1 6
03
21
0 9
c6
77
3 7
6b
0 6
13
23 8
6c
12 9
92
0 1
1c
0 0
66
Vic
iafa
ba
LB
01
10
4 8
2a
6 0
21
11 6
1a
1 7
42
20
1 5
a4
45
2 4
0a
0 1
78
6 3
6a
2 3
26
0 8
7a
0 2
90
Ga
lbra
ith
11
4 5
5b
2 7
87
8 5
8b
1 3
06
19
6 9
a4
88
2 4
1a
0 1
48
5 8
0a
0 8
06
0 8
2a
0 2
35
Tri
sM
gC
l 21
15 4
5b
5 1
94
20 3
6c
1 4
43
19
1 5
a9
10
2 9
1b
0 2
72
6 8
5a
4 6
09
1 3
0b
0 3
45
Ott
o2
1 5
6c
8 7
21
14 0
5a
7 5
89
18
4 2
a1
6 9
22 2
2a
0 5
32
4 3
5a
1 7
51
0 4
5c
0 1
8
Val
ues
are
giv
enas
mea
nan
dst
and
ard
dev
iati
on
of
the
mea
n(S
D)
of
forw
ard
scat
ter
(FS
ch
ann
elu
nit
s)s
ide
scat
ter
(SS
chan
nel
un
its)
flu
ore
scen
ce(F
Lc
han
nel
un
its)
coef
fici
ento
fv
aria
tio
no
fG
0G
1D
NA
pea
k(C
V
)b
ack
gro
un
dfa
cto
r(B
F
)an
dn
ucl
eary
ield
fact
or(Y
Fn
ucl
eisndash
1m
gndash1)
Mea
ns
foll
ow
edb
yth
esa
me
lett
er(a
bo
rc)
are
no
tsta
tist
ical
lyd
iffe
ren
tacc
ord
ing
toth
em
ult
iple
com
par
ison
Ho
lmndashS
idak
test
atPlt
00
5
Buff
er(s
)th
atp
erfo
rmed
bes
tin
each
spec
ies
are
sho
wn
inb
old
typ
e
Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM 683
significantly better than Galbraithrsquos (Fig 1C) with highermean FL intensities and lower CV values (Table 3)Figure 1C also shows the histogram obtained after nuclearisolation with TrisMgCl2 buffer In this case a G0G1 peakwith an unacceptable CV value (974 ) and considerableloss of fluorescence was obtained A similar result wasobtained for nuclei isolated with LB01
Lycopersicon esculentum
Acceptable results were obtained with all four buffersTwo buffer groups with statistically significant differencesin FL were obtained Samples prepared with Galbraithrsquosand TrisMgCl2 buffers yielded lower mean FL valuesthan their counterparts prepared with LB01 and Ottorsquosbuffers (Fig 1D) The FL values were highly heterogen-eous among buffers Ottorsquos and LB01 were the bestbuffers with Ottorsquos providing lower CV values and higherFL but lower YF and higher BF than LB01 (Table 3)
Celtis australis
Low CV values (lt30 ) and low DF (lt100 ) wereobserved for this species Nevertheless it was not easy toobtain suffiecient nuclei and the second lowest YF valueswere observed in this species With regard to FL onlyLB01 and TrisMgCl2 buffers were not statistically dif-ferent with nuclei isolated from Galbraithrsquos buffer pre-senting the lowest mean FL and Ottorsquos the highest meanFL All parameters combined TrisMgCl2 and LB01 werethe best buffers as nuclei in TrisMgCl2 presented thelowest CV values and similar FL intensity and YF valuesas in LB01 (Table 3 Fig 1E)
Pisum sativum
In this species all buffers performed reasonably wellThe lowest FL intensities were obtained for nucleiisolated with TrisMgCl2 although no statistically signi-ficant differences were observed among the tested buffersThe best buffer for this species was Ottorsquos (Table 3Fig 1F) Among the investigated species P sativum wasthe one with the highest YF
Festuca rothmaleri
With the exception of CV values and YF overall resultsfor this species were satisfying with all four buffers testedNo statistically significant differences were found regard-ing the FL of nuclei isolated in TrisMgCl2 or Galbraithrsquosbuffers as nuclei from both buffers presented low FLvalues The best buffer for this species was LB01 (Table 3Fig 1G)
Vicia faba
This species gave the lowest CV and DF values amongthose tested Generally the results were very similar tothose obtained for P sativum FL was similar for all thebuffers and no statistically significant differences wereobserved Interestingly Vicia faba was the only species
for which Ottorsquos was not the buffer with the highest FL(Fig 1H) Despite low CV values of DNA peaks thisbuffer gave the worst results with the G0G1 peak shiftedtowards the lower channels This was due to fluorescenceinstability which decreased over time In all other speciesand with the remaining buffers FL was stable after 10 minof incubation with PI (Fig 1A) Results obtained withLB01 and Galbraithrsquos buffers were similar and the best forthis species (Table 3)
Analysis of FS and SS
Generally FS and SS values differed considerablyamong the test buffers Nevertheless in most of thespecies analysis of scatter parameters revealed that two ofthe four buffers were more similar than the others and nostatistically significant differences were observed betweenthem Interestingly and with the exception of S burritobuffers that had similar FS mean values were not thosethat had similar SS values This can be seen on cytogramsof FS vs SS obtained in P sativum (Fig 2) In thisspecies and for FS no statistically significant differenceswere observed between LB01 and Galbraithrsquos buffers forSS no difference was observed for Galbraithrsquos and Ottorsquosbuffers Interestingly simultaneous analysis of FS and SSresulted in a species-specific pattern that could be used asa fingerprint of each buffer
Effect of operator and date of analysis
In most cases no statistically significant differenceswere observed between operators and dates of analysisOperators provided more homogeneous results than datesin the former statistically significant differences wereobserved only for BF and YF in more than one speciesSignificant differences between the dates of analysis weredetected in some species for FS SS CV and BF Withregard to FL one of the most important parameters inFCM analyses significant among-day differences weredetected only in C australis and differences betweenoperators occurred only in L esculentum The two speciesmore susceptible to differences were L esculentum andV faba (Table 4)
Hierarchical cluster analysis
With the exception of the results for S burrito the fourbuffers fell into two highly dissimilar and consistentclusters one with Ottorsquos buffer and the other with theremaining buffers In S burrito (Fig 3A) one cluster wasformed with Ottorsquos and Galbraithrsquos buffers while theother comprised TrisMgCl2 and LB01 buffers InC australis (Fig 3C) Galbraithrsquos buffer was more similarto LB01 and TrisMgCl2 buffers than to Ottorsquos buffer inaddition lsquoTrisMgCl2 Arsquo was more similar to LB01 thanto lsquoTrisMgCl2 Brsquo In P sativum lsquoGalbraithrsquos Arsquo wasmore related to LB01 than to lsquoGalbraithrsquos Brsquo (Fig 3D)whereas for F rothmaleri lsquoLB01 Arsquo was more similar toGalbraithrsquos than to lsquoLB01 Brsquo (Fig 3E) In L esculentumtwo groups were formed from the second clusterTrisMgCl2 and lsquoGalbraith Arsquo formed one group and
684 Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM
FS log
C
SS lo
g
1 10 100 10000middot1
0middot1
1
10
100
1000
Meanchannel
FSSS 18middot2
TrisMgCl2
37middot8735middot6960middot7()
FPCV
Meanchannel
FSSS
LB01ASS
log
0middot1
1
10
100
1000
10middot045middot4
30middot0226middot09()
FPCV
FSSS 3middot1 37middot24
29middot936middot0
Ottorsquos
FS log
D
1 10 100 10000middot1
Meanchannel ()
FPCV
FS 52middot1 28middot42SS 5middot2 39middot04
GalbraithsB
Meanchannel ()
FPCV
F I G 2 Cytograms of forward scatter (logarithmic scale FS log) vs side scatter (logarithmic scale SS log) obtained after the analysis of Pisum sativum nucleiisolated with four lysis buffers (A) LB01 (B) Galbraithrsquos (C) TrisMgCl2 and (D) Ottorsquos The mean channel number and full peak coefficient of variation
(FPCV ) are given for both parameters Note that the patterns of distributions are characteristic for each buffer
T A B L E 4 Three-way ANOVA analysis of the dates (D) and operators (O) for the parameters evaluated on each species
FS SS FL CV BF YF
Species D O D O D O D O D O D O
Sedum burrito ns ns ns ns ns ns ns ns s ns ns nsOxalis pes-caprae s ns s ns ns ns s ns ns ns ns nsLycopersicon esculentum s ns ns ns ns s s ns s s s sCeltis australis ns ns ns ns s ns ns ns s ns ns nsPisum sativum ns ns s ns ns ns s ns s ns ns nsFestuca rothmaleri s ns ns ns ns ns ns ns ns s ns nsVicia faba ns s s s ns ns ns ns ns ns s s
Forward scatter FS side scatter SS fluorescence FL coefficient of variation of the G0G1 DNA peak CV background factor BF and nuclearyield factor YF
ns not significantly different s significantly different at P lt 005
Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM 685
LB01 and lsquoGalbraith Brsquo formed the other (Fig 3B) InV faba (Fig 3F) LB01 and Galbraithrsquos formed one groupowing to greater similarities between operators thanwithin each buffer As previously stated in O pes-capraeonly two buffers (Ottorsquos and Galbraithrsquos) providedacceptable results with considerable dissimilaritiesbetween them (data not shown)
DISCUSSION
Four nuclear isolation buffers were used with a setof species that were chosen to represent different types
of leaf tissues and different nuclear DNA content(130ndash2690 pg per 2C DNA) As expected popularDNA reference standards (P sativum V faba andL esculentum) were easy to work with Neverthelessnot all buffers worked well with L esculentumpossibly owing to the presence of cytosolic compoundsHowever as the aim of the study was to compare theperformance of basic buffer formulas the use of additivesthat could counteract the negative effects of cytosol wasavoided
Overall the best results were obtained with P sativumAs its 2C nuclear DNA content is in the middle of theknown range of genome sizes in plants this observation
A
C
E
B
D
F
Dissimilarity Dissimilarity
2middot0 1middot5 1middot0 0middot5 0middot02middot0 1middot5 1middot0 0middot5 0middot0
2middot0 1middot5 1middot0 0middot5 0middot0
2middot0 1middot5 1middot0 0middot5 0middot0 2middot0 1middot5 1middot0 0middot5 0middot0
2middot0 1middot5 1middot0 0middot5 0middot0
LB01A
LB01B
LB01A
LB01B
Ottos B
Ottos A
Galbraiths B
Galbraiths A
TrisMgCl2 B
TrisMgCl2 A
Ottos B
Ottos A
Galbraiths B
Galbraiths A
Galbraiths B
Galbraiths A
TrisMgCl2 B
TrisMgCl2 A
LB01A
LB01B
Ottos B
Ottos A
TrisMgCl2 B
TrisMgCl2 A
LB01A
LB01B
LB01A
LB01B
Ottos B
Ottos A
Galbraiths A
Galbraiths B
TrisMgCl2 A
TrisMgCl2 B
Ottos B
Ottos A
Galbraiths B
Galbraiths A
Galbraiths B
Galbraiths A
TrisMgCl2 B
TrisMgCl2 A
LB01A
LB01B
Ottos B
Ottos A
TrisMgCl2 B
TrisMgCl2 A
F I G 3 Dendrograms obtained after hierarchical cluster analysis of the following species (A) Sedum burrito (B) Lycopersicon esculentum (C) Celtisaustralis (D) Pisum sativum (E) Festuca rothmaleri (F) Vicia faba according to the parameters FS SS FL CV BF and YF With the exception of S burrito
the four buffers fell into two highly dissimilar clusters of the same buffers
686 Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM
underlines its position as one of the best standards forplant DNA FCM By contrast Sedum burrito was themost difficult species to analyse due to low DNA contentoccurrence of polysomaty and high leaf water contentwhich hampered sample preparation and analysis More-over its tissues may contain tannins (J Greilhuber perscomm 2006)
In O pes-caprae cytosol of which is highly acidic(pH lt 30) measurable samples could be prepared usingonly Ottorsquos and Galbraithrsquos buffers with Ottorsquos beinghighly superior This is in accordance with the results ofEmshwiller (2002) who analysed ploidy levels in OxalisAfter testing LB01 MgSO4 and Ottorsquos buffers sheobtained measurable samples only with Ottorsquos The formertwo buffers failed presumably as a result of the acidic cellsap which may have exceeded the buffering capacity ofLB01 and MgSO4
Celtis australis was the only woody plant speciesincluded in the present study and was chosen because ofthe presence of mucilaginous compounds (Rodriguez et al2005a) which increase sample viscosity restrain nucleirelease and cause their clumping Interestingly this wasthe only species for which TrisMgCl2 was the bestperforming buffer This was probably because of a higherconcentration of the non-ionic detergent which sup-pressed the effect of mucilaginous compounds Leaftissues of F rothmaleri were particularly hard anddifficult to chop In addition preliminary experimentswith this species revealed the presence of cytosoliccompounds which would be expected to interfere withDNA staining However given the pattern of FS and SSobtained the so-called lsquotannic acid effectrsquo (Loureiro et al2006) was absent indicating that these compounds werereleased at low concentration or not at all
In order to compare the performance of nuclear isolationbuffers a set of parameters was carefully selected toevaluate sample quality Furthermore stability of fluores-cence and light scatter properties of isolated nuclei overtime were confirmed Among the parameters chosen thecoefficient of variation of DNA peaks (CV) is of majorimportance Galbraith et al (2002) considered 50 asthe maximum acceptable CV value in plant DNA flowcytometry With the exception of TrisMgCl2 buffer whenused with S burrito all mean CV values obtained hereinwere below this limit Ideally a nuclei sample should befree of cell and tissue debris Emshwiller (2002) noted acorrelation between CV and background noise In thepresent study this correlation was found only in somespecies (eg F rothmaleri) Rather the results heresuggest that the extent of background debris is determinedby the buffer itself For example a higher detergentconcentration in a buffer could lead to chloroplast lysisand consequently decrease the number of fluorescentparticles contributing to debris signals (Coba de la Penaand Brown 2001)
LB01 buffer provided very good results with theexception of O pes-caprae and S burrito low CV highFL and YF values were obtained with this buffer Highnuclei FL intensities obtained with this buffer weredefinitely an advantage as compared with TrisMgCl2 and
Galbraithrsquos buffers Galbraithrsquos buffer seems well bal-anced as acceptable results were achieved in all speciesSurprisingly the buffer gave reasonable results also withO pes-caprae which is characterized by highly acidiccytosol The presence of MOPS in the buffer may shedlight on these findings as it has a pKa of 72 and a betterbuffering capacity than TRIS with a pKa of 81 Adisadvantage of this buffer was the low fluorescenceintensity of nuclei Collectively TrisMgCl2 was the worstperforming buffer Nevertheless it provided the bestresults for C australis This may have been due to thehigher concentration of non-ionic detergent (Table 1)which counteracted the agglutinating effect of mucilagin-ous compounds and decreased sample viscosity As withGalbraithrsquos buffer low FL values were obtained withTrisMgCl2
Ottorsquos buffer is unique in that sample preparationinvolves two steps The results obtained with Ottorsquos bufferwere excellent in many species especially those withlower nuclear DNA content Dolezel and Bartos (2005)highlighted the quality of this buffer which is known toprovide DNA content histograms with unequalled resolu-tion One explanation for this may be that citric acidimproves chromatin accessibility and lsquohomogenizesrsquo chro-matin structure eliminating differences in staining intens-ity among nuclei with the same DNA content but differentchromatin state This could explain the highest FL andlowest CV values as observed here Another characteristicof this buffer was that light scatter values (especially FS)were significantly lower than those of other buffers Thismay be explained by the action of citric acid whichcauses nuclei fixation (Dolezel and Bartos 2005) It isnoteworthy that the pattern of FS vs SS distribution wassimilar to that obtained when analysing nuclei fixed withformaldehyde (Rodriguez et al 2005b) Althoughproviding excellent results this buffer gave the highestBF and lowest YF values The former can be explained bynuclei instability after the addition of Otto II After thisstep nuclei deteriorate rapidly in some species (J Dolezelunpubl res) The lowest YF values could be due to thedetergent (Tween 20) which is weaker than Triton X-100thus having a lower capacity to release nuclei
An important consequence of the observation ofdifferent fluorescence values obtained with the differentbuffers is that different fluorescence ratios may beexpected with the same species pair if the samples areprepared in different buffers In fact Dolezel et al (1998)observed differences in peak ratios obtained in differentlaboratories Further studies are needed to confirm thisobservation as it may have important consequences forestimation of genome size
The present results showed that well-defined popula-tions of nuclei could be observed on cytograms of FS vsSS With the exception of S burrito for which LB01 andTrisMgCl2 buffers did not present statistically significantdifferences for both parameters the analysis of FS and SSprovided a fingerprint pattern for each buffer Loureiroet al (2006) showed that these parameters were sensitiveto the presence of tannic acid a cytosolic compoundcommon in plants and recommended the analysis of light
Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM 687
scatter to verify suitability of particular samples for plantDNA FCM
As nuclei samples are prepared manually it wasimportant to assess the effect of date of analysis andoperator on sample quality variation Date was found to bemore significant than the effect of different operatorsespecially for FS SS CV and DF YF which depends onthe way the sample is chopped and hence on the operatordid not vary It was expected that FL which is a primarysource of data in FCM analysis would not depend on theoperator or the day of chopping This was confirmed in allspecies except L esculentum and C australis In theformer species a significant variation was obtainedbetween operators These differences could be explainedby variable results obtained with Galbraithrsquos buffer InC australis significant differences were obtained amongdates In this case significant differences were due toresults obtained on one single day Emshwiller (2002)found significant differences when multiple preparationsfrom the same plant were run on different days Thesedifferences and those found in the present study wereprobably due to instrument drift (Kudo and Kimura2001) To avoid this type of error several authors haverecommended that each measurement be repeated at leastthree times on three different days (Suda 2004 Dolezeland Bartos 2005)
This is the first study that has systematically comparednuclear isolation buffers for DNA FCM The results showthat none of the buffers works best with all species andstatistically significant differences in sample quality wereobserved among the four buffers The results obtainedwith different species and contrasting types of leaf tissuescan serve as guidelines in buffer selection Nevertheless itis recommend that a range of buffers be tested whenworking with a new species and tissue type Once the bestbuffer has been identified additives should be tested ifrequired to suppress negative effects of phenols and othercytosolic compounds
ACKNOWLEDGEMENTS
We are grateful to Prof Johann Greilhuber and Dr JanSuda for critical reading of the manuscript This work wassupported by FCT project ref POCTIAGR606722004JL was supported by the Fellowship FCTBD90032002
LITERATURE CITEDBennett MD Leitch I 2005 Genome size evolution in plants In
Gregory T ed The evolution of the genome London ElsevierAcademic Press 89ndash162
Bergounioux C Perennes C Brown SC Gadal P 1988 Cytometricanalysis of growth regulator-dependent transcription and cell cycleprogression in Petunia protoplast cultures Planta 175 500ndash505
Bergounioux C Brown SC Petit PX 1992 Flow cytometry and plantprotoplast cell biology Physiologia Plantarum 85 374ndash386
Castro S Loureiro J Santos C Ayensa G Navarro L 2005 Differentploidy levels could explain reproductive matter of invasive Oxalispes-caprae L in Mediterranean regions In Brunel S ed Invasiveplants in Mediterranean type regions of the world StrasbourgCedex France Council of Europe Publishing 235
Coba de la Pena T Brown SC 2001 Cytometry and fluorimetryIn Hawes C Satiat-Jeunemaıtre B eds Plant cell biology
a practical approach New York Oxford University Press85ndash106
Dolezel J Bartos J 2005 Plant DNA flow cytometry and estimation ofnuclear genome size Annals of Botany 95 99ndash110
Dolezel J Gohde W 1995 Sex determination in dioecious plantsMelandrium album and M rubrum using high-resolution flowcytometry Cytometry 19 103ndash106
Dolezel J Binarova P Lucretti S 1989 Analysis of nuclear DNAcontent in plant cells by flow cytometry Biologia Plantarum 31113ndash120
Dolezel J Sgorbati S Lucretti S 1992 Comparison of three DNAfluorochromes for flow cytometric estimation of nuclear DNAcontent in plants Physiologia Plantarum 85 625ndash631
Dolezel J Greilhuber J Lucretti S Meister A Lysak M Nardi LObermayer R 1998 Plant genome size estimation byflow cytometry inter-laboratory comparison Annals of Botany 8217ndash26
Dolezel J Bartos J Voglmayr H Greilhuber J 2003 Nuclear DNAcontent and genome size of trout and human Cytometry 51A127ndash128
Emshwiller E 2002 Ploidy levels among species in the lsquoOxalis tuberosaAlliancersquo as inferred by flow cytometry Annals of Botany 89741ndash753
Galbraith DW 2004 Cytometry and plant sciences a personalretrospective Cytometry 58A 37ndash44
Galbraith DW Harkins KR Maddox JM Ayres NM Sharma DPFiroozabady E 1983 Rapid flow cytometric analysis of the cell-cycle in intact plant-tissues Science 220 1049ndash1051
Galbraith DW Lambert GM Macas J Dolezel J 2002 Analysis ofnuclear DNA content and ploidy in higher plants In Robinson JPDarzynkiewicz Z Dean PN Dressler LG Rabinovitch PS StewartCV Tanke HJ Wheeless LL eds Current protocols in cytometryNew York John Wiley amp Sons 761ndash7622
Heller FO 1973 DNS-Bestimmung an Keimwurzeln von Vicia faba Lmit Hilfe der Impulscytophotometrie Bericht der DeutschenBotanischen Gesellschaft 86 437ndash441
Hintze J 2004 NCSS and PASS Kaysville Utah Number CruncherStatistical Systems
Kudo N Kimura Y 2001 Flow cytometric evidence for endopolyploidyin seedlings of some Brassica species Theoretical and AppliedGenetics 102 104ndash110
Loureiro J Rodriguez E Dolezel J Santos C 2006 Flow cytometricand microscopic analysis of the effect of tannic acid on plantnuclei and estimation of DNA content Annals of Botany 98515ndash527
Noirot M Barre P Louarn J Duperray C Hamon S 2000 Nucleusndashcytosol interactionsmdashA source of stoichiometric error in flowcytometric estimation of nuclear DNA content in plants Annals ofBotany 86 309ndash316
Noirot M Barre P Duperray C Louarn J Hamon S 2003 Effects ofcaffeine and chlorogenic acid on propidium iodide accessibility toDNA consequences on genome size evaluation in coffee treeAnnals of Botany 92 259ndash264
Otto F 1992 Preparation and staining of cells for high-resolution DNAanalysis In Radbruch A ed Flow cytometry and cell sortingBerlin Springer-Verlag 101ndash104
Pfosser M Amon A Lelley T Heberle-Bors E 1995 Evaluation ofsensitivity of flow cytometry in detecting aneuploidy in wheat usingdisomic and ditelosomic wheat-rye addition lines Cytometry 21387ndash393
Pinto G Loureiro J Lopes T Santos C 2004 Analysis of the geneticstability of Eucalyptus globulus Labill somatic embryos by flowcytometry Theoretical and Applied Genetics 109 580ndash587
Rodriguez E Gomes A Loureiro J Dolezel J Santos C 2005aEstimation of the genome size of the Iberian Peninsula Ulmaceae In9th Iberian Congress of Cytometry Book of Abstracts PortoPortugal 37
Rodriguez E Loureiro J Dolezel J Santos C 2005b The adequacy ofusing formaldehyde fixation for nuclear DNA content analyses ofplant material In 9th Iberian Congress of Cytometry Book ofAbstracts Porto Portugal PO120-PB
Shapiro H 2004 Practical flow cytometry 4th edn New YorkWiley-Liss
688 Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM
Suda J 2004 An employment of flow cytometry into plant biosystematicsPhD thesis Charles University Czech Republic Available at httpwwwibotcasczfcmsudapresentationdisertationpdf
Trotter J 2000 WinMDI version 28 Available at httpfacsscrippsedusoftwarehtml
Ulrich I Ulrich W 1986 Flow cytometric DNA analysis of plantprotoplasts with DAPI Zeitschrift fur Naturforschung 411052ndash1056
Ulrich I Fritz B Ulrich W 1988 Application of DNA fluorochromes forflow cytometric DNA analysis of plant protoplasts Plant Science 55151ndash158
Walker D Monino I Correal E 2006 Genome size inBituminaria bituminosa (L) CH Stirton (Fabaceae) populationsseparation of lsquotruersquo differences from environmental effects on DNAdetermination Environmental and Experimental Botany 55258ndash265
Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM 689
levels instrument gain was set such that the 2C peak wasapproximately on channel 100 The 2C nuclear DNAcontent was estimated as 130 6 009 pg (2C = 1271 Mbp)this is the first estimate for this species (Table 1) Com-parative analysis of the four buffers revealed that Ottorsquoswas the best in this species In general YF values were low
(the lowest values from all the test species) whereas DFvalues were high (approx 650 Table 3 Fig 1B)
Oxalis pes-caprae
Only Ottorsquos and Galbraithrsquos buffers provided acceptableresults with this species Ottorsquos was clearly and
0 75 1500
25
1023
43
3
2
2
2
2
2
2
1
1
1
1
1
PI
Peak FL CV() 1 108middot7 5middot87 2 118middot7 3middot00 3 206middot8 4middot10 4 231middot0 2middot55
TrismiddotMgCl2 bufferGreenOttorsquos bufferBlue
Peak FL CV() 1 97middot6 9middot74 2 187middot0 3middot50 3 201middot1 2middot69
TrismiddotMgCl2 bufferGreenGalbraithrsquos bufferOrangeOttorsquos bufferBlue
Peak FL CV() 1 226middot1 4middot09 2 252middot1 1middot96
TrismiddotMgCl2 bufferGreenLB01 bufferRed
Peak FL CV() 1 190middot3 2middot21 2 205middot8 3middot90
TrismiddotMgCl2 bufferGreenOttorsquos bufferBlue
Peak FL CV() 1 184middot1 2middot78 2 213middot8 3middot48
LB01 bufferRedOttorsquos bufferBlue
Peak FL CV() 1 168middot4 3middot32 2 202middot6 2middot10
Ottorsquos bufferBlueLB01 bufferRed
Peak FL CV() 1 180middot5 1middot52 2 188middot1 3middot07
Ottorsquos bufferBlueTrismiddotMgCl2 bufferGreen
0
50
75
100
125
Time (s)
Ottorsquos buffer
PI
225
0
450
0
512
128
00 PI 1023
300
Cou
nt
Cou
ntC
ount
Cou
nt
0
1024
Cou
nt
0
400
1
0
512
Cou
nt
2
1
1023PI0
512
0
Cou
nt
A B
C D
E F
G H
F I G 1 Cytogram of fluorescence intensity (PI relative channel numbers) vs time of Pisum sativum nuclei isolated with Ottorsquos buffer (A) and histograms ofrelative fluorescence intensities (BndashH) which show overlays of distributions obtained with the best and worst performing buffer for each species (B) Sedumburrito (C) Oxalis pes-caprae (D) Lycopersicon esculentum (E) Celtis australis (F) Pisum sativum (G) Festuca rothmaleri and (H) Vicia faba Relativemean channel numbers and coefficients of variation (CV ) of G0G1 peaks are given Four lysis buffers were compared LB01 (red) Galbraithrsquos (orange)
TrisMgCl2 (green) and Ottorsquos (blue)
682 Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM
TA
BL
E3
F
low
cyto
met
ric
pa
ram
eter
sa
sses
sed
inea
chsp
ecie
s
FS
(ch
ann
elu
nit
s)S
S(c
han
nel
un
its)
FL
(ch
ann
elu
nit
s)C
V(
)B
F(
)Y
F(n
ucl
eis
1m
g
1)
Sp
ecie
sB
uff
erM
ean
SD
Mea
nS
DM
ean
SD
Mea
nS
DM
ean
SD
Mea
nS
D
Sed
um
bu
rrit
oL
B0
15
76
6a
8 7
24
11 3
7a
2 4
48
11
0 9
a5
93
4 4
8a
0 4
07
69 1
5a
24 9
03
0 0
6a
0 0
04
Gal
bra
ith
41 0
6b
5 1
71
5 3
6b
2 2
99
11
2 1
a2
57
3 5
9ab
0 3
96
60 0
2a
16 6
31
0 0
5b
0 0
04
Tri
sM
gC
l 25
75
0a
8 5
56
13 3
4a
2 6
70
10
3 0
b6
19
5 6
1c
1 0
00
68 8
5a
13 8
74
0 0
5ab
0 0
04
Ott
o8 0
1c
2 1
79
2 5
8c
0 7
87
12
1 4
c3
93
3 2
7b
0 2
88
65 9
6a
6 3
30
05
ab
0 0
06
Oxa
lis
pes
-ca
pra
eL
B0
1ndash
ndashndash
ndashndash
ndashndash
ndashndash
ndashndash
ndashG
alb
rait
h1
71 3
3a
36 4
04
19 7
5a
6 9
20
17
8 5
a5
21
4 1
4a
0 7
78
7 0
5a
4 9
77
1 0
1a
0 3
64
Tri
sM
gC
l 2ndash
ndashndash
ndashndash
ndashndash
ndashndash
ndashndash
ndashO
tto
26 3
2b
4 9
33
3 9
0b
0 6
41
20
0 7
b6
24
3 0
3b
0 4
15
8 9
6a
5 9
08
0 6
2b
0 1
25
Lyc
oper
sico
nes
cule
ntu
mL
B0
11
5 4
2a
2 1
25
2 9
7a
0 4
94
25
7 9
a1
1 5
02 8
8a
0 8
80
21 2
1a
14 1
37
2 4
7a
1 5
12
Gal
bra
ith
21 5
3b
3 7
52
3 4
0a
0 7
13
20
8 5
b4
9 6
12 8
7a
0 7
12
16 8
7a
10 0
36
1 6
9ab
0 8
14
Tri
sM
gC
l 21
7 4
8a
4 9
70
4 7
6b
1 5
62
21
6 7
b2
7 2
73 7
0b
0 8
36
20 5
2a
12 9
18
1 7
2ab
1 7
45
Ott
o3 6
8c
1 5
37
1 9
5c
0 7
66
26
9 9
a1
1 1
72 1
8c
0 2
66
28 4
9b
13 2
54
0 9
1b
0 6
17
Cel
tis
au
stra
lis
LB
01
28 4
7a
4 8
47
11 4
5a
3 4
81
18
2 1
a1
3 1
33 0
2a
0 3
15
7 6
6a
3 5
65
0 5
0a
0 2
24
Gal
bra
ith
25 5
7a
1 8
42
4 5
4b
0 6
37
17
1 3
b7
76
2 8
7a
0 2
59
8 2
7a
3 4
45
0 3
5a
0 1
62
Tri
sM
gC
l 24
1 7
1b
6 9
44
16 0
8c
2 1
57
18
0 3
a1
0 4
42 8
5a
0 4
19
9 1
0b
5 0
85
0 4
9a
0 2
91
Ott
o8 2
0c
1 4
04
7 2
5b
1 7
87
19
2 8
c8
23
3 4
6b
0 4
05
23 9
6c
4 6
15
0 4
9a
0 2
00
Pis
um
sati
vum
LB
01
47 5
4a
1 5
50
10 8
7a
3 0
78
18
3 5
a5
06
2 8
1a
0 4
61
7 1
9a
2 8
23
2 3
3a
0 4
90
Gal
bra
ith
51 9
7a
3 6
24
5 2
9b
1 1
55
17
8 6
a3
36
3 0
2ab
0 4
65
6 3
4a
1 7
65
2 3
0a
0 8
76
Tri
sM
gC
l 25
9 3
7b
4 8
26
16 7
4c
2 9
14
17
7 7
a1
1 8
63 2
9b
0 5
15
6 3
9a
2 7
81
2 5
9a
0 8
94
Ott
o5 7
2c
0 9
93
4 7
9b
1 0
83
19
0 1
a6
06
1 9
4c
0 1
80
9 8
5b
2 6
68
1 1
5b
0 3
67
Fes
tuca
roth
ma
leri
LB
01
57 3
5a
1 8
25
8 5
7a
0 7
99
19
6 1
a6
07
3 2
4a
0 4
52
14 2
3ab
3 2
83
0 4
2a
0 1
82
Gal
bra
ith
61 0
3a
2 2
50
6 8
4b
0 6
49
18
2 7
b7
15
3 3
3ab
0 4
59
15 0
7a
1 9
74
0 2
7b
0 1
31
Tri
sM
gC
l 26
9 8
5a
1 6
03
17 7
2c
0 8
15
18
5 1
b8
06
3 6
6ab
0 3
12
17 3
0b
5 7
01
0 5
4a
0 1
95
Ott
o1
3 8
4b
2 8
46
8 5
5a
1 6
03
21
0 9
c6
77
3 7
6b
0 6
13
23 8
6c
12 9
92
0 1
1c
0 0
66
Vic
iafa
ba
LB
01
10
4 8
2a
6 0
21
11 6
1a
1 7
42
20
1 5
a4
45
2 4
0a
0 1
78
6 3
6a
2 3
26
0 8
7a
0 2
90
Ga
lbra
ith
11
4 5
5b
2 7
87
8 5
8b
1 3
06
19
6 9
a4
88
2 4
1a
0 1
48
5 8
0a
0 8
06
0 8
2a
0 2
35
Tri
sM
gC
l 21
15 4
5b
5 1
94
20 3
6c
1 4
43
19
1 5
a9
10
2 9
1b
0 2
72
6 8
5a
4 6
09
1 3
0b
0 3
45
Ott
o2
1 5
6c
8 7
21
14 0
5a
7 5
89
18
4 2
a1
6 9
22 2
2a
0 5
32
4 3
5a
1 7
51
0 4
5c
0 1
8
Val
ues
are
giv
enas
mea
nan
dst
and
ard
dev
iati
on
of
the
mea
n(S
D)
of
forw
ard
scat
ter
(FS
ch
ann
elu
nit
s)s
ide
scat
ter
(SS
chan
nel
un
its)
flu
ore
scen
ce(F
Lc
han
nel
un
its)
coef
fici
ento
fv
aria
tio
no
fG
0G
1D
NA
pea
k(C
V
)b
ack
gro
un
dfa
cto
r(B
F
)an
dn
ucl
eary
ield
fact
or(Y
Fn
ucl
eisndash
1m
gndash1)
Mea
ns
foll
ow
edb
yth
esa
me
lett
er(a
bo
rc)
are
no
tsta
tist
ical
lyd
iffe
ren
tacc
ord
ing
toth
em
ult
iple
com
par
ison
Ho
lmndashS
idak
test
atPlt
00
5
Buff
er(s
)th
atp
erfo
rmed
bes
tin
each
spec
ies
are
sho
wn
inb
old
typ
e
Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM 683
significantly better than Galbraithrsquos (Fig 1C) with highermean FL intensities and lower CV values (Table 3)Figure 1C also shows the histogram obtained after nuclearisolation with TrisMgCl2 buffer In this case a G0G1 peakwith an unacceptable CV value (974 ) and considerableloss of fluorescence was obtained A similar result wasobtained for nuclei isolated with LB01
Lycopersicon esculentum
Acceptable results were obtained with all four buffersTwo buffer groups with statistically significant differencesin FL were obtained Samples prepared with Galbraithrsquosand TrisMgCl2 buffers yielded lower mean FL valuesthan their counterparts prepared with LB01 and Ottorsquosbuffers (Fig 1D) The FL values were highly heterogen-eous among buffers Ottorsquos and LB01 were the bestbuffers with Ottorsquos providing lower CV values and higherFL but lower YF and higher BF than LB01 (Table 3)
Celtis australis
Low CV values (lt30 ) and low DF (lt100 ) wereobserved for this species Nevertheless it was not easy toobtain suffiecient nuclei and the second lowest YF valueswere observed in this species With regard to FL onlyLB01 and TrisMgCl2 buffers were not statistically dif-ferent with nuclei isolated from Galbraithrsquos buffer pre-senting the lowest mean FL and Ottorsquos the highest meanFL All parameters combined TrisMgCl2 and LB01 werethe best buffers as nuclei in TrisMgCl2 presented thelowest CV values and similar FL intensity and YF valuesas in LB01 (Table 3 Fig 1E)
Pisum sativum
In this species all buffers performed reasonably wellThe lowest FL intensities were obtained for nucleiisolated with TrisMgCl2 although no statistically signi-ficant differences were observed among the tested buffersThe best buffer for this species was Ottorsquos (Table 3Fig 1F) Among the investigated species P sativum wasthe one with the highest YF
Festuca rothmaleri
With the exception of CV values and YF overall resultsfor this species were satisfying with all four buffers testedNo statistically significant differences were found regard-ing the FL of nuclei isolated in TrisMgCl2 or Galbraithrsquosbuffers as nuclei from both buffers presented low FLvalues The best buffer for this species was LB01 (Table 3Fig 1G)
Vicia faba
This species gave the lowest CV and DF values amongthose tested Generally the results were very similar tothose obtained for P sativum FL was similar for all thebuffers and no statistically significant differences wereobserved Interestingly Vicia faba was the only species
for which Ottorsquos was not the buffer with the highest FL(Fig 1H) Despite low CV values of DNA peaks thisbuffer gave the worst results with the G0G1 peak shiftedtowards the lower channels This was due to fluorescenceinstability which decreased over time In all other speciesand with the remaining buffers FL was stable after 10 minof incubation with PI (Fig 1A) Results obtained withLB01 and Galbraithrsquos buffers were similar and the best forthis species (Table 3)
Analysis of FS and SS
Generally FS and SS values differed considerablyamong the test buffers Nevertheless in most of thespecies analysis of scatter parameters revealed that two ofthe four buffers were more similar than the others and nostatistically significant differences were observed betweenthem Interestingly and with the exception of S burritobuffers that had similar FS mean values were not thosethat had similar SS values This can be seen on cytogramsof FS vs SS obtained in P sativum (Fig 2) In thisspecies and for FS no statistically significant differenceswere observed between LB01 and Galbraithrsquos buffers forSS no difference was observed for Galbraithrsquos and Ottorsquosbuffers Interestingly simultaneous analysis of FS and SSresulted in a species-specific pattern that could be used asa fingerprint of each buffer
Effect of operator and date of analysis
In most cases no statistically significant differenceswere observed between operators and dates of analysisOperators provided more homogeneous results than datesin the former statistically significant differences wereobserved only for BF and YF in more than one speciesSignificant differences between the dates of analysis weredetected in some species for FS SS CV and BF Withregard to FL one of the most important parameters inFCM analyses significant among-day differences weredetected only in C australis and differences betweenoperators occurred only in L esculentum The two speciesmore susceptible to differences were L esculentum andV faba (Table 4)
Hierarchical cluster analysis
With the exception of the results for S burrito the fourbuffers fell into two highly dissimilar and consistentclusters one with Ottorsquos buffer and the other with theremaining buffers In S burrito (Fig 3A) one cluster wasformed with Ottorsquos and Galbraithrsquos buffers while theother comprised TrisMgCl2 and LB01 buffers InC australis (Fig 3C) Galbraithrsquos buffer was more similarto LB01 and TrisMgCl2 buffers than to Ottorsquos buffer inaddition lsquoTrisMgCl2 Arsquo was more similar to LB01 thanto lsquoTrisMgCl2 Brsquo In P sativum lsquoGalbraithrsquos Arsquo wasmore related to LB01 than to lsquoGalbraithrsquos Brsquo (Fig 3D)whereas for F rothmaleri lsquoLB01 Arsquo was more similar toGalbraithrsquos than to lsquoLB01 Brsquo (Fig 3E) In L esculentumtwo groups were formed from the second clusterTrisMgCl2 and lsquoGalbraith Arsquo formed one group and
684 Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM
FS log
C
SS lo
g
1 10 100 10000middot1
0middot1
1
10
100
1000
Meanchannel
FSSS 18middot2
TrisMgCl2
37middot8735middot6960middot7()
FPCV
Meanchannel
FSSS
LB01ASS
log
0middot1
1
10
100
1000
10middot045middot4
30middot0226middot09()
FPCV
FSSS 3middot1 37middot24
29middot936middot0
Ottorsquos
FS log
D
1 10 100 10000middot1
Meanchannel ()
FPCV
FS 52middot1 28middot42SS 5middot2 39middot04
GalbraithsB
Meanchannel ()
FPCV
F I G 2 Cytograms of forward scatter (logarithmic scale FS log) vs side scatter (logarithmic scale SS log) obtained after the analysis of Pisum sativum nucleiisolated with four lysis buffers (A) LB01 (B) Galbraithrsquos (C) TrisMgCl2 and (D) Ottorsquos The mean channel number and full peak coefficient of variation
(FPCV ) are given for both parameters Note that the patterns of distributions are characteristic for each buffer
T A B L E 4 Three-way ANOVA analysis of the dates (D) and operators (O) for the parameters evaluated on each species
FS SS FL CV BF YF
Species D O D O D O D O D O D O
Sedum burrito ns ns ns ns ns ns ns ns s ns ns nsOxalis pes-caprae s ns s ns ns ns s ns ns ns ns nsLycopersicon esculentum s ns ns ns ns s s ns s s s sCeltis australis ns ns ns ns s ns ns ns s ns ns nsPisum sativum ns ns s ns ns ns s ns s ns ns nsFestuca rothmaleri s ns ns ns ns ns ns ns ns s ns nsVicia faba ns s s s ns ns ns ns ns ns s s
Forward scatter FS side scatter SS fluorescence FL coefficient of variation of the G0G1 DNA peak CV background factor BF and nuclearyield factor YF
ns not significantly different s significantly different at P lt 005
Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM 685
LB01 and lsquoGalbraith Brsquo formed the other (Fig 3B) InV faba (Fig 3F) LB01 and Galbraithrsquos formed one groupowing to greater similarities between operators thanwithin each buffer As previously stated in O pes-capraeonly two buffers (Ottorsquos and Galbraithrsquos) providedacceptable results with considerable dissimilaritiesbetween them (data not shown)
DISCUSSION
Four nuclear isolation buffers were used with a setof species that were chosen to represent different types
of leaf tissues and different nuclear DNA content(130ndash2690 pg per 2C DNA) As expected popularDNA reference standards (P sativum V faba andL esculentum) were easy to work with Neverthelessnot all buffers worked well with L esculentumpossibly owing to the presence of cytosolic compoundsHowever as the aim of the study was to compare theperformance of basic buffer formulas the use of additivesthat could counteract the negative effects of cytosol wasavoided
Overall the best results were obtained with P sativumAs its 2C nuclear DNA content is in the middle of theknown range of genome sizes in plants this observation
A
C
E
B
D
F
Dissimilarity Dissimilarity
2middot0 1middot5 1middot0 0middot5 0middot02middot0 1middot5 1middot0 0middot5 0middot0
2middot0 1middot5 1middot0 0middot5 0middot0
2middot0 1middot5 1middot0 0middot5 0middot0 2middot0 1middot5 1middot0 0middot5 0middot0
2middot0 1middot5 1middot0 0middot5 0middot0
LB01A
LB01B
LB01A
LB01B
Ottos B
Ottos A
Galbraiths B
Galbraiths A
TrisMgCl2 B
TrisMgCl2 A
Ottos B
Ottos A
Galbraiths B
Galbraiths A
Galbraiths B
Galbraiths A
TrisMgCl2 B
TrisMgCl2 A
LB01A
LB01B
Ottos B
Ottos A
TrisMgCl2 B
TrisMgCl2 A
LB01A
LB01B
LB01A
LB01B
Ottos B
Ottos A
Galbraiths A
Galbraiths B
TrisMgCl2 A
TrisMgCl2 B
Ottos B
Ottos A
Galbraiths B
Galbraiths A
Galbraiths B
Galbraiths A
TrisMgCl2 B
TrisMgCl2 A
LB01A
LB01B
Ottos B
Ottos A
TrisMgCl2 B
TrisMgCl2 A
F I G 3 Dendrograms obtained after hierarchical cluster analysis of the following species (A) Sedum burrito (B) Lycopersicon esculentum (C) Celtisaustralis (D) Pisum sativum (E) Festuca rothmaleri (F) Vicia faba according to the parameters FS SS FL CV BF and YF With the exception of S burrito
the four buffers fell into two highly dissimilar clusters of the same buffers
686 Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM
underlines its position as one of the best standards forplant DNA FCM By contrast Sedum burrito was themost difficult species to analyse due to low DNA contentoccurrence of polysomaty and high leaf water contentwhich hampered sample preparation and analysis More-over its tissues may contain tannins (J Greilhuber perscomm 2006)
In O pes-caprae cytosol of which is highly acidic(pH lt 30) measurable samples could be prepared usingonly Ottorsquos and Galbraithrsquos buffers with Ottorsquos beinghighly superior This is in accordance with the results ofEmshwiller (2002) who analysed ploidy levels in OxalisAfter testing LB01 MgSO4 and Ottorsquos buffers sheobtained measurable samples only with Ottorsquos The formertwo buffers failed presumably as a result of the acidic cellsap which may have exceeded the buffering capacity ofLB01 and MgSO4
Celtis australis was the only woody plant speciesincluded in the present study and was chosen because ofthe presence of mucilaginous compounds (Rodriguez et al2005a) which increase sample viscosity restrain nucleirelease and cause their clumping Interestingly this wasthe only species for which TrisMgCl2 was the bestperforming buffer This was probably because of a higherconcentration of the non-ionic detergent which sup-pressed the effect of mucilaginous compounds Leaftissues of F rothmaleri were particularly hard anddifficult to chop In addition preliminary experimentswith this species revealed the presence of cytosoliccompounds which would be expected to interfere withDNA staining However given the pattern of FS and SSobtained the so-called lsquotannic acid effectrsquo (Loureiro et al2006) was absent indicating that these compounds werereleased at low concentration or not at all
In order to compare the performance of nuclear isolationbuffers a set of parameters was carefully selected toevaluate sample quality Furthermore stability of fluores-cence and light scatter properties of isolated nuclei overtime were confirmed Among the parameters chosen thecoefficient of variation of DNA peaks (CV) is of majorimportance Galbraith et al (2002) considered 50 asthe maximum acceptable CV value in plant DNA flowcytometry With the exception of TrisMgCl2 buffer whenused with S burrito all mean CV values obtained hereinwere below this limit Ideally a nuclei sample should befree of cell and tissue debris Emshwiller (2002) noted acorrelation between CV and background noise In thepresent study this correlation was found only in somespecies (eg F rothmaleri) Rather the results heresuggest that the extent of background debris is determinedby the buffer itself For example a higher detergentconcentration in a buffer could lead to chloroplast lysisand consequently decrease the number of fluorescentparticles contributing to debris signals (Coba de la Penaand Brown 2001)
LB01 buffer provided very good results with theexception of O pes-caprae and S burrito low CV highFL and YF values were obtained with this buffer Highnuclei FL intensities obtained with this buffer weredefinitely an advantage as compared with TrisMgCl2 and
Galbraithrsquos buffers Galbraithrsquos buffer seems well bal-anced as acceptable results were achieved in all speciesSurprisingly the buffer gave reasonable results also withO pes-caprae which is characterized by highly acidiccytosol The presence of MOPS in the buffer may shedlight on these findings as it has a pKa of 72 and a betterbuffering capacity than TRIS with a pKa of 81 Adisadvantage of this buffer was the low fluorescenceintensity of nuclei Collectively TrisMgCl2 was the worstperforming buffer Nevertheless it provided the bestresults for C australis This may have been due to thehigher concentration of non-ionic detergent (Table 1)which counteracted the agglutinating effect of mucilagin-ous compounds and decreased sample viscosity As withGalbraithrsquos buffer low FL values were obtained withTrisMgCl2
Ottorsquos buffer is unique in that sample preparationinvolves two steps The results obtained with Ottorsquos bufferwere excellent in many species especially those withlower nuclear DNA content Dolezel and Bartos (2005)highlighted the quality of this buffer which is known toprovide DNA content histograms with unequalled resolu-tion One explanation for this may be that citric acidimproves chromatin accessibility and lsquohomogenizesrsquo chro-matin structure eliminating differences in staining intens-ity among nuclei with the same DNA content but differentchromatin state This could explain the highest FL andlowest CV values as observed here Another characteristicof this buffer was that light scatter values (especially FS)were significantly lower than those of other buffers Thismay be explained by the action of citric acid whichcauses nuclei fixation (Dolezel and Bartos 2005) It isnoteworthy that the pattern of FS vs SS distribution wassimilar to that obtained when analysing nuclei fixed withformaldehyde (Rodriguez et al 2005b) Althoughproviding excellent results this buffer gave the highestBF and lowest YF values The former can be explained bynuclei instability after the addition of Otto II After thisstep nuclei deteriorate rapidly in some species (J Dolezelunpubl res) The lowest YF values could be due to thedetergent (Tween 20) which is weaker than Triton X-100thus having a lower capacity to release nuclei
An important consequence of the observation ofdifferent fluorescence values obtained with the differentbuffers is that different fluorescence ratios may beexpected with the same species pair if the samples areprepared in different buffers In fact Dolezel et al (1998)observed differences in peak ratios obtained in differentlaboratories Further studies are needed to confirm thisobservation as it may have important consequences forestimation of genome size
The present results showed that well-defined popula-tions of nuclei could be observed on cytograms of FS vsSS With the exception of S burrito for which LB01 andTrisMgCl2 buffers did not present statistically significantdifferences for both parameters the analysis of FS and SSprovided a fingerprint pattern for each buffer Loureiroet al (2006) showed that these parameters were sensitiveto the presence of tannic acid a cytosolic compoundcommon in plants and recommended the analysis of light
Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM 687
scatter to verify suitability of particular samples for plantDNA FCM
As nuclei samples are prepared manually it wasimportant to assess the effect of date of analysis andoperator on sample quality variation Date was found to bemore significant than the effect of different operatorsespecially for FS SS CV and DF YF which depends onthe way the sample is chopped and hence on the operatordid not vary It was expected that FL which is a primarysource of data in FCM analysis would not depend on theoperator or the day of chopping This was confirmed in allspecies except L esculentum and C australis In theformer species a significant variation was obtainedbetween operators These differences could be explainedby variable results obtained with Galbraithrsquos buffer InC australis significant differences were obtained amongdates In this case significant differences were due toresults obtained on one single day Emshwiller (2002)found significant differences when multiple preparationsfrom the same plant were run on different days Thesedifferences and those found in the present study wereprobably due to instrument drift (Kudo and Kimura2001) To avoid this type of error several authors haverecommended that each measurement be repeated at leastthree times on three different days (Suda 2004 Dolezeland Bartos 2005)
This is the first study that has systematically comparednuclear isolation buffers for DNA FCM The results showthat none of the buffers works best with all species andstatistically significant differences in sample quality wereobserved among the four buffers The results obtainedwith different species and contrasting types of leaf tissuescan serve as guidelines in buffer selection Nevertheless itis recommend that a range of buffers be tested whenworking with a new species and tissue type Once the bestbuffer has been identified additives should be tested ifrequired to suppress negative effects of phenols and othercytosolic compounds
ACKNOWLEDGEMENTS
We are grateful to Prof Johann Greilhuber and Dr JanSuda for critical reading of the manuscript This work wassupported by FCT project ref POCTIAGR606722004JL was supported by the Fellowship FCTBD90032002
LITERATURE CITEDBennett MD Leitch I 2005 Genome size evolution in plants In
Gregory T ed The evolution of the genome London ElsevierAcademic Press 89ndash162
Bergounioux C Perennes C Brown SC Gadal P 1988 Cytometricanalysis of growth regulator-dependent transcription and cell cycleprogression in Petunia protoplast cultures Planta 175 500ndash505
Bergounioux C Brown SC Petit PX 1992 Flow cytometry and plantprotoplast cell biology Physiologia Plantarum 85 374ndash386
Castro S Loureiro J Santos C Ayensa G Navarro L 2005 Differentploidy levels could explain reproductive matter of invasive Oxalispes-caprae L in Mediterranean regions In Brunel S ed Invasiveplants in Mediterranean type regions of the world StrasbourgCedex France Council of Europe Publishing 235
Coba de la Pena T Brown SC 2001 Cytometry and fluorimetryIn Hawes C Satiat-Jeunemaıtre B eds Plant cell biology
a practical approach New York Oxford University Press85ndash106
Dolezel J Bartos J 2005 Plant DNA flow cytometry and estimation ofnuclear genome size Annals of Botany 95 99ndash110
Dolezel J Gohde W 1995 Sex determination in dioecious plantsMelandrium album and M rubrum using high-resolution flowcytometry Cytometry 19 103ndash106
Dolezel J Binarova P Lucretti S 1989 Analysis of nuclear DNAcontent in plant cells by flow cytometry Biologia Plantarum 31113ndash120
Dolezel J Sgorbati S Lucretti S 1992 Comparison of three DNAfluorochromes for flow cytometric estimation of nuclear DNAcontent in plants Physiologia Plantarum 85 625ndash631
Dolezel J Greilhuber J Lucretti S Meister A Lysak M Nardi LObermayer R 1998 Plant genome size estimation byflow cytometry inter-laboratory comparison Annals of Botany 8217ndash26
Dolezel J Bartos J Voglmayr H Greilhuber J 2003 Nuclear DNAcontent and genome size of trout and human Cytometry 51A127ndash128
Emshwiller E 2002 Ploidy levels among species in the lsquoOxalis tuberosaAlliancersquo as inferred by flow cytometry Annals of Botany 89741ndash753
Galbraith DW 2004 Cytometry and plant sciences a personalretrospective Cytometry 58A 37ndash44
Galbraith DW Harkins KR Maddox JM Ayres NM Sharma DPFiroozabady E 1983 Rapid flow cytometric analysis of the cell-cycle in intact plant-tissues Science 220 1049ndash1051
Galbraith DW Lambert GM Macas J Dolezel J 2002 Analysis ofnuclear DNA content and ploidy in higher plants In Robinson JPDarzynkiewicz Z Dean PN Dressler LG Rabinovitch PS StewartCV Tanke HJ Wheeless LL eds Current protocols in cytometryNew York John Wiley amp Sons 761ndash7622
Heller FO 1973 DNS-Bestimmung an Keimwurzeln von Vicia faba Lmit Hilfe der Impulscytophotometrie Bericht der DeutschenBotanischen Gesellschaft 86 437ndash441
Hintze J 2004 NCSS and PASS Kaysville Utah Number CruncherStatistical Systems
Kudo N Kimura Y 2001 Flow cytometric evidence for endopolyploidyin seedlings of some Brassica species Theoretical and AppliedGenetics 102 104ndash110
Loureiro J Rodriguez E Dolezel J Santos C 2006 Flow cytometricand microscopic analysis of the effect of tannic acid on plantnuclei and estimation of DNA content Annals of Botany 98515ndash527
Noirot M Barre P Louarn J Duperray C Hamon S 2000 Nucleusndashcytosol interactionsmdashA source of stoichiometric error in flowcytometric estimation of nuclear DNA content in plants Annals ofBotany 86 309ndash316
Noirot M Barre P Duperray C Louarn J Hamon S 2003 Effects ofcaffeine and chlorogenic acid on propidium iodide accessibility toDNA consequences on genome size evaluation in coffee treeAnnals of Botany 92 259ndash264
Otto F 1992 Preparation and staining of cells for high-resolution DNAanalysis In Radbruch A ed Flow cytometry and cell sortingBerlin Springer-Verlag 101ndash104
Pfosser M Amon A Lelley T Heberle-Bors E 1995 Evaluation ofsensitivity of flow cytometry in detecting aneuploidy in wheat usingdisomic and ditelosomic wheat-rye addition lines Cytometry 21387ndash393
Pinto G Loureiro J Lopes T Santos C 2004 Analysis of the geneticstability of Eucalyptus globulus Labill somatic embryos by flowcytometry Theoretical and Applied Genetics 109 580ndash587
Rodriguez E Gomes A Loureiro J Dolezel J Santos C 2005aEstimation of the genome size of the Iberian Peninsula Ulmaceae In9th Iberian Congress of Cytometry Book of Abstracts PortoPortugal 37
Rodriguez E Loureiro J Dolezel J Santos C 2005b The adequacy ofusing formaldehyde fixation for nuclear DNA content analyses ofplant material In 9th Iberian Congress of Cytometry Book ofAbstracts Porto Portugal PO120-PB
Shapiro H 2004 Practical flow cytometry 4th edn New YorkWiley-Liss
688 Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM
Suda J 2004 An employment of flow cytometry into plant biosystematicsPhD thesis Charles University Czech Republic Available at httpwwwibotcasczfcmsudapresentationdisertationpdf
Trotter J 2000 WinMDI version 28 Available at httpfacsscrippsedusoftwarehtml
Ulrich I Ulrich W 1986 Flow cytometric DNA analysis of plantprotoplasts with DAPI Zeitschrift fur Naturforschung 411052ndash1056
Ulrich I Fritz B Ulrich W 1988 Application of DNA fluorochromes forflow cytometric DNA analysis of plant protoplasts Plant Science 55151ndash158
Walker D Monino I Correal E 2006 Genome size inBituminaria bituminosa (L) CH Stirton (Fabaceae) populationsseparation of lsquotruersquo differences from environmental effects on DNAdetermination Environmental and Experimental Botany 55258ndash265
Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM 689
TA
BL
E3
F
low
cyto
met
ric
pa
ram
eter
sa
sses
sed
inea
chsp
ecie
s
FS
(ch
ann
elu
nit
s)S
S(c
han
nel
un
its)
FL
(ch
ann
elu
nit
s)C
V(
)B
F(
)Y
F(n
ucl
eis
1m
g
1)
Sp
ecie
sB
uff
erM
ean
SD
Mea
nS
DM
ean
SD
Mea
nS
DM
ean
SD
Mea
nS
D
Sed
um
bu
rrit
oL
B0
15
76
6a
8 7
24
11 3
7a
2 4
48
11
0 9
a5
93
4 4
8a
0 4
07
69 1
5a
24 9
03
0 0
6a
0 0
04
Gal
bra
ith
41 0
6b
5 1
71
5 3
6b
2 2
99
11
2 1
a2
57
3 5
9ab
0 3
96
60 0
2a
16 6
31
0 0
5b
0 0
04
Tri
sM
gC
l 25
75
0a
8 5
56
13 3
4a
2 6
70
10
3 0
b6
19
5 6
1c
1 0
00
68 8
5a
13 8
74
0 0
5ab
0 0
04
Ott
o8 0
1c
2 1
79
2 5
8c
0 7
87
12
1 4
c3
93
3 2
7b
0 2
88
65 9
6a
6 3
30
05
ab
0 0
06
Oxa
lis
pes
-ca
pra
eL
B0
1ndash
ndashndash
ndashndash
ndashndash
ndashndash
ndashndash
ndashG
alb
rait
h1
71 3
3a
36 4
04
19 7
5a
6 9
20
17
8 5
a5
21
4 1
4a
0 7
78
7 0
5a
4 9
77
1 0
1a
0 3
64
Tri
sM
gC
l 2ndash
ndashndash
ndashndash
ndashndash
ndashndash
ndashndash
ndashO
tto
26 3
2b
4 9
33
3 9
0b
0 6
41
20
0 7
b6
24
3 0
3b
0 4
15
8 9
6a
5 9
08
0 6
2b
0 1
25
Lyc
oper
sico
nes
cule
ntu
mL
B0
11
5 4
2a
2 1
25
2 9
7a
0 4
94
25
7 9
a1
1 5
02 8
8a
0 8
80
21 2
1a
14 1
37
2 4
7a
1 5
12
Gal
bra
ith
21 5
3b
3 7
52
3 4
0a
0 7
13
20
8 5
b4
9 6
12 8
7a
0 7
12
16 8
7a
10 0
36
1 6
9ab
0 8
14
Tri
sM
gC
l 21
7 4
8a
4 9
70
4 7
6b
1 5
62
21
6 7
b2
7 2
73 7
0b
0 8
36
20 5
2a
12 9
18
1 7
2ab
1 7
45
Ott
o3 6
8c
1 5
37
1 9
5c
0 7
66
26
9 9
a1
1 1
72 1
8c
0 2
66
28 4
9b
13 2
54
0 9
1b
0 6
17
Cel
tis
au
stra
lis
LB
01
28 4
7a
4 8
47
11 4
5a
3 4
81
18
2 1
a1
3 1
33 0
2a
0 3
15
7 6
6a
3 5
65
0 5
0a
0 2
24
Gal
bra
ith
25 5
7a
1 8
42
4 5
4b
0 6
37
17
1 3
b7
76
2 8
7a
0 2
59
8 2
7a
3 4
45
0 3
5a
0 1
62
Tri
sM
gC
l 24
1 7
1b
6 9
44
16 0
8c
2 1
57
18
0 3
a1
0 4
42 8
5a
0 4
19
9 1
0b
5 0
85
0 4
9a
0 2
91
Ott
o8 2
0c
1 4
04
7 2
5b
1 7
87
19
2 8
c8
23
3 4
6b
0 4
05
23 9
6c
4 6
15
0 4
9a
0 2
00
Pis
um
sati
vum
LB
01
47 5
4a
1 5
50
10 8
7a
3 0
78
18
3 5
a5
06
2 8
1a
0 4
61
7 1
9a
2 8
23
2 3
3a
0 4
90
Gal
bra
ith
51 9
7a
3 6
24
5 2
9b
1 1
55
17
8 6
a3
36
3 0
2ab
0 4
65
6 3
4a
1 7
65
2 3
0a
0 8
76
Tri
sM
gC
l 25
9 3
7b
4 8
26
16 7
4c
2 9
14
17
7 7
a1
1 8
63 2
9b
0 5
15
6 3
9a
2 7
81
2 5
9a
0 8
94
Ott
o5 7
2c
0 9
93
4 7
9b
1 0
83
19
0 1
a6
06
1 9
4c
0 1
80
9 8
5b
2 6
68
1 1
5b
0 3
67
Fes
tuca
roth
ma
leri
LB
01
57 3
5a
1 8
25
8 5
7a
0 7
99
19
6 1
a6
07
3 2
4a
0 4
52
14 2
3ab
3 2
83
0 4
2a
0 1
82
Gal
bra
ith
61 0
3a
2 2
50
6 8
4b
0 6
49
18
2 7
b7
15
3 3
3ab
0 4
59
15 0
7a
1 9
74
0 2
7b
0 1
31
Tri
sM
gC
l 26
9 8
5a
1 6
03
17 7
2c
0 8
15
18
5 1
b8
06
3 6
6ab
0 3
12
17 3
0b
5 7
01
0 5
4a
0 1
95
Ott
o1
3 8
4b
2 8
46
8 5
5a
1 6
03
21
0 9
c6
77
3 7
6b
0 6
13
23 8
6c
12 9
92
0 1
1c
0 0
66
Vic
iafa
ba
LB
01
10
4 8
2a
6 0
21
11 6
1a
1 7
42
20
1 5
a4
45
2 4
0a
0 1
78
6 3
6a
2 3
26
0 8
7a
0 2
90
Ga
lbra
ith
11
4 5
5b
2 7
87
8 5
8b
1 3
06
19
6 9
a4
88
2 4
1a
0 1
48
5 8
0a
0 8
06
0 8
2a
0 2
35
Tri
sM
gC
l 21
15 4
5b
5 1
94
20 3
6c
1 4
43
19
1 5
a9
10
2 9
1b
0 2
72
6 8
5a
4 6
09
1 3
0b
0 3
45
Ott
o2
1 5
6c
8 7
21
14 0
5a
7 5
89
18
4 2
a1
6 9
22 2
2a
0 5
32
4 3
5a
1 7
51
0 4
5c
0 1
8
Val
ues
are
giv
enas
mea
nan
dst
and
ard
dev
iati
on
of
the
mea
n(S
D)
of
forw
ard
scat
ter
(FS
ch
ann
elu
nit
s)s
ide
scat
ter
(SS
chan
nel
un
its)
flu
ore
scen
ce(F
Lc
han
nel
un
its)
coef
fici
ento
fv
aria
tio
no
fG
0G
1D
NA
pea
k(C
V
)b
ack
gro
un
dfa
cto
r(B
F
)an
dn
ucl
eary
ield
fact
or(Y
Fn
ucl
eisndash
1m
gndash1)
Mea
ns
foll
ow
edb
yth
esa
me
lett
er(a
bo
rc)
are
no
tsta
tist
ical
lyd
iffe
ren
tacc
ord
ing
toth
em
ult
iple
com
par
ison
Ho
lmndashS
idak
test
atPlt
00
5
Buff
er(s
)th
atp
erfo
rmed
bes
tin
each
spec
ies
are
sho
wn
inb
old
typ
e
Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM 683
significantly better than Galbraithrsquos (Fig 1C) with highermean FL intensities and lower CV values (Table 3)Figure 1C also shows the histogram obtained after nuclearisolation with TrisMgCl2 buffer In this case a G0G1 peakwith an unacceptable CV value (974 ) and considerableloss of fluorescence was obtained A similar result wasobtained for nuclei isolated with LB01
Lycopersicon esculentum
Acceptable results were obtained with all four buffersTwo buffer groups with statistically significant differencesin FL were obtained Samples prepared with Galbraithrsquosand TrisMgCl2 buffers yielded lower mean FL valuesthan their counterparts prepared with LB01 and Ottorsquosbuffers (Fig 1D) The FL values were highly heterogen-eous among buffers Ottorsquos and LB01 were the bestbuffers with Ottorsquos providing lower CV values and higherFL but lower YF and higher BF than LB01 (Table 3)
Celtis australis
Low CV values (lt30 ) and low DF (lt100 ) wereobserved for this species Nevertheless it was not easy toobtain suffiecient nuclei and the second lowest YF valueswere observed in this species With regard to FL onlyLB01 and TrisMgCl2 buffers were not statistically dif-ferent with nuclei isolated from Galbraithrsquos buffer pre-senting the lowest mean FL and Ottorsquos the highest meanFL All parameters combined TrisMgCl2 and LB01 werethe best buffers as nuclei in TrisMgCl2 presented thelowest CV values and similar FL intensity and YF valuesas in LB01 (Table 3 Fig 1E)
Pisum sativum
In this species all buffers performed reasonably wellThe lowest FL intensities were obtained for nucleiisolated with TrisMgCl2 although no statistically signi-ficant differences were observed among the tested buffersThe best buffer for this species was Ottorsquos (Table 3Fig 1F) Among the investigated species P sativum wasthe one with the highest YF
Festuca rothmaleri
With the exception of CV values and YF overall resultsfor this species were satisfying with all four buffers testedNo statistically significant differences were found regard-ing the FL of nuclei isolated in TrisMgCl2 or Galbraithrsquosbuffers as nuclei from both buffers presented low FLvalues The best buffer for this species was LB01 (Table 3Fig 1G)
Vicia faba
This species gave the lowest CV and DF values amongthose tested Generally the results were very similar tothose obtained for P sativum FL was similar for all thebuffers and no statistically significant differences wereobserved Interestingly Vicia faba was the only species
for which Ottorsquos was not the buffer with the highest FL(Fig 1H) Despite low CV values of DNA peaks thisbuffer gave the worst results with the G0G1 peak shiftedtowards the lower channels This was due to fluorescenceinstability which decreased over time In all other speciesand with the remaining buffers FL was stable after 10 minof incubation with PI (Fig 1A) Results obtained withLB01 and Galbraithrsquos buffers were similar and the best forthis species (Table 3)
Analysis of FS and SS
Generally FS and SS values differed considerablyamong the test buffers Nevertheless in most of thespecies analysis of scatter parameters revealed that two ofthe four buffers were more similar than the others and nostatistically significant differences were observed betweenthem Interestingly and with the exception of S burritobuffers that had similar FS mean values were not thosethat had similar SS values This can be seen on cytogramsof FS vs SS obtained in P sativum (Fig 2) In thisspecies and for FS no statistically significant differenceswere observed between LB01 and Galbraithrsquos buffers forSS no difference was observed for Galbraithrsquos and Ottorsquosbuffers Interestingly simultaneous analysis of FS and SSresulted in a species-specific pattern that could be used asa fingerprint of each buffer
Effect of operator and date of analysis
In most cases no statistically significant differenceswere observed between operators and dates of analysisOperators provided more homogeneous results than datesin the former statistically significant differences wereobserved only for BF and YF in more than one speciesSignificant differences between the dates of analysis weredetected in some species for FS SS CV and BF Withregard to FL one of the most important parameters inFCM analyses significant among-day differences weredetected only in C australis and differences betweenoperators occurred only in L esculentum The two speciesmore susceptible to differences were L esculentum andV faba (Table 4)
Hierarchical cluster analysis
With the exception of the results for S burrito the fourbuffers fell into two highly dissimilar and consistentclusters one with Ottorsquos buffer and the other with theremaining buffers In S burrito (Fig 3A) one cluster wasformed with Ottorsquos and Galbraithrsquos buffers while theother comprised TrisMgCl2 and LB01 buffers InC australis (Fig 3C) Galbraithrsquos buffer was more similarto LB01 and TrisMgCl2 buffers than to Ottorsquos buffer inaddition lsquoTrisMgCl2 Arsquo was more similar to LB01 thanto lsquoTrisMgCl2 Brsquo In P sativum lsquoGalbraithrsquos Arsquo wasmore related to LB01 than to lsquoGalbraithrsquos Brsquo (Fig 3D)whereas for F rothmaleri lsquoLB01 Arsquo was more similar toGalbraithrsquos than to lsquoLB01 Brsquo (Fig 3E) In L esculentumtwo groups were formed from the second clusterTrisMgCl2 and lsquoGalbraith Arsquo formed one group and
684 Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM
FS log
C
SS lo
g
1 10 100 10000middot1
0middot1
1
10
100
1000
Meanchannel
FSSS 18middot2
TrisMgCl2
37middot8735middot6960middot7()
FPCV
Meanchannel
FSSS
LB01ASS
log
0middot1
1
10
100
1000
10middot045middot4
30middot0226middot09()
FPCV
FSSS 3middot1 37middot24
29middot936middot0
Ottorsquos
FS log
D
1 10 100 10000middot1
Meanchannel ()
FPCV
FS 52middot1 28middot42SS 5middot2 39middot04
GalbraithsB
Meanchannel ()
FPCV
F I G 2 Cytograms of forward scatter (logarithmic scale FS log) vs side scatter (logarithmic scale SS log) obtained after the analysis of Pisum sativum nucleiisolated with four lysis buffers (A) LB01 (B) Galbraithrsquos (C) TrisMgCl2 and (D) Ottorsquos The mean channel number and full peak coefficient of variation
(FPCV ) are given for both parameters Note that the patterns of distributions are characteristic for each buffer
T A B L E 4 Three-way ANOVA analysis of the dates (D) and operators (O) for the parameters evaluated on each species
FS SS FL CV BF YF
Species D O D O D O D O D O D O
Sedum burrito ns ns ns ns ns ns ns ns s ns ns nsOxalis pes-caprae s ns s ns ns ns s ns ns ns ns nsLycopersicon esculentum s ns ns ns ns s s ns s s s sCeltis australis ns ns ns ns s ns ns ns s ns ns nsPisum sativum ns ns s ns ns ns s ns s ns ns nsFestuca rothmaleri s ns ns ns ns ns ns ns ns s ns nsVicia faba ns s s s ns ns ns ns ns ns s s
Forward scatter FS side scatter SS fluorescence FL coefficient of variation of the G0G1 DNA peak CV background factor BF and nuclearyield factor YF
ns not significantly different s significantly different at P lt 005
Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM 685
LB01 and lsquoGalbraith Brsquo formed the other (Fig 3B) InV faba (Fig 3F) LB01 and Galbraithrsquos formed one groupowing to greater similarities between operators thanwithin each buffer As previously stated in O pes-capraeonly two buffers (Ottorsquos and Galbraithrsquos) providedacceptable results with considerable dissimilaritiesbetween them (data not shown)
DISCUSSION
Four nuclear isolation buffers were used with a setof species that were chosen to represent different types
of leaf tissues and different nuclear DNA content(130ndash2690 pg per 2C DNA) As expected popularDNA reference standards (P sativum V faba andL esculentum) were easy to work with Neverthelessnot all buffers worked well with L esculentumpossibly owing to the presence of cytosolic compoundsHowever as the aim of the study was to compare theperformance of basic buffer formulas the use of additivesthat could counteract the negative effects of cytosol wasavoided
Overall the best results were obtained with P sativumAs its 2C nuclear DNA content is in the middle of theknown range of genome sizes in plants this observation
A
C
E
B
D
F
Dissimilarity Dissimilarity
2middot0 1middot5 1middot0 0middot5 0middot02middot0 1middot5 1middot0 0middot5 0middot0
2middot0 1middot5 1middot0 0middot5 0middot0
2middot0 1middot5 1middot0 0middot5 0middot0 2middot0 1middot5 1middot0 0middot5 0middot0
2middot0 1middot5 1middot0 0middot5 0middot0
LB01A
LB01B
LB01A
LB01B
Ottos B
Ottos A
Galbraiths B
Galbraiths A
TrisMgCl2 B
TrisMgCl2 A
Ottos B
Ottos A
Galbraiths B
Galbraiths A
Galbraiths B
Galbraiths A
TrisMgCl2 B
TrisMgCl2 A
LB01A
LB01B
Ottos B
Ottos A
TrisMgCl2 B
TrisMgCl2 A
LB01A
LB01B
LB01A
LB01B
Ottos B
Ottos A
Galbraiths A
Galbraiths B
TrisMgCl2 A
TrisMgCl2 B
Ottos B
Ottos A
Galbraiths B
Galbraiths A
Galbraiths B
Galbraiths A
TrisMgCl2 B
TrisMgCl2 A
LB01A
LB01B
Ottos B
Ottos A
TrisMgCl2 B
TrisMgCl2 A
F I G 3 Dendrograms obtained after hierarchical cluster analysis of the following species (A) Sedum burrito (B) Lycopersicon esculentum (C) Celtisaustralis (D) Pisum sativum (E) Festuca rothmaleri (F) Vicia faba according to the parameters FS SS FL CV BF and YF With the exception of S burrito
the four buffers fell into two highly dissimilar clusters of the same buffers
686 Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM
underlines its position as one of the best standards forplant DNA FCM By contrast Sedum burrito was themost difficult species to analyse due to low DNA contentoccurrence of polysomaty and high leaf water contentwhich hampered sample preparation and analysis More-over its tissues may contain tannins (J Greilhuber perscomm 2006)
In O pes-caprae cytosol of which is highly acidic(pH lt 30) measurable samples could be prepared usingonly Ottorsquos and Galbraithrsquos buffers with Ottorsquos beinghighly superior This is in accordance with the results ofEmshwiller (2002) who analysed ploidy levels in OxalisAfter testing LB01 MgSO4 and Ottorsquos buffers sheobtained measurable samples only with Ottorsquos The formertwo buffers failed presumably as a result of the acidic cellsap which may have exceeded the buffering capacity ofLB01 and MgSO4
Celtis australis was the only woody plant speciesincluded in the present study and was chosen because ofthe presence of mucilaginous compounds (Rodriguez et al2005a) which increase sample viscosity restrain nucleirelease and cause their clumping Interestingly this wasthe only species for which TrisMgCl2 was the bestperforming buffer This was probably because of a higherconcentration of the non-ionic detergent which sup-pressed the effect of mucilaginous compounds Leaftissues of F rothmaleri were particularly hard anddifficult to chop In addition preliminary experimentswith this species revealed the presence of cytosoliccompounds which would be expected to interfere withDNA staining However given the pattern of FS and SSobtained the so-called lsquotannic acid effectrsquo (Loureiro et al2006) was absent indicating that these compounds werereleased at low concentration or not at all
In order to compare the performance of nuclear isolationbuffers a set of parameters was carefully selected toevaluate sample quality Furthermore stability of fluores-cence and light scatter properties of isolated nuclei overtime were confirmed Among the parameters chosen thecoefficient of variation of DNA peaks (CV) is of majorimportance Galbraith et al (2002) considered 50 asthe maximum acceptable CV value in plant DNA flowcytometry With the exception of TrisMgCl2 buffer whenused with S burrito all mean CV values obtained hereinwere below this limit Ideally a nuclei sample should befree of cell and tissue debris Emshwiller (2002) noted acorrelation between CV and background noise In thepresent study this correlation was found only in somespecies (eg F rothmaleri) Rather the results heresuggest that the extent of background debris is determinedby the buffer itself For example a higher detergentconcentration in a buffer could lead to chloroplast lysisand consequently decrease the number of fluorescentparticles contributing to debris signals (Coba de la Penaand Brown 2001)
LB01 buffer provided very good results with theexception of O pes-caprae and S burrito low CV highFL and YF values were obtained with this buffer Highnuclei FL intensities obtained with this buffer weredefinitely an advantage as compared with TrisMgCl2 and
Galbraithrsquos buffers Galbraithrsquos buffer seems well bal-anced as acceptable results were achieved in all speciesSurprisingly the buffer gave reasonable results also withO pes-caprae which is characterized by highly acidiccytosol The presence of MOPS in the buffer may shedlight on these findings as it has a pKa of 72 and a betterbuffering capacity than TRIS with a pKa of 81 Adisadvantage of this buffer was the low fluorescenceintensity of nuclei Collectively TrisMgCl2 was the worstperforming buffer Nevertheless it provided the bestresults for C australis This may have been due to thehigher concentration of non-ionic detergent (Table 1)which counteracted the agglutinating effect of mucilagin-ous compounds and decreased sample viscosity As withGalbraithrsquos buffer low FL values were obtained withTrisMgCl2
Ottorsquos buffer is unique in that sample preparationinvolves two steps The results obtained with Ottorsquos bufferwere excellent in many species especially those withlower nuclear DNA content Dolezel and Bartos (2005)highlighted the quality of this buffer which is known toprovide DNA content histograms with unequalled resolu-tion One explanation for this may be that citric acidimproves chromatin accessibility and lsquohomogenizesrsquo chro-matin structure eliminating differences in staining intens-ity among nuclei with the same DNA content but differentchromatin state This could explain the highest FL andlowest CV values as observed here Another characteristicof this buffer was that light scatter values (especially FS)were significantly lower than those of other buffers Thismay be explained by the action of citric acid whichcauses nuclei fixation (Dolezel and Bartos 2005) It isnoteworthy that the pattern of FS vs SS distribution wassimilar to that obtained when analysing nuclei fixed withformaldehyde (Rodriguez et al 2005b) Althoughproviding excellent results this buffer gave the highestBF and lowest YF values The former can be explained bynuclei instability after the addition of Otto II After thisstep nuclei deteriorate rapidly in some species (J Dolezelunpubl res) The lowest YF values could be due to thedetergent (Tween 20) which is weaker than Triton X-100thus having a lower capacity to release nuclei
An important consequence of the observation ofdifferent fluorescence values obtained with the differentbuffers is that different fluorescence ratios may beexpected with the same species pair if the samples areprepared in different buffers In fact Dolezel et al (1998)observed differences in peak ratios obtained in differentlaboratories Further studies are needed to confirm thisobservation as it may have important consequences forestimation of genome size
The present results showed that well-defined popula-tions of nuclei could be observed on cytograms of FS vsSS With the exception of S burrito for which LB01 andTrisMgCl2 buffers did not present statistically significantdifferences for both parameters the analysis of FS and SSprovided a fingerprint pattern for each buffer Loureiroet al (2006) showed that these parameters were sensitiveto the presence of tannic acid a cytosolic compoundcommon in plants and recommended the analysis of light
Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM 687
scatter to verify suitability of particular samples for plantDNA FCM
As nuclei samples are prepared manually it wasimportant to assess the effect of date of analysis andoperator on sample quality variation Date was found to bemore significant than the effect of different operatorsespecially for FS SS CV and DF YF which depends onthe way the sample is chopped and hence on the operatordid not vary It was expected that FL which is a primarysource of data in FCM analysis would not depend on theoperator or the day of chopping This was confirmed in allspecies except L esculentum and C australis In theformer species a significant variation was obtainedbetween operators These differences could be explainedby variable results obtained with Galbraithrsquos buffer InC australis significant differences were obtained amongdates In this case significant differences were due toresults obtained on one single day Emshwiller (2002)found significant differences when multiple preparationsfrom the same plant were run on different days Thesedifferences and those found in the present study wereprobably due to instrument drift (Kudo and Kimura2001) To avoid this type of error several authors haverecommended that each measurement be repeated at leastthree times on three different days (Suda 2004 Dolezeland Bartos 2005)
This is the first study that has systematically comparednuclear isolation buffers for DNA FCM The results showthat none of the buffers works best with all species andstatistically significant differences in sample quality wereobserved among the four buffers The results obtainedwith different species and contrasting types of leaf tissuescan serve as guidelines in buffer selection Nevertheless itis recommend that a range of buffers be tested whenworking with a new species and tissue type Once the bestbuffer has been identified additives should be tested ifrequired to suppress negative effects of phenols and othercytosolic compounds
ACKNOWLEDGEMENTS
We are grateful to Prof Johann Greilhuber and Dr JanSuda for critical reading of the manuscript This work wassupported by FCT project ref POCTIAGR606722004JL was supported by the Fellowship FCTBD90032002
LITERATURE CITEDBennett MD Leitch I 2005 Genome size evolution in plants In
Gregory T ed The evolution of the genome London ElsevierAcademic Press 89ndash162
Bergounioux C Perennes C Brown SC Gadal P 1988 Cytometricanalysis of growth regulator-dependent transcription and cell cycleprogression in Petunia protoplast cultures Planta 175 500ndash505
Bergounioux C Brown SC Petit PX 1992 Flow cytometry and plantprotoplast cell biology Physiologia Plantarum 85 374ndash386
Castro S Loureiro J Santos C Ayensa G Navarro L 2005 Differentploidy levels could explain reproductive matter of invasive Oxalispes-caprae L in Mediterranean regions In Brunel S ed Invasiveplants in Mediterranean type regions of the world StrasbourgCedex France Council of Europe Publishing 235
Coba de la Pena T Brown SC 2001 Cytometry and fluorimetryIn Hawes C Satiat-Jeunemaıtre B eds Plant cell biology
a practical approach New York Oxford University Press85ndash106
Dolezel J Bartos J 2005 Plant DNA flow cytometry and estimation ofnuclear genome size Annals of Botany 95 99ndash110
Dolezel J Gohde W 1995 Sex determination in dioecious plantsMelandrium album and M rubrum using high-resolution flowcytometry Cytometry 19 103ndash106
Dolezel J Binarova P Lucretti S 1989 Analysis of nuclear DNAcontent in plant cells by flow cytometry Biologia Plantarum 31113ndash120
Dolezel J Sgorbati S Lucretti S 1992 Comparison of three DNAfluorochromes for flow cytometric estimation of nuclear DNAcontent in plants Physiologia Plantarum 85 625ndash631
Dolezel J Greilhuber J Lucretti S Meister A Lysak M Nardi LObermayer R 1998 Plant genome size estimation byflow cytometry inter-laboratory comparison Annals of Botany 8217ndash26
Dolezel J Bartos J Voglmayr H Greilhuber J 2003 Nuclear DNAcontent and genome size of trout and human Cytometry 51A127ndash128
Emshwiller E 2002 Ploidy levels among species in the lsquoOxalis tuberosaAlliancersquo as inferred by flow cytometry Annals of Botany 89741ndash753
Galbraith DW 2004 Cytometry and plant sciences a personalretrospective Cytometry 58A 37ndash44
Galbraith DW Harkins KR Maddox JM Ayres NM Sharma DPFiroozabady E 1983 Rapid flow cytometric analysis of the cell-cycle in intact plant-tissues Science 220 1049ndash1051
Galbraith DW Lambert GM Macas J Dolezel J 2002 Analysis ofnuclear DNA content and ploidy in higher plants In Robinson JPDarzynkiewicz Z Dean PN Dressler LG Rabinovitch PS StewartCV Tanke HJ Wheeless LL eds Current protocols in cytometryNew York John Wiley amp Sons 761ndash7622
Heller FO 1973 DNS-Bestimmung an Keimwurzeln von Vicia faba Lmit Hilfe der Impulscytophotometrie Bericht der DeutschenBotanischen Gesellschaft 86 437ndash441
Hintze J 2004 NCSS and PASS Kaysville Utah Number CruncherStatistical Systems
Kudo N Kimura Y 2001 Flow cytometric evidence for endopolyploidyin seedlings of some Brassica species Theoretical and AppliedGenetics 102 104ndash110
Loureiro J Rodriguez E Dolezel J Santos C 2006 Flow cytometricand microscopic analysis of the effect of tannic acid on plantnuclei and estimation of DNA content Annals of Botany 98515ndash527
Noirot M Barre P Louarn J Duperray C Hamon S 2000 Nucleusndashcytosol interactionsmdashA source of stoichiometric error in flowcytometric estimation of nuclear DNA content in plants Annals ofBotany 86 309ndash316
Noirot M Barre P Duperray C Louarn J Hamon S 2003 Effects ofcaffeine and chlorogenic acid on propidium iodide accessibility toDNA consequences on genome size evaluation in coffee treeAnnals of Botany 92 259ndash264
Otto F 1992 Preparation and staining of cells for high-resolution DNAanalysis In Radbruch A ed Flow cytometry and cell sortingBerlin Springer-Verlag 101ndash104
Pfosser M Amon A Lelley T Heberle-Bors E 1995 Evaluation ofsensitivity of flow cytometry in detecting aneuploidy in wheat usingdisomic and ditelosomic wheat-rye addition lines Cytometry 21387ndash393
Pinto G Loureiro J Lopes T Santos C 2004 Analysis of the geneticstability of Eucalyptus globulus Labill somatic embryos by flowcytometry Theoretical and Applied Genetics 109 580ndash587
Rodriguez E Gomes A Loureiro J Dolezel J Santos C 2005aEstimation of the genome size of the Iberian Peninsula Ulmaceae In9th Iberian Congress of Cytometry Book of Abstracts PortoPortugal 37
Rodriguez E Loureiro J Dolezel J Santos C 2005b The adequacy ofusing formaldehyde fixation for nuclear DNA content analyses ofplant material In 9th Iberian Congress of Cytometry Book ofAbstracts Porto Portugal PO120-PB
Shapiro H 2004 Practical flow cytometry 4th edn New YorkWiley-Liss
688 Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM
Suda J 2004 An employment of flow cytometry into plant biosystematicsPhD thesis Charles University Czech Republic Available at httpwwwibotcasczfcmsudapresentationdisertationpdf
Trotter J 2000 WinMDI version 28 Available at httpfacsscrippsedusoftwarehtml
Ulrich I Ulrich W 1986 Flow cytometric DNA analysis of plantprotoplasts with DAPI Zeitschrift fur Naturforschung 411052ndash1056
Ulrich I Fritz B Ulrich W 1988 Application of DNA fluorochromes forflow cytometric DNA analysis of plant protoplasts Plant Science 55151ndash158
Walker D Monino I Correal E 2006 Genome size inBituminaria bituminosa (L) CH Stirton (Fabaceae) populationsseparation of lsquotruersquo differences from environmental effects on DNAdetermination Environmental and Experimental Botany 55258ndash265
Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM 689
significantly better than Galbraithrsquos (Fig 1C) with highermean FL intensities and lower CV values (Table 3)Figure 1C also shows the histogram obtained after nuclearisolation with TrisMgCl2 buffer In this case a G0G1 peakwith an unacceptable CV value (974 ) and considerableloss of fluorescence was obtained A similar result wasobtained for nuclei isolated with LB01
Lycopersicon esculentum
Acceptable results were obtained with all four buffersTwo buffer groups with statistically significant differencesin FL were obtained Samples prepared with Galbraithrsquosand TrisMgCl2 buffers yielded lower mean FL valuesthan their counterparts prepared with LB01 and Ottorsquosbuffers (Fig 1D) The FL values were highly heterogen-eous among buffers Ottorsquos and LB01 were the bestbuffers with Ottorsquos providing lower CV values and higherFL but lower YF and higher BF than LB01 (Table 3)
Celtis australis
Low CV values (lt30 ) and low DF (lt100 ) wereobserved for this species Nevertheless it was not easy toobtain suffiecient nuclei and the second lowest YF valueswere observed in this species With regard to FL onlyLB01 and TrisMgCl2 buffers were not statistically dif-ferent with nuclei isolated from Galbraithrsquos buffer pre-senting the lowest mean FL and Ottorsquos the highest meanFL All parameters combined TrisMgCl2 and LB01 werethe best buffers as nuclei in TrisMgCl2 presented thelowest CV values and similar FL intensity and YF valuesas in LB01 (Table 3 Fig 1E)
Pisum sativum
In this species all buffers performed reasonably wellThe lowest FL intensities were obtained for nucleiisolated with TrisMgCl2 although no statistically signi-ficant differences were observed among the tested buffersThe best buffer for this species was Ottorsquos (Table 3Fig 1F) Among the investigated species P sativum wasthe one with the highest YF
Festuca rothmaleri
With the exception of CV values and YF overall resultsfor this species were satisfying with all four buffers testedNo statistically significant differences were found regard-ing the FL of nuclei isolated in TrisMgCl2 or Galbraithrsquosbuffers as nuclei from both buffers presented low FLvalues The best buffer for this species was LB01 (Table 3Fig 1G)
Vicia faba
This species gave the lowest CV and DF values amongthose tested Generally the results were very similar tothose obtained for P sativum FL was similar for all thebuffers and no statistically significant differences wereobserved Interestingly Vicia faba was the only species
for which Ottorsquos was not the buffer with the highest FL(Fig 1H) Despite low CV values of DNA peaks thisbuffer gave the worst results with the G0G1 peak shiftedtowards the lower channels This was due to fluorescenceinstability which decreased over time In all other speciesand with the remaining buffers FL was stable after 10 minof incubation with PI (Fig 1A) Results obtained withLB01 and Galbraithrsquos buffers were similar and the best forthis species (Table 3)
Analysis of FS and SS
Generally FS and SS values differed considerablyamong the test buffers Nevertheless in most of thespecies analysis of scatter parameters revealed that two ofthe four buffers were more similar than the others and nostatistically significant differences were observed betweenthem Interestingly and with the exception of S burritobuffers that had similar FS mean values were not thosethat had similar SS values This can be seen on cytogramsof FS vs SS obtained in P sativum (Fig 2) In thisspecies and for FS no statistically significant differenceswere observed between LB01 and Galbraithrsquos buffers forSS no difference was observed for Galbraithrsquos and Ottorsquosbuffers Interestingly simultaneous analysis of FS and SSresulted in a species-specific pattern that could be used asa fingerprint of each buffer
Effect of operator and date of analysis
In most cases no statistically significant differenceswere observed between operators and dates of analysisOperators provided more homogeneous results than datesin the former statistically significant differences wereobserved only for BF and YF in more than one speciesSignificant differences between the dates of analysis weredetected in some species for FS SS CV and BF Withregard to FL one of the most important parameters inFCM analyses significant among-day differences weredetected only in C australis and differences betweenoperators occurred only in L esculentum The two speciesmore susceptible to differences were L esculentum andV faba (Table 4)
Hierarchical cluster analysis
With the exception of the results for S burrito the fourbuffers fell into two highly dissimilar and consistentclusters one with Ottorsquos buffer and the other with theremaining buffers In S burrito (Fig 3A) one cluster wasformed with Ottorsquos and Galbraithrsquos buffers while theother comprised TrisMgCl2 and LB01 buffers InC australis (Fig 3C) Galbraithrsquos buffer was more similarto LB01 and TrisMgCl2 buffers than to Ottorsquos buffer inaddition lsquoTrisMgCl2 Arsquo was more similar to LB01 thanto lsquoTrisMgCl2 Brsquo In P sativum lsquoGalbraithrsquos Arsquo wasmore related to LB01 than to lsquoGalbraithrsquos Brsquo (Fig 3D)whereas for F rothmaleri lsquoLB01 Arsquo was more similar toGalbraithrsquos than to lsquoLB01 Brsquo (Fig 3E) In L esculentumtwo groups were formed from the second clusterTrisMgCl2 and lsquoGalbraith Arsquo formed one group and
684 Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM
FS log
C
SS lo
g
1 10 100 10000middot1
0middot1
1
10
100
1000
Meanchannel
FSSS 18middot2
TrisMgCl2
37middot8735middot6960middot7()
FPCV
Meanchannel
FSSS
LB01ASS
log
0middot1
1
10
100
1000
10middot045middot4
30middot0226middot09()
FPCV
FSSS 3middot1 37middot24
29middot936middot0
Ottorsquos
FS log
D
1 10 100 10000middot1
Meanchannel ()
FPCV
FS 52middot1 28middot42SS 5middot2 39middot04
GalbraithsB
Meanchannel ()
FPCV
F I G 2 Cytograms of forward scatter (logarithmic scale FS log) vs side scatter (logarithmic scale SS log) obtained after the analysis of Pisum sativum nucleiisolated with four lysis buffers (A) LB01 (B) Galbraithrsquos (C) TrisMgCl2 and (D) Ottorsquos The mean channel number and full peak coefficient of variation
(FPCV ) are given for both parameters Note that the patterns of distributions are characteristic for each buffer
T A B L E 4 Three-way ANOVA analysis of the dates (D) and operators (O) for the parameters evaluated on each species
FS SS FL CV BF YF
Species D O D O D O D O D O D O
Sedum burrito ns ns ns ns ns ns ns ns s ns ns nsOxalis pes-caprae s ns s ns ns ns s ns ns ns ns nsLycopersicon esculentum s ns ns ns ns s s ns s s s sCeltis australis ns ns ns ns s ns ns ns s ns ns nsPisum sativum ns ns s ns ns ns s ns s ns ns nsFestuca rothmaleri s ns ns ns ns ns ns ns ns s ns nsVicia faba ns s s s ns ns ns ns ns ns s s
Forward scatter FS side scatter SS fluorescence FL coefficient of variation of the G0G1 DNA peak CV background factor BF and nuclearyield factor YF
ns not significantly different s significantly different at P lt 005
Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM 685
LB01 and lsquoGalbraith Brsquo formed the other (Fig 3B) InV faba (Fig 3F) LB01 and Galbraithrsquos formed one groupowing to greater similarities between operators thanwithin each buffer As previously stated in O pes-capraeonly two buffers (Ottorsquos and Galbraithrsquos) providedacceptable results with considerable dissimilaritiesbetween them (data not shown)
DISCUSSION
Four nuclear isolation buffers were used with a setof species that were chosen to represent different types
of leaf tissues and different nuclear DNA content(130ndash2690 pg per 2C DNA) As expected popularDNA reference standards (P sativum V faba andL esculentum) were easy to work with Neverthelessnot all buffers worked well with L esculentumpossibly owing to the presence of cytosolic compoundsHowever as the aim of the study was to compare theperformance of basic buffer formulas the use of additivesthat could counteract the negative effects of cytosol wasavoided
Overall the best results were obtained with P sativumAs its 2C nuclear DNA content is in the middle of theknown range of genome sizes in plants this observation
A
C
E
B
D
F
Dissimilarity Dissimilarity
2middot0 1middot5 1middot0 0middot5 0middot02middot0 1middot5 1middot0 0middot5 0middot0
2middot0 1middot5 1middot0 0middot5 0middot0
2middot0 1middot5 1middot0 0middot5 0middot0 2middot0 1middot5 1middot0 0middot5 0middot0
2middot0 1middot5 1middot0 0middot5 0middot0
LB01A
LB01B
LB01A
LB01B
Ottos B
Ottos A
Galbraiths B
Galbraiths A
TrisMgCl2 B
TrisMgCl2 A
Ottos B
Ottos A
Galbraiths B
Galbraiths A
Galbraiths B
Galbraiths A
TrisMgCl2 B
TrisMgCl2 A
LB01A
LB01B
Ottos B
Ottos A
TrisMgCl2 B
TrisMgCl2 A
LB01A
LB01B
LB01A
LB01B
Ottos B
Ottos A
Galbraiths A
Galbraiths B
TrisMgCl2 A
TrisMgCl2 B
Ottos B
Ottos A
Galbraiths B
Galbraiths A
Galbraiths B
Galbraiths A
TrisMgCl2 B
TrisMgCl2 A
LB01A
LB01B
Ottos B
Ottos A
TrisMgCl2 B
TrisMgCl2 A
F I G 3 Dendrograms obtained after hierarchical cluster analysis of the following species (A) Sedum burrito (B) Lycopersicon esculentum (C) Celtisaustralis (D) Pisum sativum (E) Festuca rothmaleri (F) Vicia faba according to the parameters FS SS FL CV BF and YF With the exception of S burrito
the four buffers fell into two highly dissimilar clusters of the same buffers
686 Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM
underlines its position as one of the best standards forplant DNA FCM By contrast Sedum burrito was themost difficult species to analyse due to low DNA contentoccurrence of polysomaty and high leaf water contentwhich hampered sample preparation and analysis More-over its tissues may contain tannins (J Greilhuber perscomm 2006)
In O pes-caprae cytosol of which is highly acidic(pH lt 30) measurable samples could be prepared usingonly Ottorsquos and Galbraithrsquos buffers with Ottorsquos beinghighly superior This is in accordance with the results ofEmshwiller (2002) who analysed ploidy levels in OxalisAfter testing LB01 MgSO4 and Ottorsquos buffers sheobtained measurable samples only with Ottorsquos The formertwo buffers failed presumably as a result of the acidic cellsap which may have exceeded the buffering capacity ofLB01 and MgSO4
Celtis australis was the only woody plant speciesincluded in the present study and was chosen because ofthe presence of mucilaginous compounds (Rodriguez et al2005a) which increase sample viscosity restrain nucleirelease and cause their clumping Interestingly this wasthe only species for which TrisMgCl2 was the bestperforming buffer This was probably because of a higherconcentration of the non-ionic detergent which sup-pressed the effect of mucilaginous compounds Leaftissues of F rothmaleri were particularly hard anddifficult to chop In addition preliminary experimentswith this species revealed the presence of cytosoliccompounds which would be expected to interfere withDNA staining However given the pattern of FS and SSobtained the so-called lsquotannic acid effectrsquo (Loureiro et al2006) was absent indicating that these compounds werereleased at low concentration or not at all
In order to compare the performance of nuclear isolationbuffers a set of parameters was carefully selected toevaluate sample quality Furthermore stability of fluores-cence and light scatter properties of isolated nuclei overtime were confirmed Among the parameters chosen thecoefficient of variation of DNA peaks (CV) is of majorimportance Galbraith et al (2002) considered 50 asthe maximum acceptable CV value in plant DNA flowcytometry With the exception of TrisMgCl2 buffer whenused with S burrito all mean CV values obtained hereinwere below this limit Ideally a nuclei sample should befree of cell and tissue debris Emshwiller (2002) noted acorrelation between CV and background noise In thepresent study this correlation was found only in somespecies (eg F rothmaleri) Rather the results heresuggest that the extent of background debris is determinedby the buffer itself For example a higher detergentconcentration in a buffer could lead to chloroplast lysisand consequently decrease the number of fluorescentparticles contributing to debris signals (Coba de la Penaand Brown 2001)
LB01 buffer provided very good results with theexception of O pes-caprae and S burrito low CV highFL and YF values were obtained with this buffer Highnuclei FL intensities obtained with this buffer weredefinitely an advantage as compared with TrisMgCl2 and
Galbraithrsquos buffers Galbraithrsquos buffer seems well bal-anced as acceptable results were achieved in all speciesSurprisingly the buffer gave reasonable results also withO pes-caprae which is characterized by highly acidiccytosol The presence of MOPS in the buffer may shedlight on these findings as it has a pKa of 72 and a betterbuffering capacity than TRIS with a pKa of 81 Adisadvantage of this buffer was the low fluorescenceintensity of nuclei Collectively TrisMgCl2 was the worstperforming buffer Nevertheless it provided the bestresults for C australis This may have been due to thehigher concentration of non-ionic detergent (Table 1)which counteracted the agglutinating effect of mucilagin-ous compounds and decreased sample viscosity As withGalbraithrsquos buffer low FL values were obtained withTrisMgCl2
Ottorsquos buffer is unique in that sample preparationinvolves two steps The results obtained with Ottorsquos bufferwere excellent in many species especially those withlower nuclear DNA content Dolezel and Bartos (2005)highlighted the quality of this buffer which is known toprovide DNA content histograms with unequalled resolu-tion One explanation for this may be that citric acidimproves chromatin accessibility and lsquohomogenizesrsquo chro-matin structure eliminating differences in staining intens-ity among nuclei with the same DNA content but differentchromatin state This could explain the highest FL andlowest CV values as observed here Another characteristicof this buffer was that light scatter values (especially FS)were significantly lower than those of other buffers Thismay be explained by the action of citric acid whichcauses nuclei fixation (Dolezel and Bartos 2005) It isnoteworthy that the pattern of FS vs SS distribution wassimilar to that obtained when analysing nuclei fixed withformaldehyde (Rodriguez et al 2005b) Althoughproviding excellent results this buffer gave the highestBF and lowest YF values The former can be explained bynuclei instability after the addition of Otto II After thisstep nuclei deteriorate rapidly in some species (J Dolezelunpubl res) The lowest YF values could be due to thedetergent (Tween 20) which is weaker than Triton X-100thus having a lower capacity to release nuclei
An important consequence of the observation ofdifferent fluorescence values obtained with the differentbuffers is that different fluorescence ratios may beexpected with the same species pair if the samples areprepared in different buffers In fact Dolezel et al (1998)observed differences in peak ratios obtained in differentlaboratories Further studies are needed to confirm thisobservation as it may have important consequences forestimation of genome size
The present results showed that well-defined popula-tions of nuclei could be observed on cytograms of FS vsSS With the exception of S burrito for which LB01 andTrisMgCl2 buffers did not present statistically significantdifferences for both parameters the analysis of FS and SSprovided a fingerprint pattern for each buffer Loureiroet al (2006) showed that these parameters were sensitiveto the presence of tannic acid a cytosolic compoundcommon in plants and recommended the analysis of light
Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM 687
scatter to verify suitability of particular samples for plantDNA FCM
As nuclei samples are prepared manually it wasimportant to assess the effect of date of analysis andoperator on sample quality variation Date was found to bemore significant than the effect of different operatorsespecially for FS SS CV and DF YF which depends onthe way the sample is chopped and hence on the operatordid not vary It was expected that FL which is a primarysource of data in FCM analysis would not depend on theoperator or the day of chopping This was confirmed in allspecies except L esculentum and C australis In theformer species a significant variation was obtainedbetween operators These differences could be explainedby variable results obtained with Galbraithrsquos buffer InC australis significant differences were obtained amongdates In this case significant differences were due toresults obtained on one single day Emshwiller (2002)found significant differences when multiple preparationsfrom the same plant were run on different days Thesedifferences and those found in the present study wereprobably due to instrument drift (Kudo and Kimura2001) To avoid this type of error several authors haverecommended that each measurement be repeated at leastthree times on three different days (Suda 2004 Dolezeland Bartos 2005)
This is the first study that has systematically comparednuclear isolation buffers for DNA FCM The results showthat none of the buffers works best with all species andstatistically significant differences in sample quality wereobserved among the four buffers The results obtainedwith different species and contrasting types of leaf tissuescan serve as guidelines in buffer selection Nevertheless itis recommend that a range of buffers be tested whenworking with a new species and tissue type Once the bestbuffer has been identified additives should be tested ifrequired to suppress negative effects of phenols and othercytosolic compounds
ACKNOWLEDGEMENTS
We are grateful to Prof Johann Greilhuber and Dr JanSuda for critical reading of the manuscript This work wassupported by FCT project ref POCTIAGR606722004JL was supported by the Fellowship FCTBD90032002
LITERATURE CITEDBennett MD Leitch I 2005 Genome size evolution in plants In
Gregory T ed The evolution of the genome London ElsevierAcademic Press 89ndash162
Bergounioux C Perennes C Brown SC Gadal P 1988 Cytometricanalysis of growth regulator-dependent transcription and cell cycleprogression in Petunia protoplast cultures Planta 175 500ndash505
Bergounioux C Brown SC Petit PX 1992 Flow cytometry and plantprotoplast cell biology Physiologia Plantarum 85 374ndash386
Castro S Loureiro J Santos C Ayensa G Navarro L 2005 Differentploidy levels could explain reproductive matter of invasive Oxalispes-caprae L in Mediterranean regions In Brunel S ed Invasiveplants in Mediterranean type regions of the world StrasbourgCedex France Council of Europe Publishing 235
Coba de la Pena T Brown SC 2001 Cytometry and fluorimetryIn Hawes C Satiat-Jeunemaıtre B eds Plant cell biology
a practical approach New York Oxford University Press85ndash106
Dolezel J Bartos J 2005 Plant DNA flow cytometry and estimation ofnuclear genome size Annals of Botany 95 99ndash110
Dolezel J Gohde W 1995 Sex determination in dioecious plantsMelandrium album and M rubrum using high-resolution flowcytometry Cytometry 19 103ndash106
Dolezel J Binarova P Lucretti S 1989 Analysis of nuclear DNAcontent in plant cells by flow cytometry Biologia Plantarum 31113ndash120
Dolezel J Sgorbati S Lucretti S 1992 Comparison of three DNAfluorochromes for flow cytometric estimation of nuclear DNAcontent in plants Physiologia Plantarum 85 625ndash631
Dolezel J Greilhuber J Lucretti S Meister A Lysak M Nardi LObermayer R 1998 Plant genome size estimation byflow cytometry inter-laboratory comparison Annals of Botany 8217ndash26
Dolezel J Bartos J Voglmayr H Greilhuber J 2003 Nuclear DNAcontent and genome size of trout and human Cytometry 51A127ndash128
Emshwiller E 2002 Ploidy levels among species in the lsquoOxalis tuberosaAlliancersquo as inferred by flow cytometry Annals of Botany 89741ndash753
Galbraith DW 2004 Cytometry and plant sciences a personalretrospective Cytometry 58A 37ndash44
Galbraith DW Harkins KR Maddox JM Ayres NM Sharma DPFiroozabady E 1983 Rapid flow cytometric analysis of the cell-cycle in intact plant-tissues Science 220 1049ndash1051
Galbraith DW Lambert GM Macas J Dolezel J 2002 Analysis ofnuclear DNA content and ploidy in higher plants In Robinson JPDarzynkiewicz Z Dean PN Dressler LG Rabinovitch PS StewartCV Tanke HJ Wheeless LL eds Current protocols in cytometryNew York John Wiley amp Sons 761ndash7622
Heller FO 1973 DNS-Bestimmung an Keimwurzeln von Vicia faba Lmit Hilfe der Impulscytophotometrie Bericht der DeutschenBotanischen Gesellschaft 86 437ndash441
Hintze J 2004 NCSS and PASS Kaysville Utah Number CruncherStatistical Systems
Kudo N Kimura Y 2001 Flow cytometric evidence for endopolyploidyin seedlings of some Brassica species Theoretical and AppliedGenetics 102 104ndash110
Loureiro J Rodriguez E Dolezel J Santos C 2006 Flow cytometricand microscopic analysis of the effect of tannic acid on plantnuclei and estimation of DNA content Annals of Botany 98515ndash527
Noirot M Barre P Louarn J Duperray C Hamon S 2000 Nucleusndashcytosol interactionsmdashA source of stoichiometric error in flowcytometric estimation of nuclear DNA content in plants Annals ofBotany 86 309ndash316
Noirot M Barre P Duperray C Louarn J Hamon S 2003 Effects ofcaffeine and chlorogenic acid on propidium iodide accessibility toDNA consequences on genome size evaluation in coffee treeAnnals of Botany 92 259ndash264
Otto F 1992 Preparation and staining of cells for high-resolution DNAanalysis In Radbruch A ed Flow cytometry and cell sortingBerlin Springer-Verlag 101ndash104
Pfosser M Amon A Lelley T Heberle-Bors E 1995 Evaluation ofsensitivity of flow cytometry in detecting aneuploidy in wheat usingdisomic and ditelosomic wheat-rye addition lines Cytometry 21387ndash393
Pinto G Loureiro J Lopes T Santos C 2004 Analysis of the geneticstability of Eucalyptus globulus Labill somatic embryos by flowcytometry Theoretical and Applied Genetics 109 580ndash587
Rodriguez E Gomes A Loureiro J Dolezel J Santos C 2005aEstimation of the genome size of the Iberian Peninsula Ulmaceae In9th Iberian Congress of Cytometry Book of Abstracts PortoPortugal 37
Rodriguez E Loureiro J Dolezel J Santos C 2005b The adequacy ofusing formaldehyde fixation for nuclear DNA content analyses ofplant material In 9th Iberian Congress of Cytometry Book ofAbstracts Porto Portugal PO120-PB
Shapiro H 2004 Practical flow cytometry 4th edn New YorkWiley-Liss
688 Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM
Suda J 2004 An employment of flow cytometry into plant biosystematicsPhD thesis Charles University Czech Republic Available at httpwwwibotcasczfcmsudapresentationdisertationpdf
Trotter J 2000 WinMDI version 28 Available at httpfacsscrippsedusoftwarehtml
Ulrich I Ulrich W 1986 Flow cytometric DNA analysis of plantprotoplasts with DAPI Zeitschrift fur Naturforschung 411052ndash1056
Ulrich I Fritz B Ulrich W 1988 Application of DNA fluorochromes forflow cytometric DNA analysis of plant protoplasts Plant Science 55151ndash158
Walker D Monino I Correal E 2006 Genome size inBituminaria bituminosa (L) CH Stirton (Fabaceae) populationsseparation of lsquotruersquo differences from environmental effects on DNAdetermination Environmental and Experimental Botany 55258ndash265
Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM 689
FS log
C
SS lo
g
1 10 100 10000middot1
0middot1
1
10
100
1000
Meanchannel
FSSS 18middot2
TrisMgCl2
37middot8735middot6960middot7()
FPCV
Meanchannel
FSSS
LB01ASS
log
0middot1
1
10
100
1000
10middot045middot4
30middot0226middot09()
FPCV
FSSS 3middot1 37middot24
29middot936middot0
Ottorsquos
FS log
D
1 10 100 10000middot1
Meanchannel ()
FPCV
FS 52middot1 28middot42SS 5middot2 39middot04
GalbraithsB
Meanchannel ()
FPCV
F I G 2 Cytograms of forward scatter (logarithmic scale FS log) vs side scatter (logarithmic scale SS log) obtained after the analysis of Pisum sativum nucleiisolated with four lysis buffers (A) LB01 (B) Galbraithrsquos (C) TrisMgCl2 and (D) Ottorsquos The mean channel number and full peak coefficient of variation
(FPCV ) are given for both parameters Note that the patterns of distributions are characteristic for each buffer
T A B L E 4 Three-way ANOVA analysis of the dates (D) and operators (O) for the parameters evaluated on each species
FS SS FL CV BF YF
Species D O D O D O D O D O D O
Sedum burrito ns ns ns ns ns ns ns ns s ns ns nsOxalis pes-caprae s ns s ns ns ns s ns ns ns ns nsLycopersicon esculentum s ns ns ns ns s s ns s s s sCeltis australis ns ns ns ns s ns ns ns s ns ns nsPisum sativum ns ns s ns ns ns s ns s ns ns nsFestuca rothmaleri s ns ns ns ns ns ns ns ns s ns nsVicia faba ns s s s ns ns ns ns ns ns s s
Forward scatter FS side scatter SS fluorescence FL coefficient of variation of the G0G1 DNA peak CV background factor BF and nuclearyield factor YF
ns not significantly different s significantly different at P lt 005
Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM 685
LB01 and lsquoGalbraith Brsquo formed the other (Fig 3B) InV faba (Fig 3F) LB01 and Galbraithrsquos formed one groupowing to greater similarities between operators thanwithin each buffer As previously stated in O pes-capraeonly two buffers (Ottorsquos and Galbraithrsquos) providedacceptable results with considerable dissimilaritiesbetween them (data not shown)
DISCUSSION
Four nuclear isolation buffers were used with a setof species that were chosen to represent different types
of leaf tissues and different nuclear DNA content(130ndash2690 pg per 2C DNA) As expected popularDNA reference standards (P sativum V faba andL esculentum) were easy to work with Neverthelessnot all buffers worked well with L esculentumpossibly owing to the presence of cytosolic compoundsHowever as the aim of the study was to compare theperformance of basic buffer formulas the use of additivesthat could counteract the negative effects of cytosol wasavoided
Overall the best results were obtained with P sativumAs its 2C nuclear DNA content is in the middle of theknown range of genome sizes in plants this observation
A
C
E
B
D
F
Dissimilarity Dissimilarity
2middot0 1middot5 1middot0 0middot5 0middot02middot0 1middot5 1middot0 0middot5 0middot0
2middot0 1middot5 1middot0 0middot5 0middot0
2middot0 1middot5 1middot0 0middot5 0middot0 2middot0 1middot5 1middot0 0middot5 0middot0
2middot0 1middot5 1middot0 0middot5 0middot0
LB01A
LB01B
LB01A
LB01B
Ottos B
Ottos A
Galbraiths B
Galbraiths A
TrisMgCl2 B
TrisMgCl2 A
Ottos B
Ottos A
Galbraiths B
Galbraiths A
Galbraiths B
Galbraiths A
TrisMgCl2 B
TrisMgCl2 A
LB01A
LB01B
Ottos B
Ottos A
TrisMgCl2 B
TrisMgCl2 A
LB01A
LB01B
LB01A
LB01B
Ottos B
Ottos A
Galbraiths A
Galbraiths B
TrisMgCl2 A
TrisMgCl2 B
Ottos B
Ottos A
Galbraiths B
Galbraiths A
Galbraiths B
Galbraiths A
TrisMgCl2 B
TrisMgCl2 A
LB01A
LB01B
Ottos B
Ottos A
TrisMgCl2 B
TrisMgCl2 A
F I G 3 Dendrograms obtained after hierarchical cluster analysis of the following species (A) Sedum burrito (B) Lycopersicon esculentum (C) Celtisaustralis (D) Pisum sativum (E) Festuca rothmaleri (F) Vicia faba according to the parameters FS SS FL CV BF and YF With the exception of S burrito
the four buffers fell into two highly dissimilar clusters of the same buffers
686 Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM
underlines its position as one of the best standards forplant DNA FCM By contrast Sedum burrito was themost difficult species to analyse due to low DNA contentoccurrence of polysomaty and high leaf water contentwhich hampered sample preparation and analysis More-over its tissues may contain tannins (J Greilhuber perscomm 2006)
In O pes-caprae cytosol of which is highly acidic(pH lt 30) measurable samples could be prepared usingonly Ottorsquos and Galbraithrsquos buffers with Ottorsquos beinghighly superior This is in accordance with the results ofEmshwiller (2002) who analysed ploidy levels in OxalisAfter testing LB01 MgSO4 and Ottorsquos buffers sheobtained measurable samples only with Ottorsquos The formertwo buffers failed presumably as a result of the acidic cellsap which may have exceeded the buffering capacity ofLB01 and MgSO4
Celtis australis was the only woody plant speciesincluded in the present study and was chosen because ofthe presence of mucilaginous compounds (Rodriguez et al2005a) which increase sample viscosity restrain nucleirelease and cause their clumping Interestingly this wasthe only species for which TrisMgCl2 was the bestperforming buffer This was probably because of a higherconcentration of the non-ionic detergent which sup-pressed the effect of mucilaginous compounds Leaftissues of F rothmaleri were particularly hard anddifficult to chop In addition preliminary experimentswith this species revealed the presence of cytosoliccompounds which would be expected to interfere withDNA staining However given the pattern of FS and SSobtained the so-called lsquotannic acid effectrsquo (Loureiro et al2006) was absent indicating that these compounds werereleased at low concentration or not at all
In order to compare the performance of nuclear isolationbuffers a set of parameters was carefully selected toevaluate sample quality Furthermore stability of fluores-cence and light scatter properties of isolated nuclei overtime were confirmed Among the parameters chosen thecoefficient of variation of DNA peaks (CV) is of majorimportance Galbraith et al (2002) considered 50 asthe maximum acceptable CV value in plant DNA flowcytometry With the exception of TrisMgCl2 buffer whenused with S burrito all mean CV values obtained hereinwere below this limit Ideally a nuclei sample should befree of cell and tissue debris Emshwiller (2002) noted acorrelation between CV and background noise In thepresent study this correlation was found only in somespecies (eg F rothmaleri) Rather the results heresuggest that the extent of background debris is determinedby the buffer itself For example a higher detergentconcentration in a buffer could lead to chloroplast lysisand consequently decrease the number of fluorescentparticles contributing to debris signals (Coba de la Penaand Brown 2001)
LB01 buffer provided very good results with theexception of O pes-caprae and S burrito low CV highFL and YF values were obtained with this buffer Highnuclei FL intensities obtained with this buffer weredefinitely an advantage as compared with TrisMgCl2 and
Galbraithrsquos buffers Galbraithrsquos buffer seems well bal-anced as acceptable results were achieved in all speciesSurprisingly the buffer gave reasonable results also withO pes-caprae which is characterized by highly acidiccytosol The presence of MOPS in the buffer may shedlight on these findings as it has a pKa of 72 and a betterbuffering capacity than TRIS with a pKa of 81 Adisadvantage of this buffer was the low fluorescenceintensity of nuclei Collectively TrisMgCl2 was the worstperforming buffer Nevertheless it provided the bestresults for C australis This may have been due to thehigher concentration of non-ionic detergent (Table 1)which counteracted the agglutinating effect of mucilagin-ous compounds and decreased sample viscosity As withGalbraithrsquos buffer low FL values were obtained withTrisMgCl2
Ottorsquos buffer is unique in that sample preparationinvolves two steps The results obtained with Ottorsquos bufferwere excellent in many species especially those withlower nuclear DNA content Dolezel and Bartos (2005)highlighted the quality of this buffer which is known toprovide DNA content histograms with unequalled resolu-tion One explanation for this may be that citric acidimproves chromatin accessibility and lsquohomogenizesrsquo chro-matin structure eliminating differences in staining intens-ity among nuclei with the same DNA content but differentchromatin state This could explain the highest FL andlowest CV values as observed here Another characteristicof this buffer was that light scatter values (especially FS)were significantly lower than those of other buffers Thismay be explained by the action of citric acid whichcauses nuclei fixation (Dolezel and Bartos 2005) It isnoteworthy that the pattern of FS vs SS distribution wassimilar to that obtained when analysing nuclei fixed withformaldehyde (Rodriguez et al 2005b) Althoughproviding excellent results this buffer gave the highestBF and lowest YF values The former can be explained bynuclei instability after the addition of Otto II After thisstep nuclei deteriorate rapidly in some species (J Dolezelunpubl res) The lowest YF values could be due to thedetergent (Tween 20) which is weaker than Triton X-100thus having a lower capacity to release nuclei
An important consequence of the observation ofdifferent fluorescence values obtained with the differentbuffers is that different fluorescence ratios may beexpected with the same species pair if the samples areprepared in different buffers In fact Dolezel et al (1998)observed differences in peak ratios obtained in differentlaboratories Further studies are needed to confirm thisobservation as it may have important consequences forestimation of genome size
The present results showed that well-defined popula-tions of nuclei could be observed on cytograms of FS vsSS With the exception of S burrito for which LB01 andTrisMgCl2 buffers did not present statistically significantdifferences for both parameters the analysis of FS and SSprovided a fingerprint pattern for each buffer Loureiroet al (2006) showed that these parameters were sensitiveto the presence of tannic acid a cytosolic compoundcommon in plants and recommended the analysis of light
Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM 687
scatter to verify suitability of particular samples for plantDNA FCM
As nuclei samples are prepared manually it wasimportant to assess the effect of date of analysis andoperator on sample quality variation Date was found to bemore significant than the effect of different operatorsespecially for FS SS CV and DF YF which depends onthe way the sample is chopped and hence on the operatordid not vary It was expected that FL which is a primarysource of data in FCM analysis would not depend on theoperator or the day of chopping This was confirmed in allspecies except L esculentum and C australis In theformer species a significant variation was obtainedbetween operators These differences could be explainedby variable results obtained with Galbraithrsquos buffer InC australis significant differences were obtained amongdates In this case significant differences were due toresults obtained on one single day Emshwiller (2002)found significant differences when multiple preparationsfrom the same plant were run on different days Thesedifferences and those found in the present study wereprobably due to instrument drift (Kudo and Kimura2001) To avoid this type of error several authors haverecommended that each measurement be repeated at leastthree times on three different days (Suda 2004 Dolezeland Bartos 2005)
This is the first study that has systematically comparednuclear isolation buffers for DNA FCM The results showthat none of the buffers works best with all species andstatistically significant differences in sample quality wereobserved among the four buffers The results obtainedwith different species and contrasting types of leaf tissuescan serve as guidelines in buffer selection Nevertheless itis recommend that a range of buffers be tested whenworking with a new species and tissue type Once the bestbuffer has been identified additives should be tested ifrequired to suppress negative effects of phenols and othercytosolic compounds
ACKNOWLEDGEMENTS
We are grateful to Prof Johann Greilhuber and Dr JanSuda for critical reading of the manuscript This work wassupported by FCT project ref POCTIAGR606722004JL was supported by the Fellowship FCTBD90032002
LITERATURE CITEDBennett MD Leitch I 2005 Genome size evolution in plants In
Gregory T ed The evolution of the genome London ElsevierAcademic Press 89ndash162
Bergounioux C Perennes C Brown SC Gadal P 1988 Cytometricanalysis of growth regulator-dependent transcription and cell cycleprogression in Petunia protoplast cultures Planta 175 500ndash505
Bergounioux C Brown SC Petit PX 1992 Flow cytometry and plantprotoplast cell biology Physiologia Plantarum 85 374ndash386
Castro S Loureiro J Santos C Ayensa G Navarro L 2005 Differentploidy levels could explain reproductive matter of invasive Oxalispes-caprae L in Mediterranean regions In Brunel S ed Invasiveplants in Mediterranean type regions of the world StrasbourgCedex France Council of Europe Publishing 235
Coba de la Pena T Brown SC 2001 Cytometry and fluorimetryIn Hawes C Satiat-Jeunemaıtre B eds Plant cell biology
a practical approach New York Oxford University Press85ndash106
Dolezel J Bartos J 2005 Plant DNA flow cytometry and estimation ofnuclear genome size Annals of Botany 95 99ndash110
Dolezel J Gohde W 1995 Sex determination in dioecious plantsMelandrium album and M rubrum using high-resolution flowcytometry Cytometry 19 103ndash106
Dolezel J Binarova P Lucretti S 1989 Analysis of nuclear DNAcontent in plant cells by flow cytometry Biologia Plantarum 31113ndash120
Dolezel J Sgorbati S Lucretti S 1992 Comparison of three DNAfluorochromes for flow cytometric estimation of nuclear DNAcontent in plants Physiologia Plantarum 85 625ndash631
Dolezel J Greilhuber J Lucretti S Meister A Lysak M Nardi LObermayer R 1998 Plant genome size estimation byflow cytometry inter-laboratory comparison Annals of Botany 8217ndash26
Dolezel J Bartos J Voglmayr H Greilhuber J 2003 Nuclear DNAcontent and genome size of trout and human Cytometry 51A127ndash128
Emshwiller E 2002 Ploidy levels among species in the lsquoOxalis tuberosaAlliancersquo as inferred by flow cytometry Annals of Botany 89741ndash753
Galbraith DW 2004 Cytometry and plant sciences a personalretrospective Cytometry 58A 37ndash44
Galbraith DW Harkins KR Maddox JM Ayres NM Sharma DPFiroozabady E 1983 Rapid flow cytometric analysis of the cell-cycle in intact plant-tissues Science 220 1049ndash1051
Galbraith DW Lambert GM Macas J Dolezel J 2002 Analysis ofnuclear DNA content and ploidy in higher plants In Robinson JPDarzynkiewicz Z Dean PN Dressler LG Rabinovitch PS StewartCV Tanke HJ Wheeless LL eds Current protocols in cytometryNew York John Wiley amp Sons 761ndash7622
Heller FO 1973 DNS-Bestimmung an Keimwurzeln von Vicia faba Lmit Hilfe der Impulscytophotometrie Bericht der DeutschenBotanischen Gesellschaft 86 437ndash441
Hintze J 2004 NCSS and PASS Kaysville Utah Number CruncherStatistical Systems
Kudo N Kimura Y 2001 Flow cytometric evidence for endopolyploidyin seedlings of some Brassica species Theoretical and AppliedGenetics 102 104ndash110
Loureiro J Rodriguez E Dolezel J Santos C 2006 Flow cytometricand microscopic analysis of the effect of tannic acid on plantnuclei and estimation of DNA content Annals of Botany 98515ndash527
Noirot M Barre P Louarn J Duperray C Hamon S 2000 Nucleusndashcytosol interactionsmdashA source of stoichiometric error in flowcytometric estimation of nuclear DNA content in plants Annals ofBotany 86 309ndash316
Noirot M Barre P Duperray C Louarn J Hamon S 2003 Effects ofcaffeine and chlorogenic acid on propidium iodide accessibility toDNA consequences on genome size evaluation in coffee treeAnnals of Botany 92 259ndash264
Otto F 1992 Preparation and staining of cells for high-resolution DNAanalysis In Radbruch A ed Flow cytometry and cell sortingBerlin Springer-Verlag 101ndash104
Pfosser M Amon A Lelley T Heberle-Bors E 1995 Evaluation ofsensitivity of flow cytometry in detecting aneuploidy in wheat usingdisomic and ditelosomic wheat-rye addition lines Cytometry 21387ndash393
Pinto G Loureiro J Lopes T Santos C 2004 Analysis of the geneticstability of Eucalyptus globulus Labill somatic embryos by flowcytometry Theoretical and Applied Genetics 109 580ndash587
Rodriguez E Gomes A Loureiro J Dolezel J Santos C 2005aEstimation of the genome size of the Iberian Peninsula Ulmaceae In9th Iberian Congress of Cytometry Book of Abstracts PortoPortugal 37
Rodriguez E Loureiro J Dolezel J Santos C 2005b The adequacy ofusing formaldehyde fixation for nuclear DNA content analyses ofplant material In 9th Iberian Congress of Cytometry Book ofAbstracts Porto Portugal PO120-PB
Shapiro H 2004 Practical flow cytometry 4th edn New YorkWiley-Liss
688 Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM
Suda J 2004 An employment of flow cytometry into plant biosystematicsPhD thesis Charles University Czech Republic Available at httpwwwibotcasczfcmsudapresentationdisertationpdf
Trotter J 2000 WinMDI version 28 Available at httpfacsscrippsedusoftwarehtml
Ulrich I Ulrich W 1986 Flow cytometric DNA analysis of plantprotoplasts with DAPI Zeitschrift fur Naturforschung 411052ndash1056
Ulrich I Fritz B Ulrich W 1988 Application of DNA fluorochromes forflow cytometric DNA analysis of plant protoplasts Plant Science 55151ndash158
Walker D Monino I Correal E 2006 Genome size inBituminaria bituminosa (L) CH Stirton (Fabaceae) populationsseparation of lsquotruersquo differences from environmental effects on DNAdetermination Environmental and Experimental Botany 55258ndash265
Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM 689
LB01 and lsquoGalbraith Brsquo formed the other (Fig 3B) InV faba (Fig 3F) LB01 and Galbraithrsquos formed one groupowing to greater similarities between operators thanwithin each buffer As previously stated in O pes-capraeonly two buffers (Ottorsquos and Galbraithrsquos) providedacceptable results with considerable dissimilaritiesbetween them (data not shown)
DISCUSSION
Four nuclear isolation buffers were used with a setof species that were chosen to represent different types
of leaf tissues and different nuclear DNA content(130ndash2690 pg per 2C DNA) As expected popularDNA reference standards (P sativum V faba andL esculentum) were easy to work with Neverthelessnot all buffers worked well with L esculentumpossibly owing to the presence of cytosolic compoundsHowever as the aim of the study was to compare theperformance of basic buffer formulas the use of additivesthat could counteract the negative effects of cytosol wasavoided
Overall the best results were obtained with P sativumAs its 2C nuclear DNA content is in the middle of theknown range of genome sizes in plants this observation
A
C
E
B
D
F
Dissimilarity Dissimilarity
2middot0 1middot5 1middot0 0middot5 0middot02middot0 1middot5 1middot0 0middot5 0middot0
2middot0 1middot5 1middot0 0middot5 0middot0
2middot0 1middot5 1middot0 0middot5 0middot0 2middot0 1middot5 1middot0 0middot5 0middot0
2middot0 1middot5 1middot0 0middot5 0middot0
LB01A
LB01B
LB01A
LB01B
Ottos B
Ottos A
Galbraiths B
Galbraiths A
TrisMgCl2 B
TrisMgCl2 A
Ottos B
Ottos A
Galbraiths B
Galbraiths A
Galbraiths B
Galbraiths A
TrisMgCl2 B
TrisMgCl2 A
LB01A
LB01B
Ottos B
Ottos A
TrisMgCl2 B
TrisMgCl2 A
LB01A
LB01B
LB01A
LB01B
Ottos B
Ottos A
Galbraiths A
Galbraiths B
TrisMgCl2 A
TrisMgCl2 B
Ottos B
Ottos A
Galbraiths B
Galbraiths A
Galbraiths B
Galbraiths A
TrisMgCl2 B
TrisMgCl2 A
LB01A
LB01B
Ottos B
Ottos A
TrisMgCl2 B
TrisMgCl2 A
F I G 3 Dendrograms obtained after hierarchical cluster analysis of the following species (A) Sedum burrito (B) Lycopersicon esculentum (C) Celtisaustralis (D) Pisum sativum (E) Festuca rothmaleri (F) Vicia faba according to the parameters FS SS FL CV BF and YF With the exception of S burrito
the four buffers fell into two highly dissimilar clusters of the same buffers
686 Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM
underlines its position as one of the best standards forplant DNA FCM By contrast Sedum burrito was themost difficult species to analyse due to low DNA contentoccurrence of polysomaty and high leaf water contentwhich hampered sample preparation and analysis More-over its tissues may contain tannins (J Greilhuber perscomm 2006)
In O pes-caprae cytosol of which is highly acidic(pH lt 30) measurable samples could be prepared usingonly Ottorsquos and Galbraithrsquos buffers with Ottorsquos beinghighly superior This is in accordance with the results ofEmshwiller (2002) who analysed ploidy levels in OxalisAfter testing LB01 MgSO4 and Ottorsquos buffers sheobtained measurable samples only with Ottorsquos The formertwo buffers failed presumably as a result of the acidic cellsap which may have exceeded the buffering capacity ofLB01 and MgSO4
Celtis australis was the only woody plant speciesincluded in the present study and was chosen because ofthe presence of mucilaginous compounds (Rodriguez et al2005a) which increase sample viscosity restrain nucleirelease and cause their clumping Interestingly this wasthe only species for which TrisMgCl2 was the bestperforming buffer This was probably because of a higherconcentration of the non-ionic detergent which sup-pressed the effect of mucilaginous compounds Leaftissues of F rothmaleri were particularly hard anddifficult to chop In addition preliminary experimentswith this species revealed the presence of cytosoliccompounds which would be expected to interfere withDNA staining However given the pattern of FS and SSobtained the so-called lsquotannic acid effectrsquo (Loureiro et al2006) was absent indicating that these compounds werereleased at low concentration or not at all
In order to compare the performance of nuclear isolationbuffers a set of parameters was carefully selected toevaluate sample quality Furthermore stability of fluores-cence and light scatter properties of isolated nuclei overtime were confirmed Among the parameters chosen thecoefficient of variation of DNA peaks (CV) is of majorimportance Galbraith et al (2002) considered 50 asthe maximum acceptable CV value in plant DNA flowcytometry With the exception of TrisMgCl2 buffer whenused with S burrito all mean CV values obtained hereinwere below this limit Ideally a nuclei sample should befree of cell and tissue debris Emshwiller (2002) noted acorrelation between CV and background noise In thepresent study this correlation was found only in somespecies (eg F rothmaleri) Rather the results heresuggest that the extent of background debris is determinedby the buffer itself For example a higher detergentconcentration in a buffer could lead to chloroplast lysisand consequently decrease the number of fluorescentparticles contributing to debris signals (Coba de la Penaand Brown 2001)
LB01 buffer provided very good results with theexception of O pes-caprae and S burrito low CV highFL and YF values were obtained with this buffer Highnuclei FL intensities obtained with this buffer weredefinitely an advantage as compared with TrisMgCl2 and
Galbraithrsquos buffers Galbraithrsquos buffer seems well bal-anced as acceptable results were achieved in all speciesSurprisingly the buffer gave reasonable results also withO pes-caprae which is characterized by highly acidiccytosol The presence of MOPS in the buffer may shedlight on these findings as it has a pKa of 72 and a betterbuffering capacity than TRIS with a pKa of 81 Adisadvantage of this buffer was the low fluorescenceintensity of nuclei Collectively TrisMgCl2 was the worstperforming buffer Nevertheless it provided the bestresults for C australis This may have been due to thehigher concentration of non-ionic detergent (Table 1)which counteracted the agglutinating effect of mucilagin-ous compounds and decreased sample viscosity As withGalbraithrsquos buffer low FL values were obtained withTrisMgCl2
Ottorsquos buffer is unique in that sample preparationinvolves two steps The results obtained with Ottorsquos bufferwere excellent in many species especially those withlower nuclear DNA content Dolezel and Bartos (2005)highlighted the quality of this buffer which is known toprovide DNA content histograms with unequalled resolu-tion One explanation for this may be that citric acidimproves chromatin accessibility and lsquohomogenizesrsquo chro-matin structure eliminating differences in staining intens-ity among nuclei with the same DNA content but differentchromatin state This could explain the highest FL andlowest CV values as observed here Another characteristicof this buffer was that light scatter values (especially FS)were significantly lower than those of other buffers Thismay be explained by the action of citric acid whichcauses nuclei fixation (Dolezel and Bartos 2005) It isnoteworthy that the pattern of FS vs SS distribution wassimilar to that obtained when analysing nuclei fixed withformaldehyde (Rodriguez et al 2005b) Althoughproviding excellent results this buffer gave the highestBF and lowest YF values The former can be explained bynuclei instability after the addition of Otto II After thisstep nuclei deteriorate rapidly in some species (J Dolezelunpubl res) The lowest YF values could be due to thedetergent (Tween 20) which is weaker than Triton X-100thus having a lower capacity to release nuclei
An important consequence of the observation ofdifferent fluorescence values obtained with the differentbuffers is that different fluorescence ratios may beexpected with the same species pair if the samples areprepared in different buffers In fact Dolezel et al (1998)observed differences in peak ratios obtained in differentlaboratories Further studies are needed to confirm thisobservation as it may have important consequences forestimation of genome size
The present results showed that well-defined popula-tions of nuclei could be observed on cytograms of FS vsSS With the exception of S burrito for which LB01 andTrisMgCl2 buffers did not present statistically significantdifferences for both parameters the analysis of FS and SSprovided a fingerprint pattern for each buffer Loureiroet al (2006) showed that these parameters were sensitiveto the presence of tannic acid a cytosolic compoundcommon in plants and recommended the analysis of light
Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM 687
scatter to verify suitability of particular samples for plantDNA FCM
As nuclei samples are prepared manually it wasimportant to assess the effect of date of analysis andoperator on sample quality variation Date was found to bemore significant than the effect of different operatorsespecially for FS SS CV and DF YF which depends onthe way the sample is chopped and hence on the operatordid not vary It was expected that FL which is a primarysource of data in FCM analysis would not depend on theoperator or the day of chopping This was confirmed in allspecies except L esculentum and C australis In theformer species a significant variation was obtainedbetween operators These differences could be explainedby variable results obtained with Galbraithrsquos buffer InC australis significant differences were obtained amongdates In this case significant differences were due toresults obtained on one single day Emshwiller (2002)found significant differences when multiple preparationsfrom the same plant were run on different days Thesedifferences and those found in the present study wereprobably due to instrument drift (Kudo and Kimura2001) To avoid this type of error several authors haverecommended that each measurement be repeated at leastthree times on three different days (Suda 2004 Dolezeland Bartos 2005)
This is the first study that has systematically comparednuclear isolation buffers for DNA FCM The results showthat none of the buffers works best with all species andstatistically significant differences in sample quality wereobserved among the four buffers The results obtainedwith different species and contrasting types of leaf tissuescan serve as guidelines in buffer selection Nevertheless itis recommend that a range of buffers be tested whenworking with a new species and tissue type Once the bestbuffer has been identified additives should be tested ifrequired to suppress negative effects of phenols and othercytosolic compounds
ACKNOWLEDGEMENTS
We are grateful to Prof Johann Greilhuber and Dr JanSuda for critical reading of the manuscript This work wassupported by FCT project ref POCTIAGR606722004JL was supported by the Fellowship FCTBD90032002
LITERATURE CITEDBennett MD Leitch I 2005 Genome size evolution in plants In
Gregory T ed The evolution of the genome London ElsevierAcademic Press 89ndash162
Bergounioux C Perennes C Brown SC Gadal P 1988 Cytometricanalysis of growth regulator-dependent transcription and cell cycleprogression in Petunia protoplast cultures Planta 175 500ndash505
Bergounioux C Brown SC Petit PX 1992 Flow cytometry and plantprotoplast cell biology Physiologia Plantarum 85 374ndash386
Castro S Loureiro J Santos C Ayensa G Navarro L 2005 Differentploidy levels could explain reproductive matter of invasive Oxalispes-caprae L in Mediterranean regions In Brunel S ed Invasiveplants in Mediterranean type regions of the world StrasbourgCedex France Council of Europe Publishing 235
Coba de la Pena T Brown SC 2001 Cytometry and fluorimetryIn Hawes C Satiat-Jeunemaıtre B eds Plant cell biology
a practical approach New York Oxford University Press85ndash106
Dolezel J Bartos J 2005 Plant DNA flow cytometry and estimation ofnuclear genome size Annals of Botany 95 99ndash110
Dolezel J Gohde W 1995 Sex determination in dioecious plantsMelandrium album and M rubrum using high-resolution flowcytometry Cytometry 19 103ndash106
Dolezel J Binarova P Lucretti S 1989 Analysis of nuclear DNAcontent in plant cells by flow cytometry Biologia Plantarum 31113ndash120
Dolezel J Sgorbati S Lucretti S 1992 Comparison of three DNAfluorochromes for flow cytometric estimation of nuclear DNAcontent in plants Physiologia Plantarum 85 625ndash631
Dolezel J Greilhuber J Lucretti S Meister A Lysak M Nardi LObermayer R 1998 Plant genome size estimation byflow cytometry inter-laboratory comparison Annals of Botany 8217ndash26
Dolezel J Bartos J Voglmayr H Greilhuber J 2003 Nuclear DNAcontent and genome size of trout and human Cytometry 51A127ndash128
Emshwiller E 2002 Ploidy levels among species in the lsquoOxalis tuberosaAlliancersquo as inferred by flow cytometry Annals of Botany 89741ndash753
Galbraith DW 2004 Cytometry and plant sciences a personalretrospective Cytometry 58A 37ndash44
Galbraith DW Harkins KR Maddox JM Ayres NM Sharma DPFiroozabady E 1983 Rapid flow cytometric analysis of the cell-cycle in intact plant-tissues Science 220 1049ndash1051
Galbraith DW Lambert GM Macas J Dolezel J 2002 Analysis ofnuclear DNA content and ploidy in higher plants In Robinson JPDarzynkiewicz Z Dean PN Dressler LG Rabinovitch PS StewartCV Tanke HJ Wheeless LL eds Current protocols in cytometryNew York John Wiley amp Sons 761ndash7622
Heller FO 1973 DNS-Bestimmung an Keimwurzeln von Vicia faba Lmit Hilfe der Impulscytophotometrie Bericht der DeutschenBotanischen Gesellschaft 86 437ndash441
Hintze J 2004 NCSS and PASS Kaysville Utah Number CruncherStatistical Systems
Kudo N Kimura Y 2001 Flow cytometric evidence for endopolyploidyin seedlings of some Brassica species Theoretical and AppliedGenetics 102 104ndash110
Loureiro J Rodriguez E Dolezel J Santos C 2006 Flow cytometricand microscopic analysis of the effect of tannic acid on plantnuclei and estimation of DNA content Annals of Botany 98515ndash527
Noirot M Barre P Louarn J Duperray C Hamon S 2000 Nucleusndashcytosol interactionsmdashA source of stoichiometric error in flowcytometric estimation of nuclear DNA content in plants Annals ofBotany 86 309ndash316
Noirot M Barre P Duperray C Louarn J Hamon S 2003 Effects ofcaffeine and chlorogenic acid on propidium iodide accessibility toDNA consequences on genome size evaluation in coffee treeAnnals of Botany 92 259ndash264
Otto F 1992 Preparation and staining of cells for high-resolution DNAanalysis In Radbruch A ed Flow cytometry and cell sortingBerlin Springer-Verlag 101ndash104
Pfosser M Amon A Lelley T Heberle-Bors E 1995 Evaluation ofsensitivity of flow cytometry in detecting aneuploidy in wheat usingdisomic and ditelosomic wheat-rye addition lines Cytometry 21387ndash393
Pinto G Loureiro J Lopes T Santos C 2004 Analysis of the geneticstability of Eucalyptus globulus Labill somatic embryos by flowcytometry Theoretical and Applied Genetics 109 580ndash587
Rodriguez E Gomes A Loureiro J Dolezel J Santos C 2005aEstimation of the genome size of the Iberian Peninsula Ulmaceae In9th Iberian Congress of Cytometry Book of Abstracts PortoPortugal 37
Rodriguez E Loureiro J Dolezel J Santos C 2005b The adequacy ofusing formaldehyde fixation for nuclear DNA content analyses ofplant material In 9th Iberian Congress of Cytometry Book ofAbstracts Porto Portugal PO120-PB
Shapiro H 2004 Practical flow cytometry 4th edn New YorkWiley-Liss
688 Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM
Suda J 2004 An employment of flow cytometry into plant biosystematicsPhD thesis Charles University Czech Republic Available at httpwwwibotcasczfcmsudapresentationdisertationpdf
Trotter J 2000 WinMDI version 28 Available at httpfacsscrippsedusoftwarehtml
Ulrich I Ulrich W 1986 Flow cytometric DNA analysis of plantprotoplasts with DAPI Zeitschrift fur Naturforschung 411052ndash1056
Ulrich I Fritz B Ulrich W 1988 Application of DNA fluorochromes forflow cytometric DNA analysis of plant protoplasts Plant Science 55151ndash158
Walker D Monino I Correal E 2006 Genome size inBituminaria bituminosa (L) CH Stirton (Fabaceae) populationsseparation of lsquotruersquo differences from environmental effects on DNAdetermination Environmental and Experimental Botany 55258ndash265
Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM 689
underlines its position as one of the best standards forplant DNA FCM By contrast Sedum burrito was themost difficult species to analyse due to low DNA contentoccurrence of polysomaty and high leaf water contentwhich hampered sample preparation and analysis More-over its tissues may contain tannins (J Greilhuber perscomm 2006)
In O pes-caprae cytosol of which is highly acidic(pH lt 30) measurable samples could be prepared usingonly Ottorsquos and Galbraithrsquos buffers with Ottorsquos beinghighly superior This is in accordance with the results ofEmshwiller (2002) who analysed ploidy levels in OxalisAfter testing LB01 MgSO4 and Ottorsquos buffers sheobtained measurable samples only with Ottorsquos The formertwo buffers failed presumably as a result of the acidic cellsap which may have exceeded the buffering capacity ofLB01 and MgSO4
Celtis australis was the only woody plant speciesincluded in the present study and was chosen because ofthe presence of mucilaginous compounds (Rodriguez et al2005a) which increase sample viscosity restrain nucleirelease and cause their clumping Interestingly this wasthe only species for which TrisMgCl2 was the bestperforming buffer This was probably because of a higherconcentration of the non-ionic detergent which sup-pressed the effect of mucilaginous compounds Leaftissues of F rothmaleri were particularly hard anddifficult to chop In addition preliminary experimentswith this species revealed the presence of cytosoliccompounds which would be expected to interfere withDNA staining However given the pattern of FS and SSobtained the so-called lsquotannic acid effectrsquo (Loureiro et al2006) was absent indicating that these compounds werereleased at low concentration or not at all
In order to compare the performance of nuclear isolationbuffers a set of parameters was carefully selected toevaluate sample quality Furthermore stability of fluores-cence and light scatter properties of isolated nuclei overtime were confirmed Among the parameters chosen thecoefficient of variation of DNA peaks (CV) is of majorimportance Galbraith et al (2002) considered 50 asthe maximum acceptable CV value in plant DNA flowcytometry With the exception of TrisMgCl2 buffer whenused with S burrito all mean CV values obtained hereinwere below this limit Ideally a nuclei sample should befree of cell and tissue debris Emshwiller (2002) noted acorrelation between CV and background noise In thepresent study this correlation was found only in somespecies (eg F rothmaleri) Rather the results heresuggest that the extent of background debris is determinedby the buffer itself For example a higher detergentconcentration in a buffer could lead to chloroplast lysisand consequently decrease the number of fluorescentparticles contributing to debris signals (Coba de la Penaand Brown 2001)
LB01 buffer provided very good results with theexception of O pes-caprae and S burrito low CV highFL and YF values were obtained with this buffer Highnuclei FL intensities obtained with this buffer weredefinitely an advantage as compared with TrisMgCl2 and
Galbraithrsquos buffers Galbraithrsquos buffer seems well bal-anced as acceptable results were achieved in all speciesSurprisingly the buffer gave reasonable results also withO pes-caprae which is characterized by highly acidiccytosol The presence of MOPS in the buffer may shedlight on these findings as it has a pKa of 72 and a betterbuffering capacity than TRIS with a pKa of 81 Adisadvantage of this buffer was the low fluorescenceintensity of nuclei Collectively TrisMgCl2 was the worstperforming buffer Nevertheless it provided the bestresults for C australis This may have been due to thehigher concentration of non-ionic detergent (Table 1)which counteracted the agglutinating effect of mucilagin-ous compounds and decreased sample viscosity As withGalbraithrsquos buffer low FL values were obtained withTrisMgCl2
Ottorsquos buffer is unique in that sample preparationinvolves two steps The results obtained with Ottorsquos bufferwere excellent in many species especially those withlower nuclear DNA content Dolezel and Bartos (2005)highlighted the quality of this buffer which is known toprovide DNA content histograms with unequalled resolu-tion One explanation for this may be that citric acidimproves chromatin accessibility and lsquohomogenizesrsquo chro-matin structure eliminating differences in staining intens-ity among nuclei with the same DNA content but differentchromatin state This could explain the highest FL andlowest CV values as observed here Another characteristicof this buffer was that light scatter values (especially FS)were significantly lower than those of other buffers Thismay be explained by the action of citric acid whichcauses nuclei fixation (Dolezel and Bartos 2005) It isnoteworthy that the pattern of FS vs SS distribution wassimilar to that obtained when analysing nuclei fixed withformaldehyde (Rodriguez et al 2005b) Althoughproviding excellent results this buffer gave the highestBF and lowest YF values The former can be explained bynuclei instability after the addition of Otto II After thisstep nuclei deteriorate rapidly in some species (J Dolezelunpubl res) The lowest YF values could be due to thedetergent (Tween 20) which is weaker than Triton X-100thus having a lower capacity to release nuclei
An important consequence of the observation ofdifferent fluorescence values obtained with the differentbuffers is that different fluorescence ratios may beexpected with the same species pair if the samples areprepared in different buffers In fact Dolezel et al (1998)observed differences in peak ratios obtained in differentlaboratories Further studies are needed to confirm thisobservation as it may have important consequences forestimation of genome size
The present results showed that well-defined popula-tions of nuclei could be observed on cytograms of FS vsSS With the exception of S burrito for which LB01 andTrisMgCl2 buffers did not present statistically significantdifferences for both parameters the analysis of FS and SSprovided a fingerprint pattern for each buffer Loureiroet al (2006) showed that these parameters were sensitiveto the presence of tannic acid a cytosolic compoundcommon in plants and recommended the analysis of light
Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM 687
scatter to verify suitability of particular samples for plantDNA FCM
As nuclei samples are prepared manually it wasimportant to assess the effect of date of analysis andoperator on sample quality variation Date was found to bemore significant than the effect of different operatorsespecially for FS SS CV and DF YF which depends onthe way the sample is chopped and hence on the operatordid not vary It was expected that FL which is a primarysource of data in FCM analysis would not depend on theoperator or the day of chopping This was confirmed in allspecies except L esculentum and C australis In theformer species a significant variation was obtainedbetween operators These differences could be explainedby variable results obtained with Galbraithrsquos buffer InC australis significant differences were obtained amongdates In this case significant differences were due toresults obtained on one single day Emshwiller (2002)found significant differences when multiple preparationsfrom the same plant were run on different days Thesedifferences and those found in the present study wereprobably due to instrument drift (Kudo and Kimura2001) To avoid this type of error several authors haverecommended that each measurement be repeated at leastthree times on three different days (Suda 2004 Dolezeland Bartos 2005)
This is the first study that has systematically comparednuclear isolation buffers for DNA FCM The results showthat none of the buffers works best with all species andstatistically significant differences in sample quality wereobserved among the four buffers The results obtainedwith different species and contrasting types of leaf tissuescan serve as guidelines in buffer selection Nevertheless itis recommend that a range of buffers be tested whenworking with a new species and tissue type Once the bestbuffer has been identified additives should be tested ifrequired to suppress negative effects of phenols and othercytosolic compounds
ACKNOWLEDGEMENTS
We are grateful to Prof Johann Greilhuber and Dr JanSuda for critical reading of the manuscript This work wassupported by FCT project ref POCTIAGR606722004JL was supported by the Fellowship FCTBD90032002
LITERATURE CITEDBennett MD Leitch I 2005 Genome size evolution in plants In
Gregory T ed The evolution of the genome London ElsevierAcademic Press 89ndash162
Bergounioux C Perennes C Brown SC Gadal P 1988 Cytometricanalysis of growth regulator-dependent transcription and cell cycleprogression in Petunia protoplast cultures Planta 175 500ndash505
Bergounioux C Brown SC Petit PX 1992 Flow cytometry and plantprotoplast cell biology Physiologia Plantarum 85 374ndash386
Castro S Loureiro J Santos C Ayensa G Navarro L 2005 Differentploidy levels could explain reproductive matter of invasive Oxalispes-caprae L in Mediterranean regions In Brunel S ed Invasiveplants in Mediterranean type regions of the world StrasbourgCedex France Council of Europe Publishing 235
Coba de la Pena T Brown SC 2001 Cytometry and fluorimetryIn Hawes C Satiat-Jeunemaıtre B eds Plant cell biology
a practical approach New York Oxford University Press85ndash106
Dolezel J Bartos J 2005 Plant DNA flow cytometry and estimation ofnuclear genome size Annals of Botany 95 99ndash110
Dolezel J Gohde W 1995 Sex determination in dioecious plantsMelandrium album and M rubrum using high-resolution flowcytometry Cytometry 19 103ndash106
Dolezel J Binarova P Lucretti S 1989 Analysis of nuclear DNAcontent in plant cells by flow cytometry Biologia Plantarum 31113ndash120
Dolezel J Sgorbati S Lucretti S 1992 Comparison of three DNAfluorochromes for flow cytometric estimation of nuclear DNAcontent in plants Physiologia Plantarum 85 625ndash631
Dolezel J Greilhuber J Lucretti S Meister A Lysak M Nardi LObermayer R 1998 Plant genome size estimation byflow cytometry inter-laboratory comparison Annals of Botany 8217ndash26
Dolezel J Bartos J Voglmayr H Greilhuber J 2003 Nuclear DNAcontent and genome size of trout and human Cytometry 51A127ndash128
Emshwiller E 2002 Ploidy levels among species in the lsquoOxalis tuberosaAlliancersquo as inferred by flow cytometry Annals of Botany 89741ndash753
Galbraith DW 2004 Cytometry and plant sciences a personalretrospective Cytometry 58A 37ndash44
Galbraith DW Harkins KR Maddox JM Ayres NM Sharma DPFiroozabady E 1983 Rapid flow cytometric analysis of the cell-cycle in intact plant-tissues Science 220 1049ndash1051
Galbraith DW Lambert GM Macas J Dolezel J 2002 Analysis ofnuclear DNA content and ploidy in higher plants In Robinson JPDarzynkiewicz Z Dean PN Dressler LG Rabinovitch PS StewartCV Tanke HJ Wheeless LL eds Current protocols in cytometryNew York John Wiley amp Sons 761ndash7622
Heller FO 1973 DNS-Bestimmung an Keimwurzeln von Vicia faba Lmit Hilfe der Impulscytophotometrie Bericht der DeutschenBotanischen Gesellschaft 86 437ndash441
Hintze J 2004 NCSS and PASS Kaysville Utah Number CruncherStatistical Systems
Kudo N Kimura Y 2001 Flow cytometric evidence for endopolyploidyin seedlings of some Brassica species Theoretical and AppliedGenetics 102 104ndash110
Loureiro J Rodriguez E Dolezel J Santos C 2006 Flow cytometricand microscopic analysis of the effect of tannic acid on plantnuclei and estimation of DNA content Annals of Botany 98515ndash527
Noirot M Barre P Louarn J Duperray C Hamon S 2000 Nucleusndashcytosol interactionsmdashA source of stoichiometric error in flowcytometric estimation of nuclear DNA content in plants Annals ofBotany 86 309ndash316
Noirot M Barre P Duperray C Louarn J Hamon S 2003 Effects ofcaffeine and chlorogenic acid on propidium iodide accessibility toDNA consequences on genome size evaluation in coffee treeAnnals of Botany 92 259ndash264
Otto F 1992 Preparation and staining of cells for high-resolution DNAanalysis In Radbruch A ed Flow cytometry and cell sortingBerlin Springer-Verlag 101ndash104
Pfosser M Amon A Lelley T Heberle-Bors E 1995 Evaluation ofsensitivity of flow cytometry in detecting aneuploidy in wheat usingdisomic and ditelosomic wheat-rye addition lines Cytometry 21387ndash393
Pinto G Loureiro J Lopes T Santos C 2004 Analysis of the geneticstability of Eucalyptus globulus Labill somatic embryos by flowcytometry Theoretical and Applied Genetics 109 580ndash587
Rodriguez E Gomes A Loureiro J Dolezel J Santos C 2005aEstimation of the genome size of the Iberian Peninsula Ulmaceae In9th Iberian Congress of Cytometry Book of Abstracts PortoPortugal 37
Rodriguez E Loureiro J Dolezel J Santos C 2005b The adequacy ofusing formaldehyde fixation for nuclear DNA content analyses ofplant material In 9th Iberian Congress of Cytometry Book ofAbstracts Porto Portugal PO120-PB
Shapiro H 2004 Practical flow cytometry 4th edn New YorkWiley-Liss
688 Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM
Suda J 2004 An employment of flow cytometry into plant biosystematicsPhD thesis Charles University Czech Republic Available at httpwwwibotcasczfcmsudapresentationdisertationpdf
Trotter J 2000 WinMDI version 28 Available at httpfacsscrippsedusoftwarehtml
Ulrich I Ulrich W 1986 Flow cytometric DNA analysis of plantprotoplasts with DAPI Zeitschrift fur Naturforschung 411052ndash1056
Ulrich I Fritz B Ulrich W 1988 Application of DNA fluorochromes forflow cytometric DNA analysis of plant protoplasts Plant Science 55151ndash158
Walker D Monino I Correal E 2006 Genome size inBituminaria bituminosa (L) CH Stirton (Fabaceae) populationsseparation of lsquotruersquo differences from environmental effects on DNAdetermination Environmental and Experimental Botany 55258ndash265
Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM 689
scatter to verify suitability of particular samples for plantDNA FCM
As nuclei samples are prepared manually it wasimportant to assess the effect of date of analysis andoperator on sample quality variation Date was found to bemore significant than the effect of different operatorsespecially for FS SS CV and DF YF which depends onthe way the sample is chopped and hence on the operatordid not vary It was expected that FL which is a primarysource of data in FCM analysis would not depend on theoperator or the day of chopping This was confirmed in allspecies except L esculentum and C australis In theformer species a significant variation was obtainedbetween operators These differences could be explainedby variable results obtained with Galbraithrsquos buffer InC australis significant differences were obtained amongdates In this case significant differences were due toresults obtained on one single day Emshwiller (2002)found significant differences when multiple preparationsfrom the same plant were run on different days Thesedifferences and those found in the present study wereprobably due to instrument drift (Kudo and Kimura2001) To avoid this type of error several authors haverecommended that each measurement be repeated at leastthree times on three different days (Suda 2004 Dolezeland Bartos 2005)
This is the first study that has systematically comparednuclear isolation buffers for DNA FCM The results showthat none of the buffers works best with all species andstatistically significant differences in sample quality wereobserved among the four buffers The results obtainedwith different species and contrasting types of leaf tissuescan serve as guidelines in buffer selection Nevertheless itis recommend that a range of buffers be tested whenworking with a new species and tissue type Once the bestbuffer has been identified additives should be tested ifrequired to suppress negative effects of phenols and othercytosolic compounds
ACKNOWLEDGEMENTS
We are grateful to Prof Johann Greilhuber and Dr JanSuda for critical reading of the manuscript This work wassupported by FCT project ref POCTIAGR606722004JL was supported by the Fellowship FCTBD90032002
LITERATURE CITEDBennett MD Leitch I 2005 Genome size evolution in plants In
Gregory T ed The evolution of the genome London ElsevierAcademic Press 89ndash162
Bergounioux C Perennes C Brown SC Gadal P 1988 Cytometricanalysis of growth regulator-dependent transcription and cell cycleprogression in Petunia protoplast cultures Planta 175 500ndash505
Bergounioux C Brown SC Petit PX 1992 Flow cytometry and plantprotoplast cell biology Physiologia Plantarum 85 374ndash386
Castro S Loureiro J Santos C Ayensa G Navarro L 2005 Differentploidy levels could explain reproductive matter of invasive Oxalispes-caprae L in Mediterranean regions In Brunel S ed Invasiveplants in Mediterranean type regions of the world StrasbourgCedex France Council of Europe Publishing 235
Coba de la Pena T Brown SC 2001 Cytometry and fluorimetryIn Hawes C Satiat-Jeunemaıtre B eds Plant cell biology
a practical approach New York Oxford University Press85ndash106
Dolezel J Bartos J 2005 Plant DNA flow cytometry and estimation ofnuclear genome size Annals of Botany 95 99ndash110
Dolezel J Gohde W 1995 Sex determination in dioecious plantsMelandrium album and M rubrum using high-resolution flowcytometry Cytometry 19 103ndash106
Dolezel J Binarova P Lucretti S 1989 Analysis of nuclear DNAcontent in plant cells by flow cytometry Biologia Plantarum 31113ndash120
Dolezel J Sgorbati S Lucretti S 1992 Comparison of three DNAfluorochromes for flow cytometric estimation of nuclear DNAcontent in plants Physiologia Plantarum 85 625ndash631
Dolezel J Greilhuber J Lucretti S Meister A Lysak M Nardi LObermayer R 1998 Plant genome size estimation byflow cytometry inter-laboratory comparison Annals of Botany 8217ndash26
Dolezel J Bartos J Voglmayr H Greilhuber J 2003 Nuclear DNAcontent and genome size of trout and human Cytometry 51A127ndash128
Emshwiller E 2002 Ploidy levels among species in the lsquoOxalis tuberosaAlliancersquo as inferred by flow cytometry Annals of Botany 89741ndash753
Galbraith DW 2004 Cytometry and plant sciences a personalretrospective Cytometry 58A 37ndash44
Galbraith DW Harkins KR Maddox JM Ayres NM Sharma DPFiroozabady E 1983 Rapid flow cytometric analysis of the cell-cycle in intact plant-tissues Science 220 1049ndash1051
Galbraith DW Lambert GM Macas J Dolezel J 2002 Analysis ofnuclear DNA content and ploidy in higher plants In Robinson JPDarzynkiewicz Z Dean PN Dressler LG Rabinovitch PS StewartCV Tanke HJ Wheeless LL eds Current protocols in cytometryNew York John Wiley amp Sons 761ndash7622
Heller FO 1973 DNS-Bestimmung an Keimwurzeln von Vicia faba Lmit Hilfe der Impulscytophotometrie Bericht der DeutschenBotanischen Gesellschaft 86 437ndash441
Hintze J 2004 NCSS and PASS Kaysville Utah Number CruncherStatistical Systems
Kudo N Kimura Y 2001 Flow cytometric evidence for endopolyploidyin seedlings of some Brassica species Theoretical and AppliedGenetics 102 104ndash110
Loureiro J Rodriguez E Dolezel J Santos C 2006 Flow cytometricand microscopic analysis of the effect of tannic acid on plantnuclei and estimation of DNA content Annals of Botany 98515ndash527
Noirot M Barre P Louarn J Duperray C Hamon S 2000 Nucleusndashcytosol interactionsmdashA source of stoichiometric error in flowcytometric estimation of nuclear DNA content in plants Annals ofBotany 86 309ndash316
Noirot M Barre P Duperray C Louarn J Hamon S 2003 Effects ofcaffeine and chlorogenic acid on propidium iodide accessibility toDNA consequences on genome size evaluation in coffee treeAnnals of Botany 92 259ndash264
Otto F 1992 Preparation and staining of cells for high-resolution DNAanalysis In Radbruch A ed Flow cytometry and cell sortingBerlin Springer-Verlag 101ndash104
Pfosser M Amon A Lelley T Heberle-Bors E 1995 Evaluation ofsensitivity of flow cytometry in detecting aneuploidy in wheat usingdisomic and ditelosomic wheat-rye addition lines Cytometry 21387ndash393
Pinto G Loureiro J Lopes T Santos C 2004 Analysis of the geneticstability of Eucalyptus globulus Labill somatic embryos by flowcytometry Theoretical and Applied Genetics 109 580ndash587
Rodriguez E Gomes A Loureiro J Dolezel J Santos C 2005aEstimation of the genome size of the Iberian Peninsula Ulmaceae In9th Iberian Congress of Cytometry Book of Abstracts PortoPortugal 37
Rodriguez E Loureiro J Dolezel J Santos C 2005b The adequacy ofusing formaldehyde fixation for nuclear DNA content analyses ofplant material In 9th Iberian Congress of Cytometry Book ofAbstracts Porto Portugal PO120-PB
Shapiro H 2004 Practical flow cytometry 4th edn New YorkWiley-Liss
688 Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM
Suda J 2004 An employment of flow cytometry into plant biosystematicsPhD thesis Charles University Czech Republic Available at httpwwwibotcasczfcmsudapresentationdisertationpdf
Trotter J 2000 WinMDI version 28 Available at httpfacsscrippsedusoftwarehtml
Ulrich I Ulrich W 1986 Flow cytometric DNA analysis of plantprotoplasts with DAPI Zeitschrift fur Naturforschung 411052ndash1056
Ulrich I Fritz B Ulrich W 1988 Application of DNA fluorochromes forflow cytometric DNA analysis of plant protoplasts Plant Science 55151ndash158
Walker D Monino I Correal E 2006 Genome size inBituminaria bituminosa (L) CH Stirton (Fabaceae) populationsseparation of lsquotruersquo differences from environmental effects on DNAdetermination Environmental and Experimental Botany 55258ndash265
Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM 689
Suda J 2004 An employment of flow cytometry into plant biosystematicsPhD thesis Charles University Czech Republic Available at httpwwwibotcasczfcmsudapresentationdisertationpdf
Trotter J 2000 WinMDI version 28 Available at httpfacsscrippsedusoftwarehtml
Ulrich I Ulrich W 1986 Flow cytometric DNA analysis of plantprotoplasts with DAPI Zeitschrift fur Naturforschung 411052ndash1056
Ulrich I Fritz B Ulrich W 1988 Application of DNA fluorochromes forflow cytometric DNA analysis of plant protoplasts Plant Science 55151ndash158
Walker D Monino I Correal E 2006 Genome size inBituminaria bituminosa (L) CH Stirton (Fabaceae) populationsseparation of lsquotruersquo differences from environmental effects on DNAdetermination Environmental and Experimental Botany 55258ndash265
Loureiro et al mdash Comparison of Lysis Buffers in Plant FCM 689