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Two New Nuclear Isolation Buffers for Plant DNA Flow Cytometry: A Test with 37 Species

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After the initial boom in the application of flow cytometry in plant sciences in the late 1980s and early 1990s, which was accompanied by development of many nuclear isolation buffers, only a few efforts were made to develop new buffer formulas. In this work, recent data on the performance of nuclear isolation buffers are utilized in order to develop new buffers, general purpose buffer (GPB) and woody plant buffer (WPB), for plant DNA flow cytometry. GPB and WPB were used to prepare samples for flow cytometric analysis of nuclear DNA content in a set of 37 plant species that included herbaceous and woody taxa with leaf tissues differing in structure and chemical composition. The following parameters of isolated nuclei were assessed: forward and side light scatter, propidium iodide fluorescence, coefficient of variation of DNA peaks, quantity of debris background, and the number of particles released from sample tissue. The nuclear genome size of 30 selected species was also estimated using the buffer that performed better for a given species. In unproblematic species, the use of both buffers resulted in high quality samples. The analysis of samples obtained with GPB usually resulted in histograms of DNA content with higher or similar resolution than those prepared with the WPB. In more recalcitrant tissues, such as those from woody plants, WPB performed better and GPB failed to provide acceptable results in some cases. Improved resolution of DNA content histograms in comparison with previously published buffers was achieved in most of the species analysed. WPB is a reliable buffer which is also suitable for the analysis of problematic tissues/species. Although GPB failed with some plant species, it provided high-quality DNA histograms in species from which nuclear suspensions are easy to prepare. The results indicate that even with a broad range of species, either GPB or WPB is suitable for preparation of high-quality suspensions of intact nuclei suitable for DNA flow cytometry.
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TECHNICAL ARTICLE
Two New Nuclear Isolation Buffers for Plant DNA Flow Cytometry:
A Test with 37 Species
JOA
˜
OLOUREIRO
1,
*, ELEAZAR RODRIGUEZ
1
, JAROSLAV DOLEZ
ˇ
EL
2
and CONC E I C¸A
˜
OSANTOS
1
1
Laboratory of Biotechnology and Cytomics, CESAM and Department of Biology, University of Aveiro, Campus
Universita
´
rio de Santiago, 3810-193 Aveiro, Portugal and
2
Laboratory of Molecular Cytogenetics and Cytometry, Institute
of Experimental Botany, Sokolovska
´
, Olomouc, CZ-77200, Czech Republic
Received: 20 March 2007 Returned for revision: 14 May 2007 Accepted: 8 June 2007 Published electronically: 7 August 2007
Background and Aims After the initial boom in the application of flow cytometry in plant sciences in the late 1980s
and early 1990s, which was accompanied by development of many nuclear isolation buffers, only a few efforts were
made to develop new buffer formulas. In this work, recent data on the performance of nuclear isolation buffers are
utilized in order to develop new buffers, general purpose buffer (GPB) and woody plant buffer (WPB), for plant
DNA flow cytometry.
Methods GPB and WPB were used to prepare samples for flow cytometric analysis of nuclear DNA content in a set
of 37 plant species that included herbaceous and woody taxa with leaf tissues differing in structure and chemical
composition. The following parameters of isolated nuclei were assessed: forward and side light scatter, propidium
iodide fluorescence, coefficient of variation of DNA peaks, quantity of debris background, and the number of par-
ticles released from sample tissue. The nuclear genome size of 30 selected species was also estimated using the
buffer that performed better for a given species.
Key Results In unproblematic species, the use of both buffers resulted in high quality samples. The analysis of
samples obtained with GPB usually resulted in histograms of DNA content with higher or similar resolution than
those prepared with the WPB. In more recalcitrant tissues, such as those from woody plants, WPB performed
better and GPB failed to provide acceptable results in some cases. Improved resolution of DNA content histograms
in comparison with previously published buffers was achieved in most of the species analysed.
Conclusions WPB is a reliable buffer which is also suitable for the analysis of problematic tissues/species.
Although GPB failed with some plant species, it provided high-quality DNA histograms in species from which
nuclear suspensions are easy to prepare. The results indicate that even with a broad range of species, either GPB
or WPB is suitable for preparation of high-quality suspensions of intact nuclei suitable for DNA flow cytometry.
Key words: Cytosolic compounds, flow cytometry, general purpose buffer, genome size, lysis buffers, nuclear DNA
content, nuclear DNA staining, propidium iodide, woody plant buffer.
INTRODUCTION
Since the introduction of flow cytometry to plant sciences
in the 1980s, estimation of nuclear DNA content has been
the major application of flow cytometry in research, breed-
ing and production (Dolez
ˇ
el and Bartos
ˇ
, 2005). The spread
of the method was encouraged by the relative simplicity of
sample preparation, which typically involves mechanical
homogenization of plant tissues in a nuclear isolation
buffer (Galbraith et al., 1983). The buffer should facilitate
isolation of intact nuclei free of adhering cytoplasmic
debris, maintain nuclei stability in liquid suspension and
prevent their aggregation. It ought to protect nuclear DNA
from degradation and provide an appropriate environment
for specific and stoichiometric staining of nuclear DNA,
including the minimization of negative effects of some
cytosolic compounds on DNA staining.
With the aim to fulfil these needs and to analyse nuclear DNA
content with the highest resolution, many laboratories devel-
oped their own nuclear isolation buffer formulas. The current
release of the FLOW
ER database (http://flower.web.ua.pt/)
lists 27 lysis buffers with different chemical compositions
(Loureiro et al., 2007a). The usefulness of some of the buffers
is difficult to judge as their performance has not been analysed
thoroughly, nor have they been compared with other buffers.
However, there are some exceptions and these are mainly the
most popular buffers. Thus, de Laat et al. (1987) compared
their buffer with a commercial solution, analysing the coeffi-
cient of variation (CV) of DNA peaks and the amount of
debris background. Dolez
ˇ
el et al. (1989) introduced the LB01
buffer by analysing the nuclear DNA content of leaves and in
vitro cultured calli of several plant species. Arumuganathan
and Earle (1991a) proposed a buffer containing MgSO
4
and
used it to estimate genome size in over 100 plant species
(Arumuganathan and Earle, 1991b). Marie and Brown (1993)
tested their new buffer in approx. 70 plant species. Ulrich and
Ulrich (1991) and Dolez
ˇ
el and Go
¨
hde (1995) showed the useful-
ness of so-called Otto solutions (Otto, 1990) for high resolution
analyses of DNA content. Finally, Pfosser et al. (1995) tested
Tris.MgCl
2
buffer by evaluating the sensitivity of DNA flow
cytometry to detect aneuploidy in wheat.
A systematic comparison of nuclear isolation buffers
was done only recently by Loureiro et al. (2006a) who
* For Correspondence. E-mail jloureiro@ua.pt
# The Author 2007. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.
For Permissions, please email: journals.permissions@oxfordjournals.org
Annals of Botany 100: 875–888, 2007
doi:10.1093/annbot/mcm152, available online at www.aob.oxfordjournals.org
compared four of the most common buffers differing in
chemical composition: Galbraith (Galbraith et al., 1983),
LB01 (Dolez
ˇ
el et al., 1989), Otto (Ulrich and Ulrich,
1991; Dolez
ˇ
el and Go
¨
hde, 1995) and Tris.MgCl
2
(Pfosser
et al., 1995) buffers. Among others, the results confirmed
the until then empirically known fact that due to the diver-
sity of plant tissues in structure and chemical composition,
no single buffer works well with every species (Dolez
ˇ
el and
Bartos
ˇ
, 2005). Nonetheless, Loureiro et al. (2006a) showed
that some lysis buffers consistently yielded better results
than others, at least in unproblematic species in which
high quality suspensions of isolated nuclei suitable for
DNA flow cytometry could be prepared. The same set of
buffers was evaluated while studying the effect of tannic
acid, a common phenolic compound, on isolated plant
nuclei and estimation of DNA content (Loureiro et al.,
2006b). The study revealed that tannic acid affected fluor-
escence and light scatter properties of nuclei in suspension
regardless of the isolation buffer. However, the extent of the
negative effect of tannic acid was different for each buffer.
Stimulated by the results of Loureiro et al. (2006a, b), we
decided to develop nuclear isolation buffers suitable for a
broader range of plants. This paper reports on two new
nuclear isolation buffers: general purpose buffer (GPB)
and woody plant buffer (WPB). The performance of these
buffers was evaluated by analysing a wide set of plant
species representing 37 taxa belonging to 24 different
families, including herbaceous and woody plant species,
with tissues differing in structure and chemical compo-
sition. Also the genome size of 30 out of the 37 taxa was
estimated using the buffer that performed better in a
given species of which ten are new estimations.
MATERIALS AND METHODS
Plant material
Plants of Coriandrum sativum (commercial lot), Solanum
lycopersicum ‘Stupicke
´
’, Pisum sativum ‘Ctirad’ and Vicia
faba Inovec were grown from seeds (seeds from the latter
three taxa were provided by the Institute of Experimental
Botany, Olomouc, Czech Republic). Plants of Festuca roth-
maleri, Oxalis pes-caprae and Pterospartum tridentatum
were kindly provided by Prof. Paulo Silveira, Dr
´
lvia
Castro and Eng. Armando Costa (Department of Biology,
University of Aveiro, Portugal), respectively. Plants of
Olea europaea, Quercus robur, Saintpaulia ionantha and
Vitis vinifera were available from previous studies in the
Laboratory of Biotechnology and Cytomics at University
of Aveiro. Plants of Sedum burrito were obtained from
Flo
ˆ
r do Centro Horticultural Centre (Mira, Portugal). All
plants were maintained in a greenhouse at 22 + 2 ºC, with
a photoperiod of 16 h and a light intensity of 530 +
2 mmol m
22
s
21
. Leaves from the remaining taxa were col-
lected directly from field-grown individuals in Aveiro and
Oporto districts, Portugal, and either analysed immediately
or maintained in a refrigerator on moistened paper for a
maximum of 2 d until use.
Sample preparation
In each species, 40 50 mg of young leaf tissue was used
for sample preparation. However, in Sedum burrito the
quantity of leaf material required to release a sufficient
number of nuclei had to be increased to approx. 500 mg
(Loureiro et al., 2006a). Nuclear suspensions were prepared
according to Galbraith et al. (1983) using our isolation
buffers, GPB and WPB (Table 1). In each case, 1 mL of
buffer solution was added to a Petri dish containing the
plant tissue, which was chopped using a sharp razor blade
for approx. 60 s. For genome size estimations, the buffer
that performed better in a particular species was chosen
and leaf tissue from both the sample and DNA reference
standard (Table 2) were chopped simultaneously. The
resulting homogenate was filtered through an 80-mm
nylon filter to remove large debris. Nuclei were stained
with 50 mgmL
21
propidium iodide (PI; Fluka, Buchs,
Switzerland), and 50 mgmL
21
RNase (Sigma, St Louis,
MO, USA) was added to nuclear suspension to prevent
staining of double-stranded RNA. Samples were incubated
on ice and analysed within 10 min.
Flow cytometric analyses
Samples were analysed with a Coulter EPICS XL (Beckman
Coulter
w
, Hialeah, FL, USA) flow cytometer equipped with
an air-cooled argon-ion laser tuned to 15 mW and operating
at 488 nm. Fluorescence was collected through a 645-nm
dichroic long-pass filter in reflecting mode and a 620-nm
band-pass filter. The results were acquired using the
SYSTEM II software (version 3
.
0, Beckman Coulter
w
).
The instrument settings (amplification and sample rate)
were kept constant throughout the experiment and, for the
species which had been analysed in Loureiro et al.
(2006a), they were the same as those used in that report.
The following parameters were analysed in each sample:
forward scatter (FS) as a rough measure of particle’s size,
side scatter (SS) as a measure of particle’s optical comple-
xity, fluorescence intensity of PI-stained nuclei (FL), CV of
G
0
/G
1
peaks as a measure of nuclear integrity and variation
in DNA staining, a debris background factor (DF) as a
measure of sample quality, and a nuclear yield factor (YF)
in order to compare the quantity of nuclei in suspension
TABLE 1. Chemical composition of our nuclei isolation
buffers, GPB and WPB
Buffer Composition*
GPB 0
.
5m
M spermine.4HCl, 30 mM sodium citrate, 20 mM MOPS,
80 m
M KCl, 20 mM NaCl, 0
.
5 % (v/v) Triton X-100, pH 7
.
0
WPB 0
.
2
M Tris.HCl, 4 mM MgCl
2
.6H
2
O, 2 mM EDTA Na
2
.
2H
2
O,
86 m
M NaCl, 10 mM sodium metabisulfite, 1 % PVP-10, 1 %
(v/v) Triton X-100, pH 7
.
5
* Final concentrations are given. Both buffers should be stored in
aliquots at 4 8C and remain stable for up to 3 months.
MOPS, 4-Morpholinepropane sulfonate; Tris, tris-(hydroxymethyl)-
aminomethane; EDTA, ethylenediaminetetraacetic acid.
Loureiro et al. Two New Buffers for Plant Flow Cytometry876
TABLE 2. Estimation of genome size in selected plant species
Genome size
This work Previous reports
Species Family 2C (pg) 1C (Mbp)
1
Peak CV (%) Stand. 2C (pg) Method Reference
Acer negundo Aceraceae 1
.
07 + 0
.
03 525 3
.
14 S.l. N.D.
Actinidia deliciosa Actinidaceae 4
.
80 + 0
.
06 2349 2
.
77 P.s. 4
.
45 FCM:PI Hopping, 1994
3
.
97 FCM:PI Ollitrault et al., 1994b
Allium triquetrum Alliaceae 38
.
15 + 0
.
38 18655 2
.
02 V.f. 36
.
30 Fe Jones and Rees, 1968
39
.
30 Fe Labani and Elkington, 1987
Aloysia triphylla Verbenaceae 1
.
47 + 0
.
01 720 2
.
79 S.l. N.D.
Chamaecyparis lawsoniana Cupressaceae 21
.
01 + 0
.
15 10274 2
.
95 V.f. 23
.
05 FCM:PI Hizume et al., 2001
30
.
10 Fe Ohri and Khoshoo, 1986
Citrus limon Rutaceae 0
.
84 + 0
.
005 409 3
.
74 S.l. 0
.
80 FCM:PI Ollitrault et al., 1994a
0
.
77, 0
.
80 FCM:PI Kayim et al., 1998
0
.
771
.
15 FCM:PI Iannelli et al.,1998
1
.
24, 1
.
30 FCM:PI Capparelli et al., 2004
Citrus sinensis Rutaceae 0
.
87 + 0
.
003 425 4
.
02 S.l. 0
.
75 FCM:PI Ollitrault et al., 1994a
0
.
76, 0
.
85 FCM:PI Kayim et al., 1998
0
.
76, 0
.
82 FCM:PI Arumuganathan and Earle, 1991b
1
.
20 Fe Nagl et al., 1983
1
.
24 Fe Guerra, 1984
Coriandrum sativum Apiaceae 5
.
08 + 0
.
10 2483 2
.
60 P.s. 4
.
10 Fe Olszewska and Osiecka, 1983
7
.
65, 9
.
55 Fe Das and Mallick, 1989
8
.
85, 9
.
45 Fe Chattopadhyay and Sharma, 1990
Diospyros kaki Ebenaceae 5
.
08 + 0
.
002 2482 2
.
27 P.s. N.D.
Euphorbia peplus Euphorbiaceae 0
.
69 + 0
.
004 335 4
.
50 S.l. N.D.
Ficus carica Moraceae 0
.
73 + 0
.
03 356 4
.
20 S.l. 1
.
41 Fe Ohri and Khoshoo, 1987
Forsythia intermedia Oleaceae 2
.
01 + 0
.
01 985 3
.
22 G.m. N.D.
Ginkgo biloba Ginkgoaceae 22
.
85 + 0
.
15 11172 2
.
48 V.f. 19
.
50 FCM:EB Marie and Brown, 1993
21
.
60 FCM:PI Barow and Meister, 2002
19
.
86 Fe Ohri and Khoshoo, 1986
19
.
76 Fe Greilhuber, 1988
Ilex aquifolium Aquifoliaceae 1
.
93 + 0
.
04 944 2
.
89 G.m. N.D.
Laurus nobilis Lauraceae 6
.
50 + 0
.
09 3215 2
.
26 Z.m. 6
.
10 FCM:PI Zonneveld et al., 2005
Magnolia soulangiana Magnoliaceae 9
.
83 + 0
.
002 4806 2
.
43 Z.m. 11
.
95 Fe Nagl et al., 1977
14
.
20 Fe Olszewska and Osiecka, 1983
Malus domestica Rosaceae 1
.
56 + 0
.
02 765 3
.
39 S.l. 1
.
502
.
86
2
FCM:PI Dickson et al., 1992
1
.
522
.
48
2
FCM:PI Tatum et al.,2005
Olea europaea ssp. europaea Oleaceae 3
.
24 + 0
.
02 1583 3
.
80 P.s. 4
.
40, 4
.
52 Fe Rugini et al., 1996
3
.
904
.
66 Fe Bitonti et al., 1999
2
.
973
.
07 FCM:PI Loureiro et al., 2007b
Papaver rhoeas Papaveraceae 11
.
00 + 0
.
08 5378 1
.
95 Z.m. 5
.
20
2
Fe Nagl et al., 1983
5
.
25
2
Fe Bennett and Smith, 1976
7
.
14
2
Fe Srivastava and Lavania, 1991
Pinus pinea Pinaceae 56
.
09 + 1
.
83 27429 3
.
34 V.f. 60
.
80 FCM:PI Grotkopp et al.,2004
Prunus domestica Rosaceae 0
.
66 + 0
.
01 323 4
.
10 S.l. 0
.
61 FCM:PI Arumuganathan and Earle, 1991b
Continued
Loureiro et al. Two New Buffers for Plant Flow Cytometry 877
TABLE 2. Continued
Genome size
This work Previous reports
Species Family 2C (pg) 1C (Mbp)
1
Peak CV (%) Stand. 2C (pg) Method Reference
Prunus persica Rosaceae 0
.
62 + 0
.
01 303 4
.
30 S.l. 0
.
54, 0
.
55 FCM:PI Arumuganathan and Earle, 1991b
0
.
54, 0
.
55 FCM:PI Dickson et al., 1992
0
.
570
.
64 FCM:PI Baird et al., 1994
Pterospartum tridentatum Fabaceae 4
.
64 + 0
.
05 2269 2
.
92 Z.m. N.D.
Pyrus communis Rosaceae 1
.
24 + 0
.
03 605 3
.
00 S.l. 1
.
03, 1
.
11 FCM:PI Arumuganathan and Earle, 1991b
1
.
11 FCM:PI Dickson et al., 1992
Quercus robur Fagaceae 1
.
98 + 0
.
06 968 2
.
88 G.m. 1
.
85 FCM:EB Favre and Brown, 1996
1
.
90 FCM:EB Zoldos
ˇ
et al.,1998
Rosa sp. Rosaceae 2
.
46 + 0
.
10 1204 2
.
89 Z.m. 0
.
783
.
04
2
FCM:PI Yokoya et al., 2000
0
.
201
.
65
2
FCM:PI Dickson et al., 1992
0
.
251
.
30
2
Fe Bennett and Smith, 1976
2
.
85
2
Fe Greilhuber, 1988
Saintpaulia ionantha Gesneriaceae 1
.
50 + 0
.
02 732 3
.
41 S.l. N.D.
Salix babylonica Salicaceae 1
.
61 + 0
.
01 786 2
.
65 S.l. N.D.
Tamarix africana Tamaricaceae 3
.
30 + 0
.
03 1612 2
.
66 Z.m. N.D.
Vitis vinifera Vitaceae 1
.
19 + 0
.
02 583 2
.
86 S.l. 1
.
00 FCM:PI Arumuganathan and Earle, 1991b
1
.
60 FCM:HO Faure and Nougare
`
de, 1993
0
.
861
.
00 FCM:PI Lodhi and Reisch, 1995
1
.
171
.
26 FCM:PI Leal et al., 2006
Values are given as mean and standard deviation of the mean genome size in mass values (2C, pg) and base pairs (1C, Mbp).
The coefficient of variation (Peak CV,%) of sample G
0
/G
1
peaks and the reference standard (Stand.) used to estimate the genome size in each species (S.l., Solanum lycopersicum ‘Stupicke
´
’, 2C ¼
1
.
96 pg DNA, Dolez
ˇ
el et al., 1992; G.m., Glycine max ‘Polanka’, 2C ¼ 2
.
50 pg DNA, Dolez
ˇ
el et al., 1994; Z.m., Zea mays ‘CE-777’, 2C ¼ 5
.
43 pg DNA, Lysa
´
k and Dolez
ˇ
el, 1998; P.s., Pisum sativum
‘Ctirad’, 2C ¼ 9
.
09 pg DNA, Dolez
ˇ
el et al., 1998; V.f., Vicia faba ‘Inovec’, 2C ¼ 26
.
90 pg DNA, Dolez
ˇ
el et al., 1992) are also given. For each species, previous genome size estimations together with
the used methodology (Fe, Feulgen microdensitometry; FCM, flow cytometry; PI, propidium iodide; EB, ethidium bromide; HO, Hoechst 33342) and original reference are also provided.
N.D., not determined.
1
1pg¼ 978 Mbp (Dolez
ˇ
el et al., 2003).
2
These values may reflect differences in the ploidy level.
Loureiro et al. Two New Buffers for Plant Flow Cytometry878
independently of the amount of sample tissue used. DF
and YF were determined as follows (Loureiro et al.,
2006a):
DFð%Þ¼
Total number of particles
Total number of intact nuclei
Total number of particles
100 ð1Þ
YFðnuclei s
1
mg
1
Þ¼
Total number of intact nuclei
=number of seconds of run (s)
Weight of tissue (mg)
ð2Þ
Histograms of FL obtained with each buffer were overlaid
using WinMDI software (Trotter, 2000; Fig. 1). In each
species, five replicates per buffer were performed on three
different days. In each replicate at least 5000 nuclei were
analysed.
For genome size estimations, three replicates on three
different days were made using the buffer that performed
better in a given species. The best buffer was usually
characterized by higher FL and YF and lower CV and
DF, with the main evaluating parameters being the FL
and the CV. The nuclear DNA content of each species
was calculated according to the formula:
2C nuclear DNA content of sample (pg)
¼
sample G
0
=G
1
mean FL
reference standard G
0
=G
1
mean FL
2C nuclear DNA content of reference standard
ð3Þ
Conversion of mass values into numbers of base pairs was
done according to the factor 1 pg ¼ 978 Mbp (Dolez
ˇ
el
et al., 2003).
Statistical analyses
Differences between both buffers for each parameter
were analysed using a t-test (SigmaStat for Windows
Version 3
.
1, SPSS Inc., Richmond, CA, USA).
RESULTS
Performance of the nuclear isolation buffers
Testing the two new buffers with 37 plant species revealed
pronounced differences (Table 3). Out of the seven species
that were analysed by Loureiro et al. (2006a) (highlighted
in Table 3), the use of either buffer resulted in good
DNA content histograms in Festuca rothmaleri, Oxalis pes-
caprae and Sedum burrito, and very good histograms in
Solanum lycopersicum, Pisum sativum and Vicia faba
(Fig. 1). The only exception was Celtis australis in which
measurable samples were only obtained with WPB
(Fig. 1). Out of the remaining 30 taxa, GPB yielded accep-
table histograms with CVs below 5
.
0 % and no detectable
‘tannic acid effect’ (Loureiro et al., 2006b) in only 15 of
them (i.e. 50 % success rate), while WPB worked well
with all 30 species. In most of the species where GPB
failed, an effect similar to the ‘the tannic acid effect’ was
observed. This effect was first described by Loureiro
et al. (2006b) and involved the occurrence of two new
populations of particles on cytograms of forward scatter
vs. side scatter, and side scatter vs. fluorescence (arrows
in Fig. 2). The tannic acid effect resulted in fluorescence
histograms with higher DF, higher CVs of G
0
/G
1
peaks,
and lower nuclear fluorescence (Fig. 2).
Whereas the GPB performed better than WPB in 57
.
1%
of the original set of seven species (Loureiro et al., 2006a),
in the remaining 15 taxa where both buffers worked well,
it was only better in Allium triquetrum and Euphorbia
peplus. The better-performing buffer was usually character-
ized by higher FL and YF and lower CV and DF values
(Table 3).
The yield factor was the parameter where more statisti-
cally significant differences were detected between both
buffers (47
.
6 % of the species). With the exception of
Euphorbia peplus, the differences observed were due to a
higher yield observed with WPB. Also, when statistically
significant differences were observed for FL (i.e. in
42
.
8 % of the cases), they were due to higher fluorescence
of nuclei isolated with WPB than with GPB.
In 18 species, the CVs were lower than 3
.
0 %; in the
remaining species, CVs ranged from 3
.
0 % to 5
.
0 %. The
lowest CVs were observed after analysing Allium trique-
trum nuclei isolated with WPB (mean CV ¼ 1
.
79 %).
Statistical analysis revealed that in contrast to YF and FL,
CVs were more homogenous between buffers, with signifi-
cant differences between both buffers being only detected
in four species. Major differences in CVs were detected
in Ilex aquifolium (2
.
57 % and 4
.
10 % for WPB and
GPB, respectively), and Vitis vinifera (3
.
57 % and 4
.
77 %
for WPB and GPB, respectively). Even if significant differ-
ences were detected between the two remaining species,
Olea europaea and Magnolia soulangeana, the CVs
were low (,3 %) with any buffer.
When evaluating the DF, significant differences
between the isolation buffers were only observed in five
species, Coriandrum sativum, Magnolia soulangeana,
Olea europaea, Pisum sativum and Vicia faba.Withthe
exception of Magnolia soulangeana, samples isolated
with GPB exhibited higher debris background. Although
the DF differed in Magnolia soulangeana, Pisum
sativum and Vicia faba, they were among the lowest
values obtained in this study. Contrarily, the species
with the highest background debris were Tamarix afri-
cana, Euphorbia peplus
, Chamaecyparis lawsoniana and
Salix babylonica, with values usually higher than 30 %.
In most of the other species, DF usually ranged between
10 % and 20 %.
Nuclei isolated with WPB and GPB differed more in FS
than in SS. Out of the 21 species where both buffers worked
well, FS values were significantly different in 11 species,
while only in five species was this observed for SS.
Pterospartum tridentatum, Prunus domestica and Vicia
faba were the only species with statistically significantly
differences between buffers, for both parameters.
Loureiro et al. Two New Buffers for Plant Flow Cytometry 879
FIG. 1. Histograms of relative fluorescence intensities (PI fluorescence, channel numbers) with overlays of distributions obtained with the general
purpose buffer (GPB, red) and the woody plant buffer (WPB, blue). Mean channel numbers (FL) and coefficients of variation (CV,%) of G
0
/G
1
(peaks 1 and 2) and G
2
peaks (peaks 3 and 4) are given.
Loureiro et al. Two New Buffers for Plant Flow Cytometry880
TABLE 3. Flow cytometric parameters assessed in each species
Taxa G.t. Buffer
FS (channel
units)
SS (channel units) FL (channel
units)
CV (%) DF (%) YF (nuclei
s
21
mg
21
)
Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD
Acer negundo L. W GPB
WPB 29
.
16 3
.
05 26
.
14 10
.
24 234
.
317
.
23
.
24 0
.
35 16
.
45 1
.
62 0
.
79 0
.
27
Actinidia deliciosa (A Chev.) C.F. Liang
& A.R. Ferguson
H GPB 5
.
85
a
2
.
53 10
.
56
a
2
.
29 197
.
1
a
10
.
33
.
02
a
0
.
19 14
.
00
a
4
.
15 0
.
45
a
0
.
17
WPB 16
.
60
b
3
.
93 13
.
74
a
3
.
02 210
.
2
a
8
.
42
.
76
a
0
.
36 12
.
22
a
0
.
82 0
.
84
b
0
.
56
Allium triquetrum L. H GPB 9
.
02
a
1
.
49 5
.
12
a
1
.
75 194
.
9
a
2
.
61
.
79
a
0
.
38 12
.
47
a
2
.
39 0
.
22
a
0
.
08
WPB 12
.
88
b
1
.
53 4
.
77
a
1
.
19 194
.
2
a
3
.
22
.
14
a
0
.
29 10
.
51
a
2
.
08 0
.
35
b
0
.
07
Aloysia triphylla (L’He
´
r.) Britton W GPB
WPB 10
.
13 1
.
13 6
.
88 0
.
78 203
.
211
.
62
.
93 0
.
37 25
.
68 6
.
13 0
.
63 0
.
11
Chamaecyparis lawsoniana (Murr.) Parl. W GPB 24
.
72
a
7
.
40 33
.
82
a
20
.
37 193
.
1
a
4
.
82
.
56
a
0
.
25 36
.
18
a
5
.
53 0
.
10
a
0
.
04
WPB 14
.
88
b
1
.
47 16
.
82
a
0
.
64 192
.
8
a
3
.
12
.
48
a
0
.
17 35
.
26
a
1
.
37 0
.
13
a
0
.
05
Celtis australis L. WGPB–––– ––––
WPB 54
.
25 16
.
27 9
.
59 3
.
95 208
.
013
.
82
.
99 0
.
38 21
.
31 4
.
76 0
.
30 0
.
20
Citrus limon (L.) Burm. f. W GPB 2
.
29
a
1
.
86 3
.
34
a
0
.
56 171
.
3
a
5
.
43
.
62
a
0
.
26 16
.
50
a
3
.
86 1
.
00
a
0
.
39
WPB 4
.
15
a
1
.
89 3
.
45
a
0
.
690 170
.
6
a
8
.
13
.
75
a
0
.
30 12
.
09
a
2
.
66 1
.
13
a
0
.
30
Citrus sinensis (L.) Osbeck W GPB 1
.
44
a
2
.
05 6
.
00
a
3
.
90 174
.
9
a
7
.
03
.
75
a
0
.
34 18
.
00
a
6
.
44 0
.
89
a
0
.
26
WPB 4
.
36
a
2
.
14 10
.
83
a
3
.
13 192
.
1
b
14
.
64
.
29
a
1
.
25 17
.
28
a
6
.
57 1
.
30
b
0
.
24
Coriandrum sativum L. H GPB 19
.
52
a
2
.
58 18
.
90
a
4
.
26 206
.
1
a
4
.
32
.
69
a
0
.
70 26
.
56
a
9
.
43 0
.
89
a
0
.
40
WPB 18
.
92
a
6
.
04 29
.
58
b
7
.
99 199
.
8
a
7
.
02
.
13
a
0
.
12 12
.
84
b
1
.
48 2
.
16
b
0
.
72
Diospyros kaki L. f. W GPB
WPB 21
.
28 6
.
42 13
.
96 6
.
41 205
.
74
.
82
.
09 0
.
20 26
.
36 3
.
92 0
.
68 0
.
24
Euphorbia peplus L. H GPB 15
.
10
a
3
.
49 15
.
36
a
6
.
50 202
.
2
a
3
.
03
.
66
a
0
.
13 36
.
12
a
4
.
50 2
.
74
a
0
.
76
WPB 9
.
72
b
0
.
93 12
.
26
a
1
.
95 221
.
1
b
4
.
94
.
00
a
0
.
58 38
.
80
a
2
.
43 2
.
11
b
0
.
41
Festuca rothmaleri (Litard.) Markgr.-Dann. H GPB 12
.
76
a
2
.
20 7
.
91
a
1
.
02 205
.
4
a
14
.
72
.
59
a
0
.
60 9
.
67
a
1
.
97 0
.
21
a
0
.
15
WPB 15
.
52
a
6
.
12 15
.
03
b
3
.
50 209
.
5
a
8
.
73
.
25
a
0
.
75 10
.
33
a
5
.
46 0
.
59
b
0
.
35
Ficus carica L. W GPB
WPB 20
.
60 5
.
00 8
.
77 3
.
49 214
.
94
.
64
.
16 0
.
31 26
.
98 2
.
16 0
.
46 0
.
11
Forsythia intermedia Zabel W GPB
WPB 44
.
42 2
.
31 26
.
62 8
.
78 198
.
210
.
72
.
70 0
.
36 10
.
00 1
.
01 1
.
07 0
.
34
Ginkgo biloba L. W GPB
WPB 10
.
84 2
.
14 20
.
42 4
.
35 196
.
68
.
82
.
35 0
.
39 19
.
98 5
.
06 0
.
32 0
.
22
Ilex aquifolium L. W GPB 15
.
03
a
1
.
56 20
.
70
a
6
.
29 194
.
0
a
19
.
94
.
10
a
0
.
91 18
.
48
a
4
.
64 1
.
01
a
0
.
72
WPB 9
.
50
b
1
.
65 22
.
32
a
15
.
58 271
.
4
b
13
.
62
.
57
b
0
.
34 19
.
16
a
2
.
62 1
.
20
a
0
.
63
Laurus nobilis L. W GPB
WPB 19
.
88 7
.
22 3
.
65 1
.
44 235
.
55
.
42
.
35 0
.
60 25
.
78 4
.
96 0
.
84 0
.
56
Magnolia soulangeana Soul.-Bod. W GPB 28
.
60
a
2
.
33 22
.
13
a
3
.
92 141
.
3
a
9
.
52
.
90
a
0
.
81 4
.
13
a
1
.
25 0
.
90
a
0
.
18
WPB 28
.
58
a
2
.
35 24
.
60
a
2
.
13 199
.
5
b
4
.
01
.
80
b
0
.
12 9
.
26
b
2
.
38 0
.
80
a
0
.
22
Malus domestica (Borkh.) Borkh. W GPB 8
.
83
a
2
.
45 26
.
94
a
13
.
23 202
.
8
a
2
.
33
.
70
a
0
.
66 17
.
98
a
0
.
34 1
.
10
a
0
.
38
WPB 9
.
84
a
2
.
07 15
.
14
a
2
.
30 214
.
2
b
5
.
13
.
24
a
0
.
46 16
.
82
a
3
.
80 1
.
89
a
0
.
82
Olea europaea L. ssp. europaea W GPB 16
.
56
a
4
.
37 11
.
26
a
4
.
03 178
.
1
a
10
.
52
.
97
a
0
.
56 23
.
72
a
3
.
02 0
.
16
a
0
.
06
WPB 12
.
38
a
0
.
97 12
.
22
a
1
.
66 210
.
3
b
5
.
02
.
18
b
0
.
26 17
.
28
b
1
.
30 0
.
33
b
0
.
10
Oxalis pes-caprae L. H GPB 54
.
50
a
19
.
49 9
.
00
a
5
.
10 198
.
7
a
11
.
73
.
29
a
0
.
29 8
.
94
a
2
.
48 0
.
68
a
0
.
16
WPB 66
.
19
a
34
.
88 11
.
34
a
5
.
47 206
.
3
a
5
.
03
.
80
a
1
.
06 11
.
94
a
3
.
99 0
.
64
a
0
.
15
Continued
Loureiro et al. Two New Buffers for Plant Flow Cytometry 881
TABLE 3. Continued
Taxa G.t. Buffer
FS (channel
units)
SS (channel units) FL (channel
units)
CV (%) DF (%) YF (nuclei
s
21
mg
21
)
Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD
Papaver rhoeas L. H GPB
WPB 19
.
98 3
.
33 20
.
28 6
.
26 199
.
210
.
12
.
65 0
.
53 23
.
00 14
.
39 0
.
24 0
.
10
Pinus pinea L. W GPB
WPB 57
.
58 10
.
57 98
.
14 28
.
47 185
.
612
.
43
.
09 0
.
30 22
.
88 5
.
23 0
.
03 0
.
02
Pisum sativum L. H GPB 12
.
59
a
2
.
43 4
.
50
a
2
.
43 185
.
1
a
4
.
21
.
79
a
0
.
23 11
.
98
a
2
.
62 0
.
62
a
0
.
23
WPB 32
.
34
b
2
.
95 6
.
49
a
2
.
95 195
.
5
b
5
.
71
.
92
a
0
.
18 7
.
39
b
1
.
21 1
.
15
b
0
.
28
Prunus domestica L. W GPB 4
.
25
a
0
.
98 3
.
76
a
0
.
52 189
.
9
a
1
.
74
.
35
a
0
.
46 21
.
98
a
3
.
13 1
.
64
a
0
.
38
WPB 10
.
45
b
2
.
27 6
.
04
b
1
.
38 204
.
6
a
5
.
04
.
24
a
0
.
53 22
.
98
a
4
.
76 1
.
82
a
0
.
42
Prunus persica (L.) Batsch W GPB
WPB 8
.
87 2
.
12 8
.
38 2
.
16 219
.
08
.
14
.
91 0
.
70 21
.
22 4
.
98 1
.
31 0
.
46
Pterospartum tridentatum (L.) Willk. H GPB 31
.
84
a
12
.
80 45
.
86
a
15
.
99 196
.
8
a
6
.
93
.
25
a
0
.
76 29
.
40
a
2
.
18 1
.
18
a
0
.
78
WPB 12
.
38
b
2
.
66 13
.
82
b
1
.
63 201
.
5
a
5
.
92
.
71
a
0
.
32 29
.
38
a
3
.
31 0
.
87
a
0
.
64
Pyrus communis L. W GPB
WPB 8
.
26 2
.
98 14
.
59 3
.
96 203
.
05
.
13
.
20 0
.
36 18
.
26 3
.
24 1
.
66 0
.
23
Quercus robur L. W GPB
WPB 13
.
96 1
.
75 19
.
86 0
.
90 212
.
78
.
32
.
76 0
.
75 26
.
64 5
.
19 0
.
56 0
.
41
Rosa L. sp. W GPB
WPB 20
.
44 3
.
99 12
.
17 4
.
41 200
.
15
.
52
.
46 0
.
35 18
.
98 2
.
49 1
.
52 0
.
58
Saintpaulia ionantha Wendl. H GPB
WPB 12
.
25 1
.
76 22
.
76 3
.
89 204
.
75
.
23
.
42 0
.
31 22
.
32 1
.
99 0
.
47 0
.
17
Salix babylonica L. W GPB 10
.
80
a
3
.
91 8
.
62
a
4
.
98 192
.
9
a
9
.
03
.
45
a
0
.
18 32
.
52
a
3
.
86 1
.
30
a
0
.
46
WPB 6
.
53
b
0
.
54 4
.
24
a
1
.
91 194
.
0
a
7
.
63
.
17
a
0
.
43 26
.
24
a
5
.
26 1
.
53
a
0
.
88
Sedum burrito R. Moran S GPB 8
.
34
a
1
.
90 0
.
55
a
0
.
34 114
.
0
a
3
.
23
.
00
a
0
.
50 54
.
64
a
6
.
94 0
.
10
a
0
.
03
WPB 12
.
81
b
0
.
92 0
.
58
a
0
.
16 113
.
1
a
4
.
23
.
24
a
0
.
32 49
.
96
a
11
.
97 0
.
09
a
0
.
04
Solanum lycopersicum L. H GPB 7
.
61
a
1
.
10 1
.
14
a
0
.
40 232
.
9
a
6
.
72
.
31
a
0
.
49 15
.
16
a
1
.
85 1
.
00
a
0
.
46
WPB 11
.
53
b
1
.
30 1
.
72
a
0
.
78 264
.
8
b
7
.
92
.
23
a
0
.
14 14
.
36
a
1
.
50 1
.
33
a
0
.
40
Tamarix africana Poir. W GPB
WPB 26
.
60 3
.
11 22
.
88 4
.
91 208
.
85
.
52
.
75 0
.
28 39
.
00 4
.
46 0
.
96 0
.
31
Vicia faba L. H GPB 36
.
84
a
7
.
26 4
.
46
a
1
.
04 202
.
3
a
4
.
71
.
60
a
0
.
23 7
.
25
a
2
.
44 0
.
21
a
0
.
12
WPB 74
.
35
b
11
.
33 6
.
49
b
1
.
33 212
.
3
b
5
.
21
.
72
a
0
.
18 6
.
45
b
1
.
25 1
.
03
b
0
.
35
Vitis vinifera L. W GPB 7
.
33
a
3
.
09 3
.
99
a
1
.
95 206
.
6
a
6
.
24
.
77
a
0
.
42 27
.
46
a
8
.
23 0
.
65
a
0
.
21
WPB 4
.
32
a
0
.
90 2
.
67
a
1
.
44 213
.
1
a
8
.
23
.
57
b
0
.
20 21
.
86
a
6
.
92 1
.
29
b
0
.
49
Values are given as mean and standard deviation of the mean (SD) of forward scatter (FS, channel units), side scatter (SS, channel units), fluorescence (FL, channel units), coefficient of variation of
G
0
/G
1
DNA peak (CV,%), debris background factor (DF,%) and yield factor (YF,%).
Means for the same species followed by the same letter (a or b) are not statistically different according to a t-test at P 0
.
05.
The buffer chosen for the genome size estimations in each species is shown in bold type.
G.t., Growth type; W, woody; H, herbaceous; S, succulent; GPB, general purpose buffer; WPB, woody plant buffer.
Loureiro et al. Two New Buffers for Plant Flow Cytometry882
Estimation of nuclear genome size
Table 2 lists C-values for 30 species as determined in this
study, five of which are first estimates using flow cytometry
and ten are new estimates. The buffer that performed better
with each species was selected to estimate its genome size.
As expected, mean CVs of DNA peaks (Table 2 and
Fig. 3) were generally within the range of values obtained
in the first part of the study (Table 3). Also, the standard
deviations were low, with values higher than 4 % in only
one species (Rosa sp., 4
.
06 %), indicating that the three
replicates per species on three different days yielded hom-
ogenous estimates of nuclear DNA amount.
Plant species used in this work have a wide range of
genome size, ranging from 0
.
62 pg/2C DNA in Prunus
persica to 56
.
09 pg/2C DNA in Pinus pinea. Following
the genome size classes (in C-values) of Soltis et al.
(2003), most of the species studied in this work (80
.
0%)
belong to the ‘very small’ (1
.
4 pg) or ‘small’ (.1
.
4to
3
.
5 pg) genome size categories. In four species (13
.
3%)
‘intermediate’ (.3
.
5to14
.
0 pg) genome sizes were
found and only two species (6
.
7 %) are characterized by
‘large’ (. 14
.
0to35
.
0 pg) or ‘very large’ (. 35
.
0 pg)
genomes. While in some species our assessments were in
close agreement with previous reports, considerable differ-
ences were observed in other cases with most of the
discrepancies concerning the results obtained with
Feulgen microdensitometry (Table 2).
DISCUSSION
Our recent studies (Loureiro et al., 2006a,b) provided quan-
titative data on performance of the most popular nuclear
isolation buffers and showed that none of them worked
well with all species that represented different types of
leaf tissues and different nuclear genome sizes. It was
also clear that the chemical composition was important to
cope with the negative effect of cytosolic compounds
such as tannic acid. The results of these studies prompted
us to develop improved buffers.
The popular nuclear isolation buffers are based on organic
buffers such as MOPS (Galbraith et al., 1983), Tris (Dolez
ˇ
el
et al., 1989; Pfosser et al., 1995) and 4-(hydroxymethyl)
piperazine-1-ethanesulfonic acid (HEPES) (de Laat et al.,
1987; Arumuganathan and Earle, 1991a) that stabilize pH
of the solution and keep nuclei in an intact or even sub-vital
state (Greilhuber et al., 2007). Non-ionic detergents, such
as Triton X-100 and Tween 20, are used to facilitate the
release of nuclei from cells and prevent nuclei clumping
and attachment of debris, while the nuclear chromatin is
stabilized by Mg
2þ
(Galbraith et al., 1983; Arumuganathan
FIG. 2. Cytograms of forward scatter (logarithmic scale, FS log) vs. side scatter (logarithmic scale, SS log) (A, D), histograms of PI fluorescence inten-
sity (PI fluorescence, channel numbers) (B, E), and cytograms of SS log vs. PI fluorescence (C, F) of nuclear suspensions of Rosa sp. obtained with WPB
(AC) and GPB (DF). An effect similar to the ‘tannic acid effect’ (Loureiro et al., 2006b) was observed in nuclear suspensions obtained with GPB.
Arrows indicate two additional populations of particles. The first population comprises nuclei to which weakly fluorescent particles were attached (higher
SS and FL values). The second population consists of clumps of weakly fluorescent particles (higher SS and lower FL values). Mean channel numbers
(Mean channel) and coefficients of variation (CV,%) of G
0
/G
1
peaks are given.
Loureiro et al. Two New Buffers for Plant Flow Cytometry 883
FIG. 3. Histograms of relative fluorescence intensities (PI fluorescence, channel numbers) obtained after simultaneous analysis of nuclei isolated from
sample (peak 1) and internal reference standard (peak 2) using the buffer that performed better (see Table 3). The following reference standards were
used: Solanum lycopersicum ‘Stupicke
´
’ (2C ¼ 1
.
96 pg DNA) (A, C, D, F, I, K); Glycine max ‘Polanka’ (2C ¼ 2
.
50 pg DNA) (G, L); Zea mays ‘CE-777’
(2C ¼ 5
.
43 pg DNA) (H); Pisum sativum ‘Ctirad’ (2C ¼ 9
.
09 pg DNA) (B, E); Vicia faba’Inovec’ (2C ¼ 26
.
90 pg DNA) (J). Mean channel number
(Mean), DNA index (DI ¼ mean channel number of sample/mean channel number of internal reference standard), and coefficients of variation
(CV,%) of G
0
/G
1
peaks are given.
Loureiro et al. Two New Buffers for Plant Flow Cytometry884
and Earle, 1991a) or spermine (Dolez
ˇ
el et al., 1989). In some
buffers, chelating agents (e.g. EDTA, sodium citrate) are
added to bind divalent cations, which serve as cofactors of
DNases; inorganic salts (e.g. KCl, NaCl) are used to
achieve proper ionic strength (Dolez
ˇ
el and Bartos
ˇ
, 2005).
Some buffers are supplemented with reducing agents such
as b-mercaptoethanol, metabisulfite and dithiothreitol to
prevent the action of phenolic compounds, while PVP is
added to bind the phenolics kept in a reduced state
(Greilhuber et al., 2007).
GPB was developed considering the results of Loureiro
et al. (2006a) and its chemical composition is based on
that of LB01, the buffer that performed best in that study.
As MOPS was shown to be a better buffer than Tris, this
component was used in GPB instead of Tris at the same
concentration as in the Galbraith’s buffer. Moreover, the
concentration of Triton X-100 in GPB was raised to
0
.
5 % which helped to keep isolated nuclei free from
attached debris (Loureiro et al., 2006a, b). The composition
of WPB is based on the Tris.MgCl
2
buffer, which counter-
acts the negative effects of tannic acid better than other
buffers (Loureiro et al., 2006b). The WPB formula includes
a chelating agent and inorganic salt (both from LB01
buffer) and Triton X-100 at 1
.
0 % (the highest concen-
tration reported in the literature). Although a simultaneous
inclusion of MgCl
2
and EDTA has been proposed to be
counterproductive (Greilhuber et al., 2007), preliminary
tests did not reveal any negative effect on nuclei quality
and stability, possibly due to a higher affinity of EDTA to
other metals and to a sufficient concentration of free
Mg
2þ
in the solution necessary to stabilize the chromatin
structure. Sodium metabisulfite (a reducing agent) and
PVP-10 (a phenol competitor) were added to make WPB
suitable for use in recalcitrant species such as woody
plants with tissues rich in phenols and other secondary
metabolites.
The main goal of this work was to develop new formulas
for nuclei isolation buffers based on the experience with
existing ones, generally using their components at the
same concentrations. Systematic evaluation of the effects
of different concentrations of each component was beyond
the scope of this study. However, future efforts on the
improvement of nuclei isolation buffers should consider
this aspect.
Both buffers described in this work provided good results
in many of the 37 species. However, while good samples of
isolated nuclei could be prepared from any species using
WPB, GPB failed in most woody plants. On the other
hand, in unproblematic species GPB resulted in samples
of similar or higher quality than those obtained with WPB.
Woody plants are considered recalcitrant for DNA flow
cytometry as their tissues often contain cytosolic com-
pounds that interfere with fluorescent staining of nuclear
DNA (Noirot et al., 2000, 2005; Loureiro et al., 2006b).
This was the case in most of the species where GPB
failed and where the tannic acid effect was observed. The
addition of sodium metabisulfite and PVP-10 to WPB
seemed essential for its success in species where GPB
failed and for the overall good performance of WPB.
Sodium metabisulfite, PVP, and other compounds with
similar properties (e.g. b-mercaptoethanol, ascorbic acid)
had been used previously to counteract the negative effect
of cytosolic compounds on nuclear fluorescence in oak
(Zoldos
ˇ
et al., 1998), rose (Yokoya et al., 2000) and olive
(Loureiro et al., 2007b). Antioxidants keep phenolics in a
reduced state, enabling the reversibility of the free hydrogen
bonds and its resolution by an added competitor (usually
PVP-10 or PVP-40) (Greilhuber et al., 2007).
Generally, GPB and WPB yielded better results than the
four popular buffers evaluated by Loureiro et al. (2006a).
This was evident for the CV of DNA peaks, as in most
species an improvement in peak resolution was achieved.
Improved nuclear fluorescence and less debris background
were also observed with the new buffers. Unexpectedly,
in Celtis australis measurable samples were only obtained
with WPB. Although GPB has the same concentration of
Triton X-100 as the Tris.MgCl
2
buffer (the best buffer for
this species in Loureiro et al., 2006a), it failed to surpass
the negative effect of mucilaginous compounds.
Interestingly, both GPB and WPB seem to exhibit good buf-
fering capacity, as they were suitable for isolation of nuclei
from leaf tissues of Oxalis pes-caprae with highly acidic
cell sap (Loureiro et al., 2006a; Castro et al., 2007). The
only apparent drawback of GPB and WPB was that for
some species (especially in the unproblematic ones) rather
low YF was observed. This was surprising as the concen-
tration of Triton X-100 in both buffers was increased as
compared with LB01 and Galbraith buffers. However, this
drawback can be compensated by using a higher amount
of sample tissue.
Despite their commonness and/or economical interest,
until now DNA content has not been analysed by flow cyto-
metry in 15 out of the 37 species used in this study.
Moreover, in Chamaecyparis lawsoniana (Hizume et al.,
2001), Ginkgo biloba (Marie and Brown, 1993; Barow
and Meister, 2002), Laurus nobilis (Zonneveld et al.,
2005) and Prunus domestica (Arumuganathan and Earle,
1991b), the published reports do not include DNA
content histograms and data on CV, making any compari-
son of buffer performance impossible. For the remaining
species only indirect comparisons can be made as the
experimental conditions in each work are unlike the ones
followed here. However, judging from published CVs and
DNA content histograms, with the exception of Pinus
pinea, the buffers described in the present work provided
better (e.g. Quercus robur, Malus domestica, Diospyros
kaki) or similar (e.g. Olea europaea, Vitis vinifera)
results. Particularly interesting are the high-resolution histo-
grams obtained in Quercus robur using WPB. Leaves of
this and other species from this genus contain phenolic
compounds that interfere with fluorescent staining of
nuclear DNA (Zoldos
ˇ
et al., 1998; Loureiro et al., 2005).
In order to estimate genome size in seven Quercus
species, including Quercus robur, Zoldos
ˇ
et al. (1998)
modified Galbraith’s buffer by adding metabisulfite. In
their study, CVs ranged from 4
.
2 % to 6
.
9 % for Quercus
robur, while in our work mean CVs below 3 % and low
DF values (,20 %) were achieved. In Pinus pinea, GPB
and WPB resulted in CVs around 3 %, i.e. higher than
those obtained by Grotkopp et al. (2004) who used a
Loureiro et al. Two New Buffers for Plant Flow Cytometry 885
modified Galbraith buffer to obtain CVs typically below
2 %. It should be noted, however, that we used fine
needles to prepare nuclear suspensions, while Grotkopp
et al. (2004) used a megagametophyte, from which it is
easier to prepare nuclear suspensions.
In addition to the comparison of two new nuclear isolation
buffers, this work provides data on nuclear DNA content in
30 plant species. It was noted that samples prepared from
species with small genome sizes (,1
.
0 pg/2C DNA) exhib-
ited higher CVs. Even in unproblematic species, a negative
relationship between genome size and DF was observed
(e.g. Sedum burrito and Euphorbia peplus). This was
clearly due to the presence of particles other than intact
nuclei in the samples (Galbraith et al., 2002). These
include autofluorescent chlorophyll, nuclei fragments and
non-specifically stained cellular debris, which contribute to
the background distribution over which nuclear DNA
content distribution is superimposed. Debris attached to iso-
lated nuclei then increases the variation in nuclei fluor-
escence intensity (Loureiro et al., 2006b).
For the 20 species whose genome size had been esti-
mated before, better agreement was observed for previous
results that were obtained by flow cytometry as compared
with those obtained by Feulgen microdensitometry. This
was the case of Coriandum sativum, where our estimate
of 5
.
08 pg DNA (2C) differs from earlier estimates
using the Feulgen technique that ranged from 7
.
65 pg to
9
.
55 pg (Das and Mallick, 1989; Chattopadhyay and
Sharma, 1990). Our estimates of C-values are also lower
than Feulgen-based estimates for Magnolia soulangeana
and Chamaecyparis lawsoniana (Nagl et al., 1977;
Olszewska and Osiecka 1983; Ohri and Khoshoo 1986).
However, our estimate for the latter species is similar to
that of Hizume et al. (2001) who used flow cytometry.
Another noteworthy difference concerns Ficus carica
(Moraceae), in which our estimate of 2C value is only
half of that determined by Feulgen microspectrophotome-
try (Ohri and Khoshoo, 1987). On the other hand, we
determined 2C ¼ 11
.
00 pg DNA for Papaver rhoeas
(Papaveraceae), which is double that obtained by Nagl
et al. (1983), Bennett and Smith (1976) and Srivastava
and Lavania (1991) using the Feulgen procedure. In this
species the differences in genome size may be explained
by the occurrence of minority cytotypes (Albers and
Pro
¨
bsting, 1998), with our individuals being probably
tetraploid.
The differences between flow cytometry and Feulgen
densitometry are rather unexpected as Dolez
ˇ
el et al.
(1998) showed a close agreement between both methods.
However, as noted by these authors, there are many critical
points of the Feulgen procedure (e.g. fixation, slide prep-
aration and storage, acid hydrolysis) which determine its
precision. Moreover, stoichiometry of the Feulgen pro-
cedure can be negatively affected by various components
of cytosol (Greilhuber, 1988). Some differences between
flow cytometry estimates of genome sizes in different lab-
oratories may be explained by the use of different reference
standards, sample preparation and staining protocols, and
flow cytometers (Dolez
ˇ
el et al., 1998; Dolez
ˇ
el and
Bartos
ˇ
, 2005).
This work reports the first estimates of genome size in ten
plant species. Most of the families to which these species
belong are poorly represented at the genus or species level
in the plant DNA C-values database (Bennett and Leitch,
2005). The estimates for Acer negundo (Aceraceae, 0
.
75
4
.
05 pg/2C), Aloysia triphylla (Verbenaceae, 0
.
955
.
51 pg/
2C), Forsythia intermedia (Oleaceae, 1
.
95 4
.
66 pg/2C),
Pterospartum tridentatum (Fabaceae, 1
.
0326
.
50 pg/2C)
and Saintpaulia ionantha (Gesneriaceae, 1
.
35 2
.
80 pg/2C)
are at the lower limit of the known range of genome size
for each family. Contrarily, our 2C-value for Salix babylo-
nica is near the upper limit of the known range of
2C-values in Salix sp. (0
.
700
.
96 pg/2C for diploids and
1
.
62 1
.
72 pg/2C for tetraploids). Our estimates for Ilex aqui-
folium and Euphorbia peplus are the lowest so far in
Aquifoliaceae (2
.
25 4
.
25 pg/2C) and in the Euphorbia
genus (1
.
30 28
.
70 pg/2C), respectively. By contrast, our
genome size estimation for Diospyros kaki is the highest
among the three species of Diospyros already analysed
(2
.
40 3
.
30 pg/2C). Finally, our 2C-value for Tamarix afri-
cana is close to that of Zonneveld et al. (2005) for Tamarix
tetrandra (3
.
10 pg/2C), which was until now the only
species analysed in Tamaricaceae.
In conclusion, the present results show that in species
relatively free of cytosolic compounds, GPB provides
similar and, in some cases, better results than WPB, and
may be preferred. With problematic tissues, GPB usually
performs less well than WPB, which is more suitable for
the recalcitrant samples characterized, among other, by
the presence of phenolics and mucilaginous compounds.
When compared with other nuclear isolation buffers, the
use of WPB results in improved histogram quality.
Therefore it is recommended as the first choice when pro-
blematic tissues/species are to be analysed for DNA
content using flow cytometry.
ACKNOWLEDGEMENTS
Thanks are due to Prof. Paulo Silveira, Dr Sı
´
lvia Castro and
Eng. Armando Costa for providing some of the plant
material used in this study and to three anonymous
reviewers for their useful comments and suggestions. This
work was supported by FCT project ref. POCTI/AGR/
60672/2004. J.L. was supported by Fellowship FCT/BD/
9003/2002 and E.R. by Fellowship FCT/BD/27467/2006.
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... We ground approximately five not fully developed fresh leaves from each sample to pieces using liquid nitrogen in a centrifuge tube. We added 1 mL of nuclei extraction woody plant buffer (Loureiro et al. 2007) to the centrifuge tube, and incubated the sample for 5 min. We filtered the mixture through a 300-mesh sieve, which we then centrifuged at 10,000 rpm and 4 °C for 10 min. ...
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The hazelnut industry requires further development given the gap between production and demand. However, polyploid plantlet induction has not been reported in hazelnut varieties. As such, our aim in this study was to enrich the hazelnut germplasm resources. We used the shoot tips of in vitro propagated plantlets of Corylus heterophylla × Corylus avellana '84–226' as the material for inducing polyploidy. We supplemented the culture medium with 0, 100, 200, and 300 mg/L of colchicine, and we treated shoot tips for 20, 25, or 30 days. We found that the induction rate of tetraploids was the highest in treatments with 300 mg/L for 25 days; it was substantially higher by up to 20% than in the other treatments. In diploid and tetraploid tissue-cultured seedlings, tetraploid plantlets were much shorter, thicker, and stronger, as well as greener, than the diploid plantlets. The acquisition of polyploid hazelnuts provides an important prerequisite for hazelnut breeding.
... In another study, Sadhu et al. [22] used 1% of PVP in different extraction buffers, which yielded good-quality nuclei from plants of different genera of the same family from both root and shoot tissues. In earlier reports, it has been reported that PVP reduced the effect of polyphenols by changing their conformational structure, maintaining the cell compounds in a reduced state and making hydrogen bonds [63][64][65]. Thus, our result revealed that the two extraction buffers viz., GB and MB01, could be used for the extraction of nuclei for estimation of genome size as no variation in genome size within Acacia species has been observed as compared to other extraction buffers used in this study. ...
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Acacias are widely distributed in tropical and subtropical regions of the world and have both economic as well as medicinal value. The estimation of genome size is very important as it changes due to the change in noncoding DNA sequence as well as genome duplication among organisms for their evolutionary aspects. Three potential species of the genus Acacia including Acacia etbaica, Acacia johnwoodii and Acacia origena, which are threatened and nearly endemic to Saudi Arabia, were collected. The present study was carried out to determine the genomes’ size (2C DNA contents), total phenolic content (TPC), total flavonoid (TFC) and some bioactive compounds in these species for their comparison. The genome size ranged from 1.90 pg (A. etbaica) to 2.45 pg/2C (A. origena) among the Acacia species, which correspond to genome sizes 1858.2–2396.1 Mbp, respectively. The variation was observed in genome size within Acacia species as nuclei were extracted using different extraction buffers except for GB and MB01 buffers. The FTIR analysis revealed the presence of various functional groups in compounds that might be responsible for different types of phytochemicals in these Acacia species. Total flavonoid content (TFC) ranged from 0.647 (A. origena) to 1.084 mg QE /g DW (A. johnwoodii), whereas the total phenolic f content (TPC) ranged between 15.322 (A. origena) to 28.849 (A. johnwoodii) mg/g DW of GAE. HPLC analysis revealed the presence of quercetin 3-β-glucoside and luteolin 7-rutinoside in the leaves of all three Acacia species in considerable amounts, and these might have good health-promoting effects. This is our first study on genome size (2C DNA content) using flow cytometry and phytochemical profiling on these Acacias. Thus, estimated genome size and phytochemical study of these species could help to understand the biosynthesis of secondary metabolites under various genes and the evolutionary relationships among them.
... Mb and 124 L50 count of n=5 scaffolds (Supplementary Table 1). After selecting the chromosomes only, our final 125 chromosomal assembly consists of 8 chromosomes of >20Mb each, totaling 267.2 Mb, in agreement with 126 previous chromosome squashes and previous flow cytometry analyses (Fasihi et al.;Loureiro et al. 2007) 127 (Figure 2A). Benchmarking of universal, single-copy orthologs (BUSCO) analysis of those 128 chromosomes indicated a mostly complete assembly with 98.5% of Embryophyta BUSCO orthologs 129 identified, most of which (95.5%) were single-copy (Supplementary Table 2 We masked repeats in the genome using RepeatModeler and RepeatMasker, which led to masking of 144 57.66% of the nucleotides in all scaffolds and 48.55% of the nucleotides in the assembled chromosomes. ...
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Euphorbia peplus (petty spurge) is a small, fast-growing plant that is native to Eurasia and has become a naturalized weed in North America and Australia. E. peplus is not only medicinally valuable, serving as a source for the skin cancer drug ingenol mebutate, but also has great potential as a model for latex production owing to its small size, ease of manipulation in the laboratory, and rapid reproductive cycle. To help establish E. peplus as a new model, we generated a 267.2 Mb HiC-anchored PacBio HiFi nuclear genome assembly with an embryophyta BUSCO score of 98.5%, a genome annotation based on RNA-seq data from six tissues, and publicly accessible tools including a genome browser and an interactive organ-specific expression atlas. Chromosome number is highly variable across Euphorbia species. Using a comparative analysis of our newly sequenced E. peplus genome with other Euphorbiaceae genomes, we show that variation in Euphorbia chromosome number is likely due to fragmentation and rearrangement rather than aneuploidy. Moreover, we found that the E. peplus genome is relatively compact compared to related members of the genus in part due to restricted expansion of the Ty3 transposon family. Finally, we identify a large gene cluster that contains many previously identified enzymes in the putative ingenol mebutate biosynthesis pathway, along with additional gene candidates for this biosynthetic pathway. The genomic resources we have created for E. peplus will help advance research on latex production and ingenol mebutate biosynthesis in the commercially important Euphorbiaceae family. Significance statement Euphorbia is one of the five largest genera in the plant kingdom. Despite an impressive phenotypic and metabolic diversity in this genus, only one Euphorbia genome has been sequenced so far, restricting insights into Euphorbia biology. Euphorbia peplus has excellent potential as a model species due to its latex production, fast growth rate and production of the anticancer drug ingenol mebutate. Here, we present a chromosome-level E. peplus genome assembly and publicly accessible resources to support molecular research for this unique species and the broader genus. We also provide an explanation of one reason the genome is so small, and identify more candidate genes for the anticancer drug and related compounds.
... The relative nuclear DNA content was determined by using a CytoFLEX flow cytometer (Beckman Coulter, Suzhou, China) equipped with a 488 nm argon laser using the protocol proposed by Zhang et al. [9]. After main veins were removed, about 50 mg of fresh leaf tissue was rapidly chopped using a sharp razor blade in 350 µL of cold woody plant buffer (WPB), as described by Loureiro et al. [28] for releasing nuclei. The resulting crude solution was filtered through a 40 µm nylon mesh into a 5 mL sample loading tube to eliminate cell debris, and then 300 µL of fluorochrome stain (50 µg mL −1 ) composed of propidium iodide (PI) and RNase A were added to the tube. ...
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Aneuploids are valuable materials of genetic diversity for genetic analysis and improvement in diverse plant species, which can be propagated mainly via in vitro culture methods. However, somaclonal variation is common in tissue culture-derived plants including euploid caladium. In the present study, the genetic stability of in vitro-propagated plants from the leaf cultures of two types of caladium (Caladium × hortulanum Birdsey) aneuploids obtained previously was analyzed morphologically, cytologically, and molecularly. Out of the randomly selected 23 and 8 plants regenerated from the diploid aneuploid SVT9 (2n = 2x − 2 = 28) and the tetraploid aneuploid SVT14 (2n = 4x − 6 = 54), respectively, 5 plants from the SVT9 and 3 plants from the SVT14 exhibited morphological differences from their corresponding parent. Stomatal analysis indicated that both the SVT9-derived variants and the SVT14-originated plants showed significant differences in stomatal guard cell length and width. In addition, the variants from the SVT14 were observed to have rounder and thicker leaves with larger stomatal guard cells and significantly reduced stomatal density compared with the regenerants of the SVT9. Amongst the established plants from the SVT9, two morphological variants containing 3.14–3.58% less mean fluorescence intensity (MFI) lost one chromosome, and four variants containing 4.55–11.02% more MFI gained one or two chromosomes. As for the plants regenerated from the SVT14, one variant with significantly higher MFI gained two chromosomes and three plants having significantly lower MFI resulted in losing four chromosomes. Three, out of the twelve, simple sequence repeat (SSR) markers identified DNA band profile changes in four variants from the SVT9, whereas no polymorphism was detected among the SVT14 and its regenerants. These results indicated that a relatively high frequency of somaclonal variation occurred in the in vitro-propagated plants from caladium aneuploids, especially for the tetraploid aneuploid caladium. Newly produced aneuploid plants are highly valuable germplasm for future genetic improvement and research in caladium.
... The best results were obtained using the first or second pair of leaves of young Cannabis plants. Additionally, different flow cytometry buffers (LB01 [105], Ebihara [106], Cystain Ox Protect and PI Absolute buffers (Sysmex-Partec GmbH)) were tested as well, before choosing the general purpose buffer GPB [107] supplemented with 3% PVP-40 [108] as the most appropriate one. Additional measures, such as reducing chopping intensity and working in ice-cold conditions, were taken to reduce the potential effects of secondary metabolites. ...
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Cannabis sativa has been used for millennia in traditional medicine for ritual purposes and for the production of food and fibres, thus, providing important and versatile services to humans. The species, which currently has a worldwide distribution, strikes out for displaying a huge morphological and chemical diversity. Differences in Cannabis genome size have also been found, suggesting it could be a useful character to differentiate between accessions. We used flow cytometry to investigate the extent of genome size diversity across 483 individuals belonging to 84 accessions, with a wide range of wild/feral, landrace, and cultivated accessions. We also carried out sex determination using the MADC2 marker and investigated the potential of flow cytometry as a method for early sex determination. All individuals were diploid, with genome sizes ranging from 1.810 up to 2.152 pg/2C (1.189-fold variation), apart from a triploid, with 2.884 pg/2C. Our results suggest that the geographical expansion of Cannabis and its domestication had little impact on its overall genome size. We found significant differences between the genome size of male and female individuals. Unfortunately, differences were, however, too small to be discriminated using flow cytometry through the direct processing of combined male and female individuals.
... To accurately determine the genome size of C. pubescens, we followed the one-step flow cytometry procedure [32], with modifications as described in Pellicer et al. [33]. Freshly collected tissue from the same individual sampled for DNA and RNA sequencing was measured together with Oryza sativa L. 'IR-36' as the calibration standard (0.49 Gbp/1C [33 ]) using General Purpose Buffer [34] supplemented with 3% PVP-40 and -mercaptoethanol [35]. The samples were analysed on a Partec Cyflow SL3 flow cytometer (Partec GmbH, Münster, Germany) fitted with a 100 mW green solid-state laser (532 nm, Cobolt Samba, Solna, Sweden). ...
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The Andean fever tree (Cinchona L.; Rubiaceae) is a source of bioactive quinine alkaloids used to treat malaria. C. pubescens Vahl is a valuable cash crop within its native range in northwestern South America, however, genomic resources are lacking. Here we provide the first highly contiguous and annotated nuclear and plastid genome assemblies using Oxford Nanopore PromethION-derived long-read and Illumina short-read data. Our nuclear genome assembly comprises 603 scaffolds with a total length of 904 Mbp (∼82% of the full genome based on a genome size of 1.1 Gbp/1C). Using a combination of de novo and reference-based transcriptome assemblies we annotated 72,305 coding sequences comprising 83% of the BUSCO gene set and 4.6% fragmented sequences. Using additional plastid and nuclear datasets we place C. pubescens in the Gentianales order. This first genomic resource for C. pubescens opens new research avenues, including the analysis of alkaloid biosynthesis in the fever tree.
... The young leaves were taken from seedlings for flow cytometric analysis. The leaf fragments of the sample plant and the standard plant (Zea mays L., 2C= 5.48 pg) were chopped using a razor blade in 1 mL of woody plant buffer (0.2 M Tris HCl, 4 mM MgCl 2 ·6H 2 O, 2 mM EDTA, Na 2 ·2H 2 O, 86 mM NaCl, 10 mM K 2 S 2 O 5 , 1% PVP-10, 1% (v/v) Triton X-100, pH 7.5) (Loureiro et al. 2007) supplemented with 50 µg mL 1 propidium iodide and 50 µg mL 1 DNAse-free RNase, filtered through a 30 µm mesh and stored on ice, in dark, until measurement. Three independent samples were extracted, filtered, and measured on the same day. ...
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Irano-Turanian floristic region in Turkey appears to be the center of origin of Crepis foetida subsp. rhoeadiflolia (Asteraceae). In this study, karyotype and flow cytometric analyses of six Irano-Turanian populations of C. foetida subsp. rhoeadiflolia in Turkey were performed. The cytogenetic characteristics of the populations using multivariate analyses (cluster analysis, principal components analysis) were evaluated in a cytogeographic context. Two main groups are found in C. foetida subsp. rhoeadiflolia based on cytogenetic characteristics among populations based on distribution pattern. The significant differences among populations mainly concern the long length of chromosome, centromeric index, and genome size. The ecological and evolutionary value of cytogenetic data are discussed within the framework of the results obtained.
Chapter
Mangosteen is one of the most popular tropical fruits in Southeast Asia. It is called ‘The Queen of Tropical Fruits’ as its thick sepals collectively resemble a crown. Mangosteen fruits contain white and juicy edible pulp with a sweet flavour and pleasant aroma. They are rich in beneficial phytochemicals such as xanthones, which make mangosteen a potential medicinal plant. Traditionally, mangosteen has been used to treat fever, diarrhoea, and wounds. In recent studies, researchers found that mangosteen has anti-cancer and anti-diabetic properties. However, mangosteen is still an underutilised crop due to its slow growth rate with a long juvenile period that usually takes eight to ten years to bear fruit. It is also an obligative apomict with asexual reproduction, hence producing clones of progenies with low genetic variations. Therefore, the breeding programme of mangosteen is challenging with a very low success rate. Furthermore, genetic information on mangosteen accessions in different countries is limited to unravel its lineage and parental history. Other constraints in mangosteen improvement include low viability of recalcitrant seeds and the lack of a rapid propagation method. Efforts have been made to understand this crop through functional genomic studies. Recent genomic studies of mangosteen, including genome sequencing, genome survey, genome size estimation, and cytogenetic analysis, are highlighted in this chapter.KeywordsCytogeneticsGarciniaGenome sequencingGenome sizeMangosteen
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A rapid and efficient in vitro micropropagation system was developed to conserve Tripleurospermum fissurale (Sosn.) E.Hossain (Asteraceae), a rare endemic species for Turkey. Murashige and Skoog basal medium (MS) supplemented with 4.7 µM KIN was found to be the most appropriate basal medium with a 60% germination rate. MS medium supplemented with 4.9 µM 2iP and 0.5 µM IBA gave the highest shoot length of 54.3 ± 3.53 mm. Furthermore, 4.4 µM 6-BA combined with 0.5 µM IBA was superior for the highest shoot number with 3.4 ± 0.49 after 4-wk culture. Cytogenetic analyses indicated that all propagated plants have the same DNA ploidy level (x) and chromosome number (2n = 18) compared with the mother plants. After 4 wk, the rooting percentage achieved 100% in all tested rooting media. MS medium supplemented with 2.7 µM NAA favored the highest root number, root length, and secondary root number with 3.44 ± 0.5, 170.1 ± 4.91 mm, and 26.1 ± 1.65, respectively. Rooted plantlets were initially acclimatized and then transferred in greenhouse conditions. This method could be evaluated for ex situ conservation of rare endemic and endangered plant species.
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Pimpinella species are annual, biennial, and perennial semibushy aromatic plants cultivated for folk medicine, pharmaceuticals, food, and spices. The karyology and genome size of 17 populations of 16 different Pimpinella species collected from different locations in Iran were analyzed for inter-specific karyotypic and genome size variations. For karyological studies, root tips were squashed and painted with a DAPI solution (1 mg/ml). For flow cytometric measurements, fresh leaves of the standard reference (Solanum lycopersicum cv. Stupick, 2C DNA = 1.96 pg) and the Pimpinella samples were stained with propidium iodide. We identified two ploidy levels: diploid (2x) and tetraploid (4x), as well as five metaphase chromosomal counts of 18, 20, 22, 24, and 40. 2n = 24 is reported for the first time in the Pimpinella genus, and the presence of a B-chromosome is reported for one species. The nuclear DNA content ranged from 2C = 2.48 to 2C = 5.50 pg, along with a wide range of genome sizes between 1212.72 and 2689.50 Mbp. The average monoploid genome size and the average value of 2C DNA/chromosome were not proportional to ploidy. There were considerable positive correlations between 2C DNA and total chromatin length and total chromosomal volume. The present study results enable us to classify the genus Pimpinella with a high degree of morphological variation in Iran. In addition, cytological studies demonstrate karyotypic differences between P. anthriscoides and other species of Pimpinella, which may be utilized as a novel identification key to affiliate into a distinct, new genus – Pseudopimpinella.
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Laser flow cytometry was used to analyze nuclear DNA contents (2C values) of five genera (Severinia Ten., Atalantia Correa, Fortunella Swing., Poncirus Raf., and Citrus L.) taxonomically grouped in subtribe Citrinae (citrus fruit trees) of the Rutaceae. The genotypes analyzed had 2C values ranging from 0.67 pg for diploid Severinia buxifolia (Poir.) Ten. to 1.27 pg for tetraploid Hongkong Fortunella hindsii Swing. There was no significant difference in the 2C values within the sexually compatible diploid species of 11 'true citrus fruit trees' [Citrus aurantium L., C. grandis (L.) Osbeck, C. limon (L.) Burm. f., C. limonia Osbeck, C. paradisi Macf., C. reshni Hort. ex Tanaka, C. sinensis (L.) Osbeck, C. volkameriana Ten. and Pasq., Poncirus trifoliata (L.) Raf., and the intergeneric hybrid C. sinensis x P. trifoliata]. The species Atalantia ceylanica (Arn.) Oliv. (a 'near-citrus fruit tree'), sexually incompatible with Citrus spp., had a 2C value significantly different from those of the true citrus fruit tree species. The 2C value of Severinia buxifolia (a 'primitive citrus fruit tree'), another species sexually incompatible with the Citrus spp., also differed from those of some of the true citrus fruit tree species. The data largely corresponds with taxonomical differences between a) the genera Citrus and Poncirus and b) the genera Severinia and Atalantia, all assigned to subtribe Citrinae.
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The genome sizes of 13 species of Taxodiaceae, 19 species of Cupressaceae s.s. and Sciadopitys verticillata were determined by flow cytometry of isolated nuclei stained with propidium iodide, using Hordeum vulgare nuclei as an internal standard. In Taxodiaceae, the genomes of Cunninghamia lanceolata (28.34pg/2C) and Taiwania species (25.78, 26.80 pg/2C) were larger than those of other genera/species, which ranged from 19.85 to 22.87 pg/2C. In Cupressaceae s.s., genome size ranged from 20.03 to 27.93 pg/2C among 16 species. The Calocedrus species and Thujopsis had a larger genome than most other species. Sciadopitys verticillata had a large genome of 41.60 pg/2C. After comparing the diversity in genome size with previously reported cladograms constructed using nucleotide sequence data, the tendency of changes in genome size with phylogenetic differentiation is discussed.
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In 23 species of Papaver L., 2C nuclear DNA amounts range from 4.64 pg in Papaver persicum (2n = 14) to 22.43 pg in Papaver orientale (2n = 42), revealing a fivefold variation within the genus. However, such variation is limited to only twofold among the species that have the same chromosome number (2n = 14). The distribution of DNA is discontinuously spread over six groups in the genus. A strong positive correlation exists between nuclear DNA content and metaphase chromosome length. Viewed in the context of evolutionary divergence, it is revealed that DNA reduction has taken place in conjunction with speciation. This is achieved by equal reduction to each chromosome independent of chromosome size, as apparent from the estimated DNA values for individual chromosomes within the complements. The diminution in DNA amount with evolutionary specialisation appears to be a genomic strategy to dispense with the less important DNA associated with heterochromatic segments. The uniform distribution of such dispensible DNA throughout the complement is probably nucleotypically conducive to allow the genomic loss to be adaptationally operative, lest it affects the very survival of the evolving species.Key words: Papaver, evolution, DNA content, DNA systematics.
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The amount and spatial organization of the heterochromatin in nuclei of the shoot meristem and the frequency in the nuclear DNA of sequences belonging to a family of tandem repeats were investigated in cultivars of Olea europaea and related species. Significant differences between Olea species and between cultivars of O. europaea were observed: (i) in the spatial organization of the heterochromatin in interphase nuclei as determined by the number and surface area of the chromocentres; (ii) in genome size; and (iii) in the amoun