Annals of Botany 86: 929±934, 2000
doi:10.1006/anbo.2000.1255, available online at http://www.idealibrary.com on
Sun¯ower (Helianthus annuus) Leaves Contain Compounds that Reduce Nuclear
Propidium Iodide Fluorescence
H . J A M E S P R I C E , G E O R G E H O D N E T T and J . S P E N C E R J O H N S TO N
Departments of Soil & Crop Sciences and of Entomology, Texas A&M University, College Station, Texas 77843
Received: 25 April 2000 Returned for revision: 6 June 2000 Accepted: 7 July 2000
Published electronically: 30 August 2000
Sun¯ower leaves have unidenti®ed compounds that interfere with propidium iodide (PI) intercalation and/or
¯uorescence. Independently prepared pea leaf nuclei show greater PI ¯uorescence than nuclei from pea leaves
simultaneously processed (co-chopped) with sun¯ower leaves. Di€erences in ¯uorescence persist after mixing the
PI-stained pea and the co-chopped pea/sun¯ower samples, i.e. PI staining protects the nuclei from the e€ects of the
inhibitor. The current results are signi®cant to practical ¯ow cytometric determination of plant nuclear DNA content.
They show: (1) simultaneous processing of nuclear samples from the target and the standard species is necessary to
obtain reliable DNA estimates; (2) a test for the presence of inhibitors should be conducted; and (3) when inhibitors
are present caution should be taken in interpreting di€erences in estimated DNA content. The previously reported
environmentally-induced variation in DNA content in sun¯ower populations is most simply explained by variation in
the amount of environmentally-induced inhibitor that interferes with intercalation and/or ¯uorescence of PI.
Intraspeci®c variation of DNA content for Helianthus annuus needs to be re-evaluated using best practice techniques
comparing physiologically uniform tissues that are free of inhibitors. The best estimate for 2C DNA content of
# 2000 Annals of Botany Company
H. annuus used in this study is 7.3 pg.
Key words: Helianthus annuus, DNA content, ¯ow cytometry, propidium iodide, endogenous inhibitors.
For several decades there has been considerable interest in
the roles of varying DNA content in plant adaptation and
evolution (Bennett and Smith, 1976; Price, 1976, 1988;
Bennett, 1998). Genome size varies by nearly an order of
magnitude among diploid congeneric species. DNA content
positively correlates with physical parameters such as
chromosome size, nuclear volume, and mitotic cycle time
(Price, 1976; Bennett, 1998).
There have been numerous reports of intraspeci®c
variation in genome size in plants (Bennett, 1985; Price,
1988). One notable example is Zea mays, where the observed
37 % variation in DNA content has been correlated with the
number and size of heterochromatic knobs (Laurie and
Bennett, 1985; Rayburn et al., 1985). Another well established example is ¯ax, Linum usitatissimum (Durrant, 1962;
Evans, 1968), where increases and decreases in DNA
content over an approximate 15 % range can be environmentally induced by varying amounts of phosphorous and
nitrogen in the fertilizer. In ¯ax, the variation in DNA
content results from di€erential ampli®cation of repetitive
sequences (Cullis, 1985; Cullis and Cleary, 1986).
Most reports of intraspeci®c DNA content variation have
not been independently con®rmed. This has led to questions concerning the frequency and magnitude of intraspeci®c DNA content changes (Greilhuber, 1998). Since
there are both technical and biological sources of variation
that can lead to quantitative di€erences in binding and/or
¯uorescence of ¯uorochromes and in Feulgen staining
(Johnston et al., 1996; Greilhuber, 1998) artifactual
di€erences in DNA content may be reported and real
di€erences may go undetected.
0305-7364/00/110929+06 $35.00/00
Sun¯ower, Helianthus annuus, is an example of a species
in which intraspeci®c variation in DNA content determined
by Feulgen microspectrophotometry has been reported by
several di€erent laboratories. Nagl and Capesius (1976),
Olszewska and Osiecka (1983) and Cavillini et al. (1986,
1989) reported up to 60 % variation in DNA content
among H. annuus varieties. Cavillini et al. (1986, 1989) also
reported that the nuclear DNA content was higher in
sun¯ower seedlings grown from achenes from the periphery
of the in¯orescence and progressively decreased in achenes
positioned towards the centre of the head.
Well documented limitations to the acquisition of DNA
contents by Feulgen microspectrophotometry include
problems with standardization, chromatin compaction,
light absorbancy (Price et al., 1980; Price and Johnston,
1996a), and inhibitory e€ects of compounds such as
endogenous tannins (Grelhuber, 1986, 1988). In an attempt
to avoid the limitations of the Feulgen technique, Michaelson et al. (1991b) initiated a study of DNA content
variation in H. annuus using laser ¯ow cytometry of
propidium iodide (PI) stained nuclei. Flow cytometric
estimates of DNA content variation among leaves and
among plants of the same sun¯ower variety resulted in
extensive variation from approx. 3±8.5 pg (Michaelson
et al., 1991b; Johnston et al., 1996; Price and Johnston,
1996a). This range in DNA content is similar to that
reported among sun¯ower plants from other laboratories
(Arumuganathan and Earle, 1991). Although the cytometric technique of Michaelson et al. (1991b), Johnston
et al. (1996) and Price and Johnston (1996b) utilized a
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930
Price et al.ÐInhibitors of PI Fluorescence in Sun¯ower Leaves
barley or pea standard, it was independently processed and
stained before being added to the sun¯ower samples.
Johnston et al. (1996) and Price and Johnston (1996b)
reported that PI-based ¯ow cytometrically detected DNA
content variation among sun¯ower plants in growth
chambers and a greenhouse correlated with the quality
and quantity of light under which the plants were grown.
These DNA content di€erences were also apparent from
microspectrophotometric measurements and image analysis
of Feulgen-stained nuclei (Johnston et al., 1996). At that
time it appeared that the DNA content of the sun¯ower
changed during development in response to light quality
and quantity. This hypothesis was further supported by the
measurement of nuclear DNA content of sun¯ower plants
of a natural population where the plants received various
proportions of direct sunlight and irradiance re¯ected from
neighbouring vegetation (Price et al., 1998). However,
factors independent of DNA content could lead to di€erences in the ¯uorescence of PI-stained nuclei. Both
Greilhuber (1998) and Price et al. (1998) suggested that
the observed di€erences in PI ¯uorescence in sun¯ower may
be related to secondary plant products that interfere with
the intercalation and/or ¯uorescence of propidium iodide.
Here we report technical and physiological factors that
quantitatively in¯uence the outcome of ¯ow cytometry
using PI-stained sun¯ower nuclei. It is concluded that the
reported light-induced di€erences in mean PI ¯uorescence
among sun¯ower plants are due to varying levels of
environmentally-induced inhibitor(s) of PI intercalation
and/or ¯uorescence, and do not represent di€erences in
DNA content.
M AT E R I A L S A N D M E T H O D S
Plant materials
The H. annuus plants used in this study were from a wild
population located on the west side of Building 955 on the
Texas A&M campus, College Station, Texas. Pisum sativum
`Minerva Maple' leaves were from plants grown in a growth
chamber adjusted to a 16/8 h, 28/208C day/night cycle.
Laser ¯ow cytometry
Samples of nuclei for ¯ow cytometry were obtained from
leaves. Each sample was chopped with a new razor blade in
a modi®ed Galbraith et al. (1983) bu€er as described by
Price et al. (1998). The bu€er consisted of, per litre, 4.26 g
MgCl2 , 8.84 g sodium citrate, 4.2 g 3-[morpholino]propane
sulfonic acid, 1 ml Triton X-100, 1 mg boiled ribonuclease
A, pH 7.2. The resulting slurry was ®ltered through a 53 mm
nylon ®lter, and propidium iodide (PI) was added to a ®nal
concentration of 50 ppm. The stained samples were stored
in the dark on ice and analysed by ¯ow cytometry after 1 h.
The ¯ow cytometer was a Coulter Epics Elite (Coulter
Electronics, Hialeah FL) equipped with a water-cooled
laser tuned at 514 nm and 500 mW. Fluorescence emission
at 4615 nm was detected with a photomultiplier screened
by a long pass ®lter. O€set and linearity of the ¯ow
cytometer were checked with ¯uorescent check beads
(Coulter Electronics, Hialeah, FL) as recommended by
the manufacturer.
Test for inhibitors
Sun¯ower extracts were tested for unidenti®ed compounds that reduce PI ¯uorescence of pea nuclei as follows.
Nuclei were released from an approximate 40 mm 20 mm
sun¯ower leaf and one-half of a pea leaf that were simultaneously processed (co-chopped) and stained with PI
(sample A). Sample B consisted of PI-stained nuclei from
the independently processed and stained other half of the
pea leaf used in sample A. After staining for 1 h, samples A
and B were individually measured for mean PI ¯uorescence,
after which they were mixed and measured up to 17 min, at
60 min, and at 120 min. The experiment was replicated
three times. Reduced ¯uorescence of nuclei from pea leaves
simultaneously processed with sun¯ower leaves, compared
to nuclei from independently processed pea leaves gave
evidence of sun¯ower inhibitors. A second similar experiment was conducted to test the stability of PI ¯uorescence
of prestained pea nuclei after being added to a stained
nuclear sample from simultaneously processed sun¯ower
and pea leaves. Five replicates were measured for PI
¯uorescence during the ®rst 15 min after mixing and
again after 20 h.
Test for concentration e€ects of inhibitors
The e€ects of the concentration of sun¯ower extract on
PI ¯uorescence of pea nuclei were tested. Approximately
13 mm square segments were cut from sun¯ower and pea
leaves and processed separately in 3 ml chopping bu€er.
Four di€erent amounts of the unstained, processed sun¯ower sample (adjusted to 2 ml with bu€er) were added to
0.5 ml unstained pea sample to produce a 40-fold range of
sun¯ower/pea extract concentration. PI was added after the
samples were mixed and red ¯uorescence quanti®ed after
1 h. The experiment consisted of three replicates.
The e€ect of bu€er volume on PI ¯uorescence of nuclei from
simultaneously processed sun¯ower and pea leaves
An experiment was designed to test whether or not the
inhibitory e€ect of sun¯ower extracts on pea nuclei PI
¯uorescence could be diluted out. In this experiment,
approx. 13 mm square segments of sun¯ower and pea
leaves were simultaneously processed in 1.5, 15.0 and
30.0 ml bu€er. The samples were stained with PI and the
mean ¯uorescence of the sun¯ower and pea nuclei recorded.
This experiment represented a 20-fold dilution of sample. In
normal practice, 13 mm square leaf segments would
typically be processed in about 1.5±3.0 ml bu€er.
R E S U LT S
Compound(s) in sun¯ower leaves inhibit PI-¯uorescence
Helianthus annuus leaves contain substance(s) that inhibit
PI ¯uorescence of pea nuclei. This is apparent in the ¯ow
Number of nuclei
Price et al.ÐInhibitors of PI Fluorescence in Sun¯ower Leaves
B
50
D
C
A
25
200
400
600
Relative PI fluorescence
F I G . 1. Flow cytogram of ¯uorescence of PI-stained nuclei from
simultaneously processed sun¯ower (B) and pea (C) leaves to which PIstained nuclei from independently processed pea (D) leaves were added.
PI ¯uorescence was measured 20 h after mixing the samples. Peak (A)
was produced by ¯uorescent calibration beads
cytogram shown in Fig. 1. In this cytogram the lowest peak
(A) is produced by 0.6 mm 10 % ¯uorescence calibration
beads (Molecular Probes, Eugene, Oregon). Peaks B and C
represent nuclei of sun¯ower and pea leaves that were
simultaneously processed prior to PI staining. Peak D is
nuclei from independently processed and PI-stained pea
leaves that were added and measured after 20 h. The
di€erences between the two pea peaks, one inhibited (C)
and the other not inhibited (D) are relatively stable over
time.
Table 1 presents a time course of the ratio of the mean
¯uorescence of nuclei of pea leaves, simultaneously
processed and stained with sun¯ower leaves, to the mean
¯uorescence of added PI-stained nuclei from independently
processed pea leaves. In the 2 h after mixing there was a
progressive, but not signi®cant, increase in the ratio from
0.70 to 0.76 (Table 1). The second experiment showed no
di€erence in the ratio during the ®rst 15 min after mixing
and after 20 h (Table 2).
Concentration e€ects of inhibitors
The e€ect of concentration of inhibitor(s) was tested by
processing same size pea leaf sections in varying concentrations (40-fold range) of bu€er (containing independently
processed sun¯ower leaves) prior to PI staining. The PI
¯uorescence of both sun¯ower and pea nuclei increased
with decreasing concentration of sun¯ower extract
(Table 3). However, the ¯uorescence of pea nuclei in the
most dilute solution increased to only 89 % of that of the
control pea nuclei. The mean sun¯ower nuclei ¯uorescence,
expressed as DNA content, relative to the ¯uorescence of
pea nuclei, was similar in the samples containing 0.05, 0.25
and 0.50 ml of sun¯ower slurry. The sample containing
2 ml sun¯ower slurry had a signi®cantly lower estimated
DNA content (Table 3).
931
T A B L E 1. Time course of the ratio of the mean (+s.d.) PI
¯uorescence of nuclei (pea 1) of pea leaves simultaneously
processed and stained with sun¯ower leaves to the mean
¯uorescence of added PI-stained stained nuclei ( pea 2) from
independently processed pea leaves
Time after mixing (min)
Pea 1/Pea 2*
0±2.5
2.5±5.0
5.0±7.5
7.5±10.0
10.0±12.5
12.5±15.0
15.0±17.0
60
120
0.70 + 0.05
0.71 + 0.05
0.72 + 0.04
0.73 + 0.04
0.73 + 0.04
0.73 + 0.04
0.74 + 0.03
0.75 + 0.02
0.76 + 0.03
* Each ratio is the mean of three replicates. No signi®cant di€erences
(P ˆ 0.05) were observed among the means using a Duncan multiple
range test.
T A B L E 2. Ratio of mean (+s.d.) PI ¯uorescence of nuclei
( pea 1) of pea leaves simultaneously processed and stained
with sun¯ower leaves to the mean ¯uorescence of added
PI-stained nuclei (pea 2) from independently processed
leaves
Time after mixing
Pea 1/Pea 2
1.0±15.0 min
20.0 h
0.77 + 0.033
0.77 + 0.036
Each ratio is the mean of ®ve replicates. Measurements were taken at
1.0 to 15 min and at 20 h after mixing.
E€ect of bu€er volume on PI ¯uorescence of nuclei from
simultaneously processed sun¯ower and pea leaves
Testing the e€ect of inhibitor dilution on PI ¯uorescence
involved simultaneously processing sun¯ower and pea
leaves in bu€er volumes ranging over a 20-fold range
prior to PI staining (Table 4). In this experiment the ratio of
the means of sun¯ower and pea nuclei ¯uorescence
remained similar when leaves were processed in 1.5, 15
and 30 ml bu€er. The PI ¯uorescence of independently
processed pea nuclei decreased with increased volume of
chopping bu€er. The estimated DNA content of sun¯ower
nuclei (7.3±7.4 pg) from leaves processed with pea leaves in
this experiment (Table 4) is much higher than the
4.5±5.3 pg in the experiment in which sun¯ower and pea
leaves were independently processed and then mixed prior
to staining (Table 3).
DISCUSSION
The current research provides evidence for the presence of
inhibitor(s) of PI intercalation and/or ¯uorescence in
sun¯ower leaves. The quantity of inhibitor(s) is apparently
environmentally regulated and the inhibitory e€ect correlates with light quantity and quality (Price and Johnston,
1996b; Price et al., 1998). The mode of action of the
932
Price et al.ÐInhibitors of PI Fluorescence in Sun¯ower Leaves
T A B L E 3. PI ¯uorescence of nuclei from mixtures of independently-chopped sun¯ower and pea leaf samples*
Mean PI ¯uorescence
Composition{
Sun¯ower
Pea
Sun¯ower/pea ratio
Sun¯ower DNA
content ( pg){
Duncan's grouping}
2 ml sun¯ower
0.5 ml pea
176.7 + 25.5
372.5 + 17.7
0.47
4.5
A
0.5 ml sun¯ower
0.5 ml bu€er
0.5 ml pea
272.7 + 4.7
490.5 + 13.6
0.56
5.3
B
0.25 ml sun¯ower
1.75 ml bu€er
296.7 + 3.7
538.5 + 12.5
0.55
5.3
B
0.05 ml sun¯ower
1.95 ml bu€er
0.5 ml pea
332.3 + 4.1
615.4 + 14.3
0.54
5.2
B
689.4 + 8.5
0 ml sun¯ower
2.00 ml bu€er
0.5 ml pea
* All values are the means of three replicates +s.d.
{ Slurries containing the pea and sun¯ower nuclei were mixed before the propidium iodide was added.
{ The mean DNA content of the sun¯ower was calculated by the formula: DNA content ˆ (mean ¯uorescence sun¯ower/mean ¯uorescence
pea)(mean DNA content of the pea standard). The 2C DNA content of pea is 9.56 pg (Johnston et al., 1999).
} Multiple range test groupings (P ˆ 0.05). Means with the same letter are not signi®cantly di€erent.
T A B L E 4. Mean PI ¯uorescence of nuclei from pea and
sun¯ower leaves simultaneously processed in di€erent
volumes of bu€er*
Mean PI ¯uorescence
Bu€er volume
Sun¯ower
Pea
Sun¯ower/
pea ratio
Pea ‡ sun¯ower
1.5 ml
15 ml
30 ml
414 + 10
407 + 37
461 + 28
534 + 20
532 + 45
601 + 32
0.78
0.77
0.77
Pea controls
1.5 ml
15 ml
30 ml
Sun¯ower
DNA content
( pg){
7.4
7.3
7.3
800 + 18
719 + 31
707 + 20
* All values are means of ®ve replicates +s.d.
{ The mean 2C DNA content of the sun¯ower was calculated by the
formula: DNA content ˆ (mean ¯uorescence sun¯ower/mean ¯uorescence pea)(mean DNA content of the pea standard). The 2C DNA
content of pea is 9.56pg (Johnston et al., 1999).
inhibitor(s) is unknown, but appears to involve either an
intercalation into the DNA of the nuclei or interaction with
the proteins of chromatin that inhibits the access of PI to
DNA. It is also possible that the inhibitor acts directly on
the PI molecule and interferes with its ¯uorescence. This
hypothesis would require that intercalated PI be less
sensitive to the inhibitor than free PI.
The chemical nature of the inhibitor(s) of PI ¯uorescence
is not known but probably involves one or more of the
numerous secondary metabolites produced by plants. Our
preliminary experiments indicate that antioxidants such as
polyvinylpyrrolidine, ascorbate, 2-mercaptoethanol, and
dithiothreitol that are sometimes used in chopping bu€ers
(Bharathan et al., 1994) have no measurable e€ect on
reducing the inhibition. Greilhuber (1998) showed that
tannins interfere with Feulgen staining. It is possible that
they could inhibit PI ¯uorescence as well. However, we have
shown that soluble tannins isolated from sun¯ower leaves
increase the variance of the peaks but do not inhibit PI
¯uorescence of pea nuclei (Price, Hodnett and Johnston,
unpubl. res.). Therefore, tannins are apparently not among
the compounds that interfere with PI ¯uorescence and/or
intercalation.
The presence of inhibitors compromises the reliability of
estimated DNA content, particularly if detection of small
di€erences in DNA content is desired. Combined processing of the target and standard species reduces potential
variability and improves standardization (DolezÏel, 1991;
Baranyi and Greilhuber, 1995). This procedure may
minimize but not entirely eliminate the e€ect of inhibitors
on estimated DNA content. The practice of measuring
mixed, independently-processed and stained nuclei from
standard and target species, although not uncommon in the
literature (O'Brien et al., 1996; Price and Johnston, 1996b;
Rayburn et al., 1997; and others), may result in greatly
exaggerated di€erences in ¯uorochrome ¯uorescence that
may not be due to di€erences in DNA amount. This is
apparent when comparing the large di€erences between
estimated sun¯ower DNA content in Table 3 (4.5±5.3 pg)
and Table 4 (7.3±7.4 pg). The values in Table 3 involved
mixing independently processed pea and sun¯ower samples
before staining with PI. The sun¯ower DNA contents of
Table 4 were calculated from nuclei of simultaneously
processed and stained sun¯ower and pea leaves. Our best
estimate for the 2C nuclear DNA content of H. annuus used
in the current study is 7.3 pg.
Environmentally-induced DNA content di€erences such
as those reported by Price and Johnston (1996b) and
Price et al.ÐInhibitors of PI Fluorescence in Sun¯ower Leaves
Price et al. (1998) are most simply explained as due to
environmentally-induced variation in the amount of
inhibitors that interfere with the intercalation and/or
¯uorescence of PI. Due to e€ects of endogenous inhibitors,
intraspeci®c variation in DNA content of H. annuus needs
to be re-evaluated by comparing physiologically uniform
tissues that are free of inhibitors and using best practice
¯ow cytometry procedures.
Flow cytometry of PI-stained nuclei is generally considered to be a reliable method for estimating plant DNA
content (Michaelson, 1991a; Price and Johnston, 1996a;
DolezÏel et al., 1998). However, the best practice techniques
for ¯ow cytometry are still being developed. For example,
Johnston et al. (1999) emphasized the importance of using
internationally accepted plant species as standards in the
determination of plant DNA contents. The results of the
current study are important to best practice ¯ow cytometry.
First, it is apparent that the simultaneous processing of
tissue of the target and standard species is absolutely
necessary to obtain reliable DNA content estimates. The
current study also establishes that compounds in sun¯ower
leaves greatly inhibit the PI ¯uorescence of the standard pea
nuclei. The proportion of plant species with natural inhibitors of PI intercalation or ¯uorescence remains to be determined. A preliminary study detected inhibitors in four out
of ten randomly chosen angiosperm species (Bennett, pers.
comm.). It is likely that naturally occurring inhibitors that
decrease ¯uorochrome ¯uorescence of plant nuclei are common. Therefore, a test for the presence of inhibitors should
be used in all ¯ow cytometry studies. This can be done
simply by comparing the mean PI ¯uorescence of the
standard species nuclei, from samples containing simultaneously processed target and standard tissues, with the
mean ¯uorescence of standard nuclei from tissues that have
been processed in the absence of target species tissue. A
lower mean ¯uorescence of the standard nuclei in samples
of simultaneously processed standard and target tissues
indicates the presence of at least one inhibitor. When
inhibitors are present, additional caution should be taken in
interpreting di€erences in DNA content among plant
samples.
L I T E R AT U R E C I T E D
Arumuganathan K, Earle ED. 1991. Nuclear DNA content of some
important plant species. Plant Molecular Biology Reporter 9:
208±218.
Baranyi M, Greilhuber J. 1995. Flow cytometric analysis of genome
size variation in cultivated and wild Pisum sativum (Fabaceae).
Plant Systematics and Evolution 194: 231±239.
Bharathan G, Lambert G, Galbraith DW. 1994. Nuclear DNA content
of monocotyledons and related taxa. American Journal of Botany
81: 381±386.
Bennett MD. 1985. Intraspeci®c variation in DNA amount and the
nucleotypic dimension in plant genetics. In: Freeling M, ed. Plant
genetics. New York: Alan R. Liss, 283±302.
Bennett MD. 1998. Plant genome values: How much do we know?.
Proceedings of the National Academy of Sciences, USA 95:
2011±2016.
Bennett MD, Smith JB. 1976. Nuclear DNA amounts in angiosperms.
Philosophical Transactions of the Royal Society of London B 274:
227±274.
933
Cavillini A, Zol®no C, Natali L, Cionini G, Cionini PG. 1989. Nuclear
changes within Helianthus annuus L.: origin and control mechanism. Theoretical and Applied Genetics 77: 12±16.
Cavillini A, Zol®no C, Cremonini G, Natali L, Sassoli O, Cionini PG.
1986. DNA changes within Helianthus annuus L.: cytophotometric, karyotypic and biochemical analysis. Theoretical and
Applied Genetics 73: 20±26.
Cullis CA. 1985. Environmentally induced changes in genome size. In:
Cavalier-Smith T, ed. The evolution of genome size. New York:
John Wiley, 197±201.
Cullis CA, Cleary W. 1986. Rapidly varying DNA sequences in ¯ax.
Canadian Journal of Genetics and Cytology 28: 252±259.
DolezÏel J. 1991. Flow cytometric analysis of nuclear DNA content in
higher plants. Phytochemical Analysis 2: 143±154.
DolezÏel J, Greilhuber J, Lucretti S, Meister A, Lysak MA, Nardi L,
Obermayer R. 1998. Plant genome size estimation by ¯ow
cytometry: Inter-laboratory comparison. Annals of Botany
82 (Suppl. A): 17±26.
Durrant A. 1962. The environmental induction of heritable changes in
Linum. Heredity 17: 27±61.
Evans GM. 1968. Nuclear changes in ¯ax. Heredity 23: 25±38.
Galbraith DW, Harkins KR, Maddox JM, Ayres NM, Sharma DP,
Firoozabady E. 1983. Rapid ¯ow cytometric analysis of the cell
cycle in intact plant tissues. Science 220: 1049±1051.
Greilhuber J. 1986. Severely distorted Feulgen DNA amounts in Pinus
(Coniferophytina) after nonadditive ®xations as a result of
meristematic self-tanning with vacuole contents. Canadian Journal
of Genetics and Cytology 28: 409±415.
Greilhuber J. 1988. `Self-tanning'ÐA new and important source of
stoichiometric error in cytophotometric determination of nuclear
DNA content in plants. Plant Systematics and Evolution 158:
87±96.
Greilhuber J. 1998. Intraspeci®c variation in genome size: A critical
reassessment. Annals of Botany 82 (Suppl. A): 27±35.
Johnston JS, Jenson A, Czeschin DG Jr., Price HJ. 1996. Environmentally induced nuclear 2C DNA content variation in
Helianthus annuus. American Journal of Botany 83: 1113±1120.
Johnston JS, Bennett MD, Rayburn AL, Galbraith DW, Price HJ.
1999. Reference standards for determination of DNA content of
plant nuclei. American Journal of Botany 86: 609±613.
Laurie DA, Bennett MD. 1985. Nuclear DNA content in the genera
Zea and Sorghum. Intergeneric, interspeci®c and intraspeci®c
variation. Heredity 55: 307±313.
Michaelson MJ, Price HJ, Ellison JR, Johnston JS. 1991a. Comparison of plant DNA contents determined by Feulgen microspectrophotometry and laser ¯ow cytometry. American Journal of
Botany 78: 183±188.
Michaelson MJ, Price HJ, Johnston JS, Ellison JR. 1991b. Variation
of nuclear DNA content in Helianthus annuus (Asteraceae).
American Journal of Botany 78: 1238±1243.
Nagl W, Capesius I. 1976. Molecular and cytological characteristics of
nuclear DNA and chromatin for angiosperm systematics: basic
data for Helianthus annuus. Plant Systematics and Evolution 126:
221±237.
O'Brien IE, Smith DR, Gardner RC, Murray BG. 1996. Flow
cytometric determination of genome size in Pinus. Plant Science
115: 91±99.
Olszewska MJ, Osiecka R. 1983. The relationship between 2C DNA
content, life cycle type, systematic position, and the dynamics of
DNA endoreduplication in parenchyma nuclei during growth and
di€erentiation of roots in some dicotyledonous herbaceous
species. Biochemie und Physiologie der P¯anzen 78: 581±599.
Price HJ. 1976. Evolution of DNA content in higher plants. The
Botanical Review 42: 27±52.
Price HJ. 1988. Nuclear DNA content variation within angiosperm
species. Evolutionary Trends in Plants 2: 53±60.
Price HJ, Johnston JS. 1996a. Analysis of plant DNA content by
Feulgen microspectrophotometry and ¯ow cytometry. In: Jauhar
PP, ed. Methods of genome analysis: their merits and pitfalls. Boca
Raton: CRC Press, Inc., 115±132.
Price HJ, Johnston JS. 1996b. In¯uence of light on DNA content of
Helianthus annuus. Proceedings of the National Academy of
Sciences, USA 93: 11264±11267.
934
Price et al.ÐInhibitors of PI Fluorescence in Sun¯ower Leaves
Price HJ, Morgan PW, Johnston JS. 1998. Environmentally correlated
variation in 2C nuclear DNA content measurements in Helianthus
annuus L. Annals of Botany 82 (Suppl. A): 95±98.
Price HJ, Bachmann K, Chambers KL, Riggs J. 1980. Detection of
intraspeci®c variation in nuclear DNA content in Microseris
douglasii. Botanical Gazette 141: 195±198.
Rayburn AL, Price HJ, Smith JD, Gold JR. 1985. C-band heterochromatin and DNA content in Zea mays L. American Journal of
Botany 72: 1610±1617.
Rayburn AL, Birdari DP, Bullock DG, Nelson RL, Gourmet C, Wentzel
JB. 1997. Nuclear DNA content diversity in Chinese soybean
introductions. Annals of Botany 80: 321±325.