Annals of Botany 85: 351±357, 2000
doi:10.1006/anbo.1999.1071, available online at http://www.idealibrary.com on
Allium cepa L. Cultivars from Four Continents Compared by Flow Cytometry show
Nuclear DNA Constancy
M I C H A E L D . B E N N E T T *{, SP E N C E R JO HN STO N{, GE O R G E L . H O D N E T T }
and H . J A M E S P R I C E }
{Royal Botanic Gardens Kew, Richmond, Surrey TW9 3DS, UK, Departments of {Entomology and
}Soil and Crop Science, Texas A&M University, College Station, Texas 77843-2474, USA
Received: 27 August 1999 Returned for revision: 23 September 1999 Accepted: 11 November 1999
In 1965 Van't Hof estimated the nuclear DNA amount of an unidenti®ed Allium cepa L. cultivar as 2C ˆ 33.55 pg
(Experimental Cell Research 39: 8±58). This value has been adopted by common usage as the main calibration
standard for angiosperm DNA C-value estimations. However, di€erent cultivars have been used while assuming
species DNA C-value constancy. Surprisingly this assumption has never been tested. A. cepa is an outbreeder with
telomeric heterochromatic segments, so intraspeci®c variation in C-value, possibly correlated with environmental
factors as seen in Zea mays L., might be expected. We used laser ¯ow cytometry to compare nuclear DNA amounts in
roots of six A. cepa cultivars used as calibration standards or from di€erent environments. Tissues from one cultivar,
or similar volumes of tissue from two cultivars, were run and the variance between nuclei in 2C peaks compared. Only
one shoulderless 2C peak was seen for all pairs of co-chopped cultivars. Thus, no large di€erences in C-value between
cultivars from di€erent environments were found. Moreover, comparing cultivars run singly or as pairs showed no
evidence for increased variation in 2C peaks in the latter, and hence of critical di€erences in DNA amounts between
`Ailsa Craig' and another cultivar. Such variation was insucient to make their use as alternative calibration
standards, or the practice of imputing Van't Hof's original C-value estimate to them, unacceptable for most practical
purposes. Given the mechanisms known which can generate genome size variation, the degree of constancy in DNA
# 2000 Annals of Botany Company
C-value found seems remarkable.
Key words: Allium cepa, onion cultivars, calibration standards, DNA C-value constancy, ¯ow cytometry.
I N T RO D U C T I O N
In 1965 Van't Hof published an estimate of the nuclear
DNA amount of Allium cepa (2C ˆ 33.55 pg) carefully
obtained using chemical methods. This was later used as a
calibration standard by his colleagues from Brookhaven
(e.g. Price and Bachmann, 1975), and by other groups
including Rees and colleagues at Aberystwyth (e.g. Jones
and Rees, 1968), Bennett and Smith (1976) in Cambridge,
and Greilhuber (1977) in Vienna. Indeed, Allium cepa has
been informally adopted by common usage as the main
calibration standard for C-value estimations in angiosperms. At least 143 (ˆ 46.7%) out of 306 original sources
of data listed in Bennett et al. (1997) used A. cepa with an
assumed 4C DNA amount of 67.1 [or 67.0] pg as a
calibration standard. The crop was chosen for this purpose
because it is widely grown, easy to obtain as seed or bulbs,
and highly amenable to cytology.
If Van't Hof's original absolute estimate for A. cepa was
inaccurate, then so are all the estimates for other taxa which
use it as a calibration standard (Bennett and Smith, 1976).
Such estimates would all deviate from reality by a constant
proportion, but the relative di€erences between taxa would
be una€ected. However, there is good reason for believing
that the absolute 4C value for A. cepa (67.1 pg) measured
* For correspondence. Fax ‡44 (0)208 332 5311, e-mail m.bennett
@rbgkew.org.uk
0305-7364/00/030351+07 $35.00/00
by Van't Hof (1965) is tolerably accurate. Thus, three
separate studies using animal nuclei as calibration standards have independently obtained very similar results;
namely Arumuganathan and Earle (1991) using Gallus
domesticus (65.4 pg), Ulrich et al. (1988) using both Homo
sapiens and G. domesticus (67.5 pg), and Dolezel, Sgorbati
and Lucretti (1992) using H. sapiens (69.5 pg). The mean
4C DNA estimate for these three studies (67.46 pg) is within
1% of Van't Hof's original estimate of 67.1 pg. This
con®rmed an earlier conclusion by Greilhuber et al. (1983)
that estimates for several animals, including man, obtained
assuming Van't Hof's value for A. cepa (1C ˆ 16.75) are
consistent with a number of independent, absolute DNA
content determinations reported for those species. Thus,
it is reasonable to assume that absolute DNA estimates
for other taxa made using Allium cepa (assuming
2C ˆ 33.55 pg) as a calibration standard are tolerably
accurate, if other aspects of best practice were followed.
Another concern regarding the use of Allium cepa as a
calibration standard stems from possible variation in
C-values between cultivars. Its consequence would be
serious as estimates for unknowns would be di€erentially
a€ected by non-constant and unknown proportions, and
the relative di€erences between species estimates disturbed.
Intraspeci®c variation in C-values has been reported in
many plant species (Bennett, 1985; Greilhuber, 1998)
ranging from a few to a few hundred per cent. The cause
# 2000 Annals of Botany Company
352
Bennett et al.ÐNuclear DNA Constancy in Allium cepa Cultivars
of such variation is hotly disputed, and in many cases is
shown to be artifactual in origin (Greilhuber, 1988, 1998).
However, intraspeci®c variation is clear and undisputed in
some species which display variation in the number and
size of heterochromatic segments between cultivars. For
example, C-values vary by over 30% between lines of Zea
mays with di€erent knob numbers (Laurie and Bennett,
1985; Rayburn et al., 1985; Poggio and Naranjo, 1991).
While Van't Hof's (1965) estimate for Allium cepa
(2C ˆ 33.55 pg) has been widely accepted as a calibration
standard, the cultivar used to determine this C-value was
not stated, and is unknown (Van't Hof, pers. comm).
However, workers have used several di€erent cultivars for
this purpose on the implicit or explicit assumption that
there is no critical variation in C-values between them.
Surprisingly this assumption has never been rigorously
tested. The three di€erent estimates (65.4 pg, 67.5 pg and
69.5 pg, see above) obtained using two animal standards
di€er by up to 6.3% which might indicate the occurrence of
intraspeci®c variation in A. cepa. Moreover, A. cepa is an
outbreeding species with blocks of heterochromatin at most
telomeres on its chromosomes (Stack and Comings, 1979).
Thus, it seemed possible that signi®cant detectable intraspeci®c variation in C-value might commonly occur
between its cultivars, such as occurs in other outbreeders,
e.g. Secale cereale L. (Bennett, 1985) and Zea mays (Laurie
and Bennett, 1985; Rayburn et al., 1985; Tito et al., 1991).
Moreover, such variation in Z. mays may correlate with
environmental factors such as latitude and altitude (see
Table 2 in Poggio et al., 1998). Perhaps C-values of onion
cultivars adapted to the low latitudes of India and Texas
di€er from those of cultivars adapted to the higher latitudes
of the UK or Canada. If so, and if locally adapted cultivars
are mainly used as calibration standards, it could introduce
systematic errors in DNA estimates for the other taxa in
di€erent locations. We tested these possibilities by comparing several onion cultivars either listed as calibration
standards in published papers by international practitioners
in the ®eld, and/or adapted to grow in di€erent environments.
M AT E R I A L S A ND M E T H O D S
Selection of plant materials
Table 1 lists six A. cepa cultivars compared in the present
work. Van't Hof (1965) did not name the cultivar used to
estimate the C-value for A. cepa. The British cultivar `Ailsa
Craig' was selected because it has been recommended and
widely used as a calibration standard for over 24 years
(Bennett and Smith, 1976). Bennett and Leitch (1995, 1997)
listed several other A. cepa cultivars used as calibration
standards by various authors since 1981. Two were chosen
which have been well used as calibration standards, and are
adapted to widely di€erent environments, namely `Stuttgarter Riesen' from Germany, and `Nazik Red' from India.
`Canterbury Longkeeper' from the South Island of New
Zealand was included as a representative from the Southern
Hemisphere. Two North American cultivars from di€erent
latitudes were included, namely `Precedent', recommended
for cultivation near the great lakes in Canada and the USA,
and `TG-1015Y', grown in south Texas. These cultivars
from six countries in four continents include three which
have often been used as calibration standards (Table 1), and
a range adapted for cultivation at di€erent latitudes in
North America or Eurasia. Comparing them should
indicate if there is critical intraspeci®c variation in DNA
T A B L E 1. Cultivars of Allium cepa L. compared in the present work, their country of origin and approximate latitude of
cultivation, source of supply, and references citing their use as C-value calibration standards
Cultivation
Cultivar
Country
Latitude
Supplier/Source
Reference(s) to use as a
calibration standard
`Ailsa Craig'
UK
approx. 558N
M. D. Bennett, Royal
Botanic Gardens Kew
Bennett and Smith, 1976;
Li and Liu, 1996
`Nazik Red'
India
approx. 258N
USDA ARS PGRU,
Cornell University
Ohri and Kumar, 1986;
Hiremath and Salimath,
1991
`Stuttgarter
Riesen'
Germany
approx. 488N
Konrad Bachmann
Gatersleben
Greilhuber, 1988;
Baranyi and
Greilhuber, 1996
`Canterbury
Longkeeper'
New Zealand
approx. 438S
Brian Murray
University of Auckland
Ðseed from Blenheim,
South Island
Ð
`Precedent'
(ˆ`Sunex 1460')
USA/ Canada
approx. 458N
Stokes Seeds Inc,
Bu€alo, New York
Ð
`TG-1015Y'
USA
approx. 308N
Leonard Pike,
Texas A & M
University
Ð
Bennett et al.ÐNuclear DNA Constancy in Allium cepa Cultivars
C-value in A. cepa, and if so, whether it is related to
latitude.
Sample preparation
Samples of nuclei for ¯ow cytometry were obtained either
from bulbs or young seedlings. Fifty seeds of a known
A. cepa cultivar were placed in 25 ml of deionized water at
48C to imbibe for about 24 h. They were then plated out on
treble-thickness ®lter paper moistened with deionized water
in a Petri-dish to germinate in the dark either at room
temperature (15±248C), or in an incubator at 238C. After
5±10 d either young roots 10±20 mm long, or seedlings
(minus the seed coat) with roots 15±20 mm long were
excised using a scalpel in a drop of ice-cold Galbraith bu€er
(Galbraith et al., 1983) on a microscope slide, and
transferred to a 5 cm plastic Petri dish containing 2 ml of
ice cold Galbraith bu€er for chopping. The bu€er ( pH 7.2)
consisted of, per litre, 4.26 g MgCl2 , 8.84 g sodium citrate,
4.2 g 3-[N-morpholino] propane sulfonic acid, 1 ml Triton
X-100 and 1 mg boiled ribonuclease A, as described by
Galbraith et al., (1983) and Price and Johnston (1996).
Bulbs grown in Texas from seed of Allium cepa L. `Ailsa
Craig' supplied from stock held at the Royal Botanic
Gardens, Kew were suspended above deionized water in
darkness at room temperature (15±248C). The base of each
bulb, one per beaker, just touched the water which was
changed every 24±48 h. After 2±7 d, root tips 5±10 mm
long were excised from roots 10±20 mm long as above and
chopped in ice-cold bu€er for ¯ow cytometry. Occasionally,
a segment of leaf tissue about 10 mm long was excised from
an etiolated young leaf projecting 20±50 mm from the top
of a bulb.
The ratio of tissue to bu€er and the minimum volume of
bu€er needed to obtain satisfactory yields of nuclei were
determined in preliminary experiments. These showed that
three±®ve root-tips or one leaf segment from a bulb, or
®ve±ten roots from seedlings chopped in 2 ml of bu€er,
typically yielded 2500±10 000 nuclei in runs lasting 5±
18 min using 1 ml samples recovered after ®ltering.
Plants of Pisum sativum L. `Minerva Maple' were grown
in pots in a growth chamber given 16 h d at 228C and 8 h
nights at 138C. Healthy young lea¯ets were sampled for
calibration checks against Allium cepa `Ailsa Craig' using
¯ow cytometry. One or two lea¯ets were chopped in 2 ml
ice-cold bu€er without Allium, whereas half a lea¯et cochopped with Allium tissue as above normally gave similar
yields of Pisum and Allium 2C nuclei, and hence similarlysized peaks.
Comparisons of nuclear DNA amount
DNA content was determined from young leaf or root
tissue excised from bulbs or seeds using laser ¯ow cytometry with propidium iodide (PI) as the ¯uorochrome. The
healthy young leaf and/or root tissues excised from bulbs or
seeds were chopped in 2 ml of ice-cold bu€er using a new
razor-blade for each sample. The chopped tissues were
®ltered through a 53 mm nylon ®lter. One ml of ®ltrate was
pipetted into a microfuge tube to which ice cold propidium
353
iodide was added to a ®nal concentration of 50 ppm PI. The
samples were stored on ice in a dark refrigerator, and
analysed after 1 to 8 h. The DNA content for four to
12 replicates for each cultivar, or pair of cultivars, was
determined by a protocol adapted from Price and Johnston
(1996) using a Coulter Epic Elite ¯ow cytometer (Coulter
Electronics, Hialeah, Florida, USA) equipped with a watercooled laser tuned at 514 nm and 500 mW. Fluorescence
emission at 4615 nm was detected with a photomultiplier
screened by a long pass ®lter. The ¯ow cytometer was tuned
to produce minimal variance with Coulter Flow-Check2
¯uorospheres (Coulter Corporation, Miami, Florida, USA)
and mean ¯uorescence of A. cepa `Ailsa Craig' 2C nuclei
between 370 and 410.
The following approach was used to test whether
cultivars di€er in DNA C-values. First, nuclei of single
cultivars were run and the coecient of variation (CV)
among 2C nuclei was noted. Second, nuclei from pairs of
known co-chopped cultivars were run together and the
corresponding statistic noted for 2C nuclei. When pairs of
cultivars were co-chopped, equal volumes of tissue from
each cultivar were used. If two cultivars di€er greatly in
C-values (i.e. 45%) ¯ow cytometry should show separate
2C peaks. Given only a small di€erence in C-value (i.e.
42%) the shape of a 2C peak should be detectably broader
for the mixtures than for either cultivar run alone
(Hopping, 1994). Thus, if two cultivars di€ered even so
little in C-value, the variance for the 2C peak should be
higher for a co-chopped mixture than for either single
cultivar. The statistic chosen for this comparison, given by
the ¯ow cytometer printout was the half peak CV
comparing similarly sized populations of 2C nuclei. Both
the coecients of variation and arc sine root transformed
CVs were compared using the orthogonal contrast statement from PROG GLM in v6.12 SAS (SAS Institute, Inc.
Cary, NC, USA).
R E S ULT S
The material and methods used gave acceptable yields of
nuclei for ¯ow cytometry for all the A. cepa cultivars
studied. However, long runs, usually 10 + 5 min, were
needed to acquire estimates for populations typically of
about 5000 nuclei. Figure 1A and B shows typical plots for
two Eurasian cultivars run alone, while Fig. 1C shows a
typical plot for the same two cultivars co-chopped and run
together. Figure 2 shows a similar comparison for two
American cultivars run alone (A and B) or together (C).
Fluorescence pro®les for root-tip nuclei typically had a
much larger 2C than 4C peak with little or no evidence of
an 8C or higher peak (Figs 1 and 2). The C-value status of
these peaks was initially veri®ed in a few preliminary comparisons with peaks for nuclei from Pisum sativum lea¯ets,
whose pro®le and 2C values were known from our previous
work (e.g. Johnston et al., 1999), run separately or together
using the same FC setting.
The ¯uorescence peaks from young leaf tissue from bulbs
had a di€erent pro®le. The 2C peak was largest, but a 4C
peak was relatively larger than in root tissue. Other smaller
peaks gave clear evidence of 8C and 16C populations (data
354
Bennett et al.ÐNuclear DNA Constancy in Allium cepa Cultivars
F I G . 1. Relative red ¯uorescence of PI-stained nuclei of `Ailsa Craig'
(A) and `Nazik Red' (B) run alone, and (C) together.
F I G . 2. Relative red ¯uorescence of PI-stained nuclei of `TG-1015Y'
(A) and `Precedent' (B) run alone, and (C) together.
not shown). The 8C peaks may provide a useful calibration
standard with 134.2 pg for estimating unknown DNA
amounts in species with high C-values in the range 100±
200 pg. However, a drawback with the use of such leaf
tissue was a tendency for material in the sample to clog and
slow the ¯ow, requiring cleaning and resetting of the
cytometer. This problem also occurred using roots, but to a
much lesser degree. Nevertheless, the long durations of
runs, combined with an unpredictable need to clean and
reset the FC, made it impossible to compare all the present
materials in one experiment, or sometimes to complete all
the replicates intended on a single day. The critical test was
between known cultivars run individually or together in
pairs in the same experiment, so sensitivity was not
precisely standardized between all experiments. Thus the
photomultiplier was set to give 2C peaks for `Ailsa Craig'
around 400 (Table 2) or 370 (Table 4), but arbitrarily at a
higher range around 450 (Table 3) in expt 4 with `Canterbury Longkeeper'.
Most importantly, given the main reason for this study
(i.e. the possibility that cultivars of A. cepa may have
critical di€erences in C-value), no evidence of two separate
2C peaks was seen in any of the six combinations of two cochopped cultivars examined, i.e. `Ailsa Craig' plus each one
of the ®ve other cultivars studied, or `TG-1015Y' plus
`Precedent' (Tables 2±5). Similarly, no repeatable evidence
of a shoulder on the peak containing 2C nuclei was noted in
any replicate for any one of these combinations (Figs 1 and
2). Thus, no evidence for large di€erences in C-value, such
as commonly occur between cultivars of Zea mays from
di€erent environments, was found for any of the present
A. cepa materials grown in di€erent regions and environments.
As noted above, comparisons of the shapes of peaks for
populations of 2C nuclei for pairs of cultivars run singly or
together should provide a telling test for small but still
unacceptably large di€erences in C-values between cultivars
used as calibration standards. The orthogonal contrasts
showed no signi®cant di€erences in CVs for nuclei of 2C
peaks for singletons and co-chopped pairs of cultivars
including some from di€erent latitudes where di€erences
might have been expected (Figs 1 and 2; Tables 2±5). Thus,
the mean CVs (Table 5) for six mixtures (2.25) and six
single cultivars (2.20) were virtually identical (F1 ,56 ˆ 0.71;
Bennett et al.ÐNuclear DNA Constancy in Allium cepa Cultivars
355
T A B L E 2. Mean red ¯uorescence from PI-stained 2C nuclei of three cultivars of A. cepa used as calibration standards,
chopped alone, or in pairs including `Ailsa Craig'
Cultivar(s)
`Ailsa Craig'
`Nazik Red'
`Ailsa Craig' ‡ `Nazik
Red'
`Stuttgarter Riesen'
`Ailsa Craig' ‡
`Stuttgarter Riesen'
Replicate
Mean
CV
Mean
CV
Mean
CV
Mean
CV
Mean
CV
Experiment 1
1
2
3
387.7
415.0
400.1
2.12
1.54
2.21
388.0
392.9
398.5
1.89
2.35
1.80
398.0
397.5
400.0
1.97
2.39
1.72
395.6
413.4
399.7
2.47
2.02
1.91
417.2
412.9
409.8
1.96
1.83
1.55
Experiment 2
4
5
6
398.0
400.3
389.9
2.17
2.95
3.18
395.8
382.3
391.5
1.87
1.56
2.18
399.7
407.9
404.4
1.83
1.44
1.40
404.7
405.0
402.5
2.41
1.75
2.08
415.8
418.5
418.2
2.09
2.13
2.37
Mean
398.5
2.36
391.5
1.94
401.3
1.79
403.5
2.11
415.4
1.99
T A B L E 3. Mean red ¯uorescence from PI-stained 2C nuclei of `Ailsa Craig' and `Canterbury Longkeeper' chopped alone
or together
Cultivar(s)
`Ailsa Craig'
`Canterbury Longkeeper'
`Ailsa Craig' ‡ `Canterbuty Longkeeper'
Replicate
Mean
CV
Mean
CV
Mean
CV
Experiment 3
1
2
3
4
403.1
416.7
Ð
Ð
2.41
2.36
Ð
Ð
414.4
396.9
Ð
Ð
2.30
2.31
Ð
Ð
418.6
416.5
426.8
425.1
2.50
2.15
2.69
2.35
Experiment 4
5
6
Ð
Ð
Ð
Ð
452.3*
452.8*
2.73
2.01
462.5*
434.4*
1.94
2.19
409.9
2.39
429.1*
(405.5)
2.34
430.7*
(421.8)
2.30
Mean
* The mean red ¯uorescence for 2C peaks obtained in expt 4 re¯ects a FC sensitivity giving values uniquely above those obtained for these and
other cultivars within the normal range of settings in other experiments or days. Consequently, means for values omitting this anomalous setting
are also given (in brackets) to aid comparison.
T A B L E 4. Mean red ¯uorescence from PI-stained 2C nuclei of three cultivars of A. cepa chopped alone, or in pairs
Cultivar(s)
`Ailsa Craig'
`TG-1015Y'
`Precedent'
`Ailsa Craig' ‡
`TG-1015Y'
`Ailsa Craig' ‡
`Precedent'
`TG-1015Y' ‡
`Precedent'
Replicate
Mean
CV
Mean
CV
Mean
CV
Mean
CV
Mean
CV
Mean
CV
Experiment 5
1
2
372.7
Ð
1.95
Ð
365.4
367.6
2.55
2.17
375.5
367.5
1.97
2.58
364.6
348.1
2.19
1.69
Ð
Ð
Ð
Ð
368.8
371.5
2.24
1.95
Experiment 6
3
4
5
6
371.0
371.3
373.6
Ð
2.15
3.15
2.86
Ð
356.7
363.6
369.0
Ð
1.02
3.30
2.69
Ð
368.6
368.5
363.3
363.2
2.81
2.36
3.61
2.72
380.8
375.9
358.8
372.7
2.17
2.31
2.51
2.07
367.2
374.4
368.8
370.8
2.19
2.36
2.82
2.04
367.6
376.8
369.1
Ð
2.09
2.63
2.58
Ð
Mean
372.2
2.53
364.5
2.35
367.8
2.68
366.8
2.16
370.4
2.35
370.8
2.30
356
Bennett et al.ÐNuclear DNA Constancy in Allium cepa Cultivars
T A B L E 5. Mean CV for 2C peaks in six A. cepa cultivars chopped alone or co-chopped in pairs
Single cultivars
CV
`Nazik Red'
`Stuttgarter Riesen'
`TG-1015Y'
`Precedent'
`Canterbury Longkeeper'
2.36 (6)
2.39 (2)
2.53 (4)
2.42 (12)
1.94 (6)
1.79 (6)
2.35 (5)
2.68 (6)
2.34 (4)
Mean
2.25 (6)
`Ailsa Craig'
Paired cultivars
CV
`Ailsa Craig' ‡ `Nazik Red'
2.11 (6)
`Ailsa Craig' ‡ `Stuttgarter Riesen'
`Ailsa Craig' ‡ `TG-1015Y'
`Ailsa Craig' ‡ `Precedent'
`TG-1015Y' ‡ `Precedent'
`Ailsa Craig' ‡ `Canterbury Longkeeper'
1.99 (6)
2.16 (6)
2.35 (4)
2.30 (5)
2.30 (6)
2.20 (6)
The number of replicates are given in parentheses for each mean.
P 4 0.40). Surprisingly, on average, the 2C peaks for
mixtures were just as sharp as for single cultivars. The mean
CVs (Table 2) of 2C peaks for `Ailsa Craig' (2.36) from the
UK (approx. 558N) and `Nazik Red' (1.94) from India
(approx 258N) were not signi®cantly di€erent from their cochopped mixture (2.11) (F1 ,24 ˆ 0.05; P 4 0.817). Similarly, the mean CVs (Table 4) of 2C peaks for `TG-1015Y'
(2.35) from the southern USA (approx. 308N) and
`Precedent' (2.68) from the Great Lakes (approx. 458N)
were not signi®cantly di€erent from that of their cochopped mixture (2.30) (F1 ,23 ˆ 0.56; P 4 0.46). Thus, the
present work provides no evidence of a latitudinal cline for
DNA C-value in A. cepa, unlike that reported for Zea mays
(Laurie and Bennett, 1985; Rayburn et al., 1985).
DISCUSSION
As noted previously (Bennett and Smith, 1976; Bennett and
Leitch, 1995, 1997) various workers have used several
di€erent cultivars of Allium cepa as calibration standards
on the explicit or implicit assumption that no critical
variation in DNA C-value exists between them. However,
this assumption had never been widely tested, although
some comparisons of cultivars from the same region
showed no di€erence (Waldherr, 1992). The present work
compared six cultivars from widely di€erent geographical
locations and adapted to di€erent environments, including
two from Europe (`Ailsa Craig' and `Stuttgarter Riesen')
and one from India (`Nazik Red') previously used as
calibration standards. Comparing 2C curves for known
pairs of cultivars run either singly or together showed no
evidence of increased variance in the latter, and hence no
evidence for critical di€erences in nuclear DNA amounts
between the test cultivars.
Thus the present results for A. cepa parallel previous
work by Bennett and Smith (1976) which found no critical
di€erences between cultivars of Vicia faba L., and of
Hordeum vulgare L. Moreover, it supports the extensive
recent results by Greilhuber and colleagues showing little or
no signi®cant variation between cultivars of Pisum sativum
(Greilhuber and Ebert, 1994; Baranyi and Greilhuber, 1995,
1996) and Glycine max (L.) Merr. (Greilhuber and
Obermayer, 1997). Clearly a tendency towards a global
constancy for C-values within many species is not
uncommon.
To obtain perfect, absolute C-values the ideal calibration
standard should exhibit no variation between individuals.
In the real world of biodiversity and error variation this
ideal is approached by using best practice to minimize real
and error variation, and to produce acceptable C-value
estimates whose accuracy re¯ects need and resources.
In order to minimize error due to biodiversity in A. cepa
it is desirable to use just one cultivar of the species as a
calibration standard. Moreover, to minimize the possibility
of variance due to environment, it is best to use seed from a
large population grown in one place and harvested at one
time. The use of such seed of A. cepa `Ailsa Craig', distributed widely for this purpose since 1976, has presumably
tended to minimize such errors, and is still best practice to
approach the ideal. Nevertheless, the present work indicates
that best practice would not be seriously prejudiced by
using the other cultivars compared in the present work.
Thus, it is now reasonable to presume that their past use to
estimate DNA C-values for other species caused no
unacceptable errors.
It is not suggested that no intraspeci®c variation in DNA
C-value occurs between individuals or cultivars in A. cepa.
Vosa's observation (1973) that most chromosomes were
heteromorphic for C-band patterns in an Allium species
suggests otherwise. Rather we note that the level of such
variation between `Ailsa Craig' and ®ve other cultivars
compared in the present work was not so large as to make
their use as alternative calibration standards unacceptable,
or to render the practice of imputing to them the C-value
originally estimated for an unidenti®ed cultivar of A. cepa
by Van't Hof (1965) untenable, for most practical purposes.
Recent results for 14 A. cepa cultivars including `Stuttgarter
Riesen' estimated by Feulgen microdensitometry or ¯ow
cytometry using propidium iodide strongly support this
view (Baranyi and Greilhuber, 1999). Thus, their assumption of 2C ˆ 33.50 for `Stuttgarter Riesen' as a calibration
standard gave 2C DNA estimates ranging from only 33.04
to 34.60 for the 13 other cultivars.
The variation in C-values between the present onion
cultivars was clearly small, perhaps surprisingly so, given
the very di€erent environments in which they are cultivated.
Clearly mechanisms exist which can rapidly generate
Bennett et al.ÐNuclear DNA Constancy in Allium cepa Cultivars
considerable variation in DNA C-value within individuals
and even in individual cells. In view of the molecular
mechanisms now known which can generate variation in
genome size (Kubis et al., 1998; Sanmiguel and Bennetzen,
1998), the degree of C-value constancy found in many
species, including A. cepa, is remarkable, and needs
explanation. Indeed, it is arguable that such constancy
would not be expected without some mechanism(s) to select
for constancy (or against drift) in C-value, which thereby
controls variation in DNA C-value back towards some
encoded norm for each species. Were it not so, the
frequency and extent of intraspeci®c variation in DNA
amount would surely be much larger, and the observed
degree of species DNA constancy would present a new Cvalue paradox.
Genome size is widely perceived as free to vary, changes
being undetected and uncorrected by internal control
mechanisms. The present results challenge this view,
suggesting instead that DNA amount may normally be
subject to innate controls by `counting' mechanisms which
somehow detect, quantify and regulate genomic size
characters within quite tightly de®ned or preselected limits.
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