Euphytica 111: 105110, 2000.

2000 Kluwer Academic Publishers. Printed in the Netherlands.

105

Flow cytometric analysis of DNA content in Hosta reveals ploidy chimeras

Ben J.M. Zonneveld & Frank Van Iren
Institute of Molecular Plant Sciences, Leiden University, PO Box 9505, 2300 RA Leiden, The Netherlands
Received 11 June 1998; accepted 17 June 1999

Key words: flow cytometry, Hosta, ploidy chimeras, propidium iodide

Summary
Nuclear DNA content in 84 Hosta cultivars was measured by flow cytometry. As an internal standard we used the
closely related Agave americana, the latter was calibrated against other reference species. Propidium iodide values
were 20% higher than DAPI values, indicating a lower AT/GC ratio for Hosta. Ten new tetraploid plants were
found in addition to the only tetraploid known so far; H. ventricosa. Small forms of the tetraploid H. ventricosa
turned out to be triploid hybrids. Three other triploids were discovered. Three supposed tetraploids turned out to be
diploid. In five plants the ploidy levels of the germ layers L1 and L3 appeared to be different. In these cases thick
leaves coincides with a tetraploid DNA content. The DNA contents as measured in these ploidy chimeras coincide
with differences in leaf colour, thus giving an opportunity to analyze the build-up of Hosta plants. In general,
analysis of the nuclear DNA content can be used as a simple means to increase the possibilities for creating new
cultivars in this economically important perennial.

Introduction
Hosta Tratt. the number one selling perennial in the
USA has some interesting characteristics for the study
of cell lineage. It is generally accepted that dicotyledons are basically built from 3 germ layers named
L1 (forming mainly the epidermis), L2 (gametes and
some subepidermal tissues) and L3 (ground tissue and
vascular tissues) (Hanstein, 1868; Satina et al., 1940;
Poehtig, 1989). Stewart & Dermen (1979) demonstrated that also many monocotyledons are composed
of three germ layers in plants where L2 differs in
chloroplast or in DNA content from L1 and L3. Therefore, we assume the monocotyledon Hosta to have a
similar build-up.
Hosta is rather unique in that both chloroplastand ploidy-chimerism occur sometimes in the same
plant. In those cases the different germ layers can be
followed not only by determining DNA content, but
also by observing the differently coloured parts of the
leaves. Moreover Hosta can be multiplied vegetatively
enabling the chimeras to be propagated permanently.
Chimeras, viz plants with genetically different
germ layers have since long interested researchers and

breeders alike (Winkler, 1907; Tillney-Bassett, 1978;

Steward & Dermen, 1979). Chloroplast chimeras are
important in ornamental plants as they result in plants
with variegated leaves. Ploidy is one of the parameters
that influences cell size; higher ploidy levels usually
resulting in proportionally larger cells. So polyploid
plants and ploidy chimeras may have thicker leaves
and larger fruits and are therefore of interest to the
breeders of ornamental plants and the fruit growing
industry (Neilson-Jones, 1969). Hosta is grown on
a large scale for their showy leaves in a tremendous
array of colours.
Up to now, ploidy has not played an important
role in selection in Hosta, because no substantiated
changes in ploidy have been reported. Tetraploid chromosome numbers have been reported for H. ventricosa
and triploid numbers for H. clausa (Kaneko, 1968).
Here we report the analysis by flow cytometry of the
nuclear DNA content of 84 Hosta cultivars.
Generally, microscopical Feulgen densitometry is
considered the most reliable estimate of the DNA content of a single nucleus (Bennett & Leitch, 1995). Several authors indeed found that flow cytometric DAPI
measurements correlated badly with Feulgen data in

106
wide cross sections of the angiosperms (Doleel et al.,
1998). However, it seems likely that within a lower
taxon like a genus the differences in AT content are
usually much smaller (Buitendijk et al., 1997).
DAPI is a widely used DNA stain in flow cytometry of cell nuclei. Because fluorescence of the
unbound stain is relatively low, background problems
are less. However, because of its AT preference, estimates depend strongly on the base composition and
this varies widely throughout the plant kingdom. So
it can be used only when comparing DNA contents of
samples with the same AT/GC ratio.
Propidium iodide is the preferred stain for absolute nuclear DNA content measurements, as it has
no base preference and it has been demonstrated to
correlate well with DNA contents determined with
Feulgen staining (Doleel et al., 1998; Buitendijk et
al., 1997). However, because of substantial fluorescence of the unbound dye and lower specificity of the
staining, there are more background problems than
with DAPI. We applied both methods, in order to obtain accurate values for DNA amounts per nucleus (2C
content), and to see whether AT/GC differences are
present throughout the measured Hosta cultivars.

Materials and methods

Plant material
Plants were from our collection or obtained from the
Dutch nurseries of P. Warmerdam at Noordwijk, M.
Fransen at Ter Aar, J. van der Top at Barneveld, the
English nursery of A. and R. Bowden at Okehampton
and the American nurseries of Walters Gardens Inc.
Holland Michigan and Plant Delight Nursery, Raleigh,
N. Carolina. In most cases leaves were used, but
occasionally also roots were measured.
Flow cytometry of nuclear DNA content
Propidium iodide
For the isolation of nuclei, about 0.5 cm2 of an adult
leaf was chopped with a new razor blade in a Petri dish
in 0.25 ml nuclei-isolation buffer A of the Partec high
resolution DNA kit (Partec, Mnster) to which per ml
0.25 mg RNAse was added. Agave americana was
used as an internal standard (see below). After adding
0.75 ml PI solution (50 mg/l in water) the suspension
with nuclei was filtered through a 30 m mesh nylon
filter. The fluorescence of the nuclei was measured using a Partec CA-II flow cytometer. The optical path

contained the filters KG1, BG12, the dichroic mirror

TK500, and OG570 with a Leitz 50 1 water immersion objective. Relative fluorescence intensity of the
nuclei was analyzed using Partec DPAC software. At
least three different samples were measured for each
plant. Most histograms revealed a CV of less than 5%.
DAPI
For the isolation of nuclei about 0.5 cm2 of an adult
leaf was chopped with a new razor blade in a Petri
dish in a few drops of Partec buffer containing DAPI
(4,6-diamidino-2-phenylindole) (De Laat et al., 1987).
After adding 2 ml more DAPI buffer the suspension
with nuclei was filtered through a 30 m mesh size
nylon filter. Fluorescence of the nuclei was measured using a Partec CA-II flow cytometer within 30
minutes. The optical path contained the filters KG1,
BG38 and UG1, the dichroic miffor TK420, and
GG435 with a 20 0.66 Partec objective. Relative
fluorescence intensity of the nuclei was analyzed using Partec DPAC software. At least three, but in most
cases more than five different samples were measured
for each plant. Most histograms revealed a CV of less
than 5%.
Internal standard
In order to eliminate differences in signal intensities
due to light absorption, quenching and other variables,
a piece of Agave americana as an internal standard
was always chopped together with the material to be
analyzed. Agave americana was chosen as it is closely
related (Bogler & Simpson, 1995) and has about the
same DNA content. It has the same basic number
and size of chromosomes as Hosta and is year-round
available.
Pisum sativum and Hordeum vulgare were used to
calibrate our Agave americana. Baranyi & Greilhuber
(1996) found no significant differences in the nuclear DNA content of 38 accessions of Pisum sativum.
We measured the commercially available cultivars
Rondo (also included in the Baranyi & Greilhuber
study) and Frisson. When chopped together with,
and measured against Hordeum vulgare Sultan (obtained from I. Leitch, Kew Gardens Eng.), we also
found no difference; in both cases the ratio of the
propidium signals from P. sativum and H. vulgare was
0.872.
In a recent and thorough study by four laboratories
(Doleel et al., 1998) the nuclear DNA contents of P.
sativum and H. vulgare were calculated to be 8.75 and
10.04 pg respectively. The ratio between these values

107
Table 1. DNA content in Hosta cultivars measured
with Propidium Iodide and ratio in DNA content as
measured with Propidium Iodide and DAPI

Diploid cultivars
Beatrice
Bella
Bunter Babette
Butter Rim
Blue Cadet
Diamond Tiara
Elata
Fire Works
Fall Bouquet
Fortunei Albopicta
Francee
Fragrant Bouquet
Francis Williams
Ginko Craig
Golden Tiara
Gold Edger
Gold Standard
Great Expectations
Hadspen Blue
Hakyo
Halcyon
Hirao Majesty
Hirao Supreme
Hirao Tetra
Hydon Sunset
Inaho
Invincible
Kabitan
Kirishima
Little Jim
Little Wonder
Love Pat
Maruba
Masquerade
Neat Splash
On Stage
Out House Delight
Purple Profusion
Regal Rhubarb
Royal Standard
Sagae
Shining Tot
Sitting Pretty
Snowcap
Snow Flakes
Sparky

pg DNA per
2C nucleus

PI/DAPI
ratio

25.7
24.8
25.7
25.2
23.4
22.3
26.7
26.0
28.3
26.3
25.0
25.9
24.2
25.9
22.4
20.9
26.7
24.2
27.1
23.9
27.2
24.4
24.6
23.8
24.1
25.2
26.0
23.4
23.9
23.0
20.2
24.5
27.8
23.2
23.6
25.9
24.2
26.4
25.8
26.4
25.7
24.1
24.9
27.5
24.9
25.7

1.17
1.16
1.23
1.22
1.28
1.13
1.20
1.20
1.22
1.18
1.20
1.25
1.18
1.20
1.12
1.12
1.26
1.13
1.24
1.21
1.27
1.22
1.24
1.17
1.18
1.27
1.26
1.12
1.21
1.13
1.10
1.17
1.18
1.18
1.25
1.19
1.18
1.28
1.22
1.27
1.25
1.26
1.20
1.19
1.14
1.21

Table 1. Continued.
pg DNA per
2C nucleus

PI/DAPI
ratio

Sweet Susan
Tardiflora Shining Tot
Theos Blue
Urajiro Hachyo
Ultravoilet Light
William Lachman
White Tachi

25.9
26.7
24.9
24.1
22.3
24.9
24.3

1.22
1.25
1.22
1.14
1.23
1.18
1.25

Triploid cultivars
Hollys Honey
Peedee Elfin Bells
Venucosa
Tuckers Valentine
clausa ventricosa
venusta ventricosa
clausa v clausa
Sum and Substance

30.7
33.1
32.7
32.3
32.3
30.7
31.3
39.0

1.20
1.22
1.24
1.14
1.21
1.22
1.18
1.16

Tetraploid cultivars
F. Minuteman
F. Twilight
F. Patriot 3
F. Patriot 4
F. Patriot Green
F. Whirlwind
F. Second Wind
Grand Tiara
ventricosa Aureomarginata
Jolly Green Dwarf
Little Blue

53.0
52.8
51.2
50.3
50.3
49.7
49.8
43.4
41.6
40.8
38.7

1.17
1.13
1.26
1.20
1.22
1.13
1.14
1.20
1.17
1.16
1.21

is the same as we found, viz., 0.871. We adopted their

figures for the nuclear DNA content. Whether we cochopped with P. sativum, or with H. vulgare, the result
for our internal standard Agave americana was 15.9 pg
per nucleus (2C value) as measured with PI.

Results and discussion

Many Hosta cultivars have yellow or white coloured
leaf edges or leaf centres. These leaf parts were analyzed separately to reveal possible differences in nuclear DNA content. If there is no difference in DNA
content between the edge or center of the leaf only a
single value is given. Table 1 shows the DNA content
of a large number of hosta cultivars ranging from about
2028 pg in the diploids, 3133 pg in the triploids and

108
Table 2. DNA content in ploidy chimeras of Hosta, measured
with Propidium Iodide
Hosta cultivars

Royal Super

Cheatin Hart

U. Middle Ridge

Fortunei Patriot 1

Fortunei Patriot 2

plantaginea
Heaven Scent

pg DNA per nucleus

2C
4C
8C
leaf edge
leaf center
root
leaf edge
leaf center
root
leaf edge
leaf center
root
leaf edge
leaf center
root
leaf edge
leaf center
root
leaf edge
leaf center
root

26.1
26.5

23.0
22.5

25.4
24.8

26.7
27.7

27.6
27.7

30.0
28.5

52.6
53.6

44.4
44.8
52.3
51.2
51.6
52.8
54.8
55.9
52.3
52.8
52.8
53.2
56.7
54.1

105.6

91.3

a = ploidy level absent.

Figure 1. Histograms of fluorescence intensity of nuclei from leaf

tissue of Agave americana (peak 1) and root tissue of Hosta Patriot
1 (peak 2 and 3). Nuclei were isolated and stained simultaneously.
A: stained with propidium iodide and B: stained with DAPI.

4153 pg in the tetraploids. If different ploidy levels

within a single sample were present, as in roots and
in ploidy chimeras (Table 2), more than one DNA
content is presented.
Due to endoreduplication many dicotyledons show
multiple DNA contents in leaves, especially in the
vascular tissue. Therefore, also the veins of the leaf
were analyzed separately. Because no difference with
the rest of the leaf was found, it was concluded that
endoreduplication is absent in Hosta leaves as it is
in the internal standard Agave. This is in accordance
with the results of Bharathan et al., (1994) for other
monocotyledons.
Diploid cultivars
The PI/DAPI ratio demonstrates that with PI the DNA
contents are about 20% higher compared with the
DAPI measurements (also shown in Figure 1). So it
appears likely that Agave americana (published values
for four other Agave species about 55% AT, Cavallini

et al., 1996) has a higher AT content than Hosta. No

clear patterns in the ratios are found between Hosta
with a lower and those with a higher DNA content.
This suggests that the higher DNA content in some
cultivars was due to an increase with DNA of a similar
AT content as found in the plants with the lower DNA
content.

Triploid cultivars
H. Hollys Honey, Peedee Elfin Bells, Tuckers Valentine, and Venucosa are reported to be
small forms or hybrids of the apomictic H. ventricosa
(Schmid, 1991). The DNA values clearly show all
of them to be triploid. The relatively low amount
of DNA of the above mentioned triploids and the
semi-bellshaped flowers, suggests that they are indeed
hybrids with the tetraploid H. ventricosa, just as the
two triploid species hybrids (made by the first author)
in Table 2. Based on its low pollen stainability and
seemingly aneuploid offspring Sum and Substance
must be considered a triploid. However, the high DNA
content indicates that H. ventricosa is not one of the
parents.

109
Tetraploid cultivars
Some Hosta plants have been described with leaves
thicker than usual (The genus Hosta. List of registered
cultivars, 1993 and additions).
Several plants with reportedly thick leaves were
measured and eleven turned out to be new tetraploids
(Table 1). H. Little Blue, small, with bell-shaped
flowers like H. ventricosa, and H. Jolly Green Dwarf
with thick flowerstalks, have a DNA amount slightly
lower than H. ventricosa. This lower DNA content and
low pollen fertility (Zonneveld, unpublished) suggest
that they are aneuploids.
Hirao (1981) reported that he treated Hosta with
colchicine inferring that the resultant plants were tetraploid. The amount of DNA found in H. Hirao
Tetra, Hirao Supreme, and Hirao Majesty are,
however, clearly at the diploid level. Moreover, they
all have a low pollen stainability (data not shown). So
despite the colchicine treatment they have not become
tetraploid.
Roots
The roots of Hosta always show 25% to 50% cells
with double the DNA content irrespective whether
the plants are diploid, triploid or tetraploid (Figure 1). Usually this doubling is ascribed to G2 arrest
(dAmato, 1964; Torrey, 1961). The proportion of G2
cells also varies within a single root. After stripping
the cortex from the stele, the G2 arrested cells with
double DNA content turned out to be present in the
cortex only. This phenomenon has been found in roots
in many other plants also mainly if not exclusively in
the cortex.
Ploidy- or chromosomal chimeras (Table 2)
H. Royal Super is thick leaved with a yellow leaf
edge and a sport from the all green H. Royal Standard. The yellow leaf edge, containing only epidermal
L1 cells, has a diploid DNA content. The center of
the leaf blade is tetraploid, apart from a small fraction of diploid cells derived from the epidermis. The
tetraploidy of the L3 is corroborated by the roots in
which only tetraploid (4C) and 8C cells are present.
Diploid cells (2C) are absent. This also indicates that
L1 does not participate in root formation. Obviously,
a tetraploid L3 in an otherwise diploid leaf was sufficient to lead to a thickened leaf. The fact that the plant
is sterile indicates that the L2 is diploid. H. Cheatin
Hart turned out to have the same composition, with a
diploid L1 and a tetraploid L3.

H. Undulata Middle Ridge a plant with a white

leaf center was just the opposite to H. Royal Super
with respect to its ploidy distribution. In the green leaf
edge nuclei were found to have double the amount of
DNA with respect to the white leaf center. The tetraploid DNA content in 25% of the cells of the roots
is not dissimilar from wholly diploid plants indicating
again that the L1 does not contribute to the roots.
Also H. plantaginea Heaven Scent a sport from
H. plantaginea has a tetraploid yellow edge and a
diploid green leaf center.
The same combination of a tetraploid L1 and diploid L3 was found in two accessions of H. Patriot and
both have diploid roots (mainly 2C, Figure 1). H. Patriot is, just as the tetraploid H. Minuteman a sport
from the diploid H. Francee. Their much broader
white edges as compared to the small white edges of
H. Francee are clearly due to the L1 being tetraploid
in both sports. On the other hand, two other accessions
of H. Patriot and also an unnamed green sport of it
turned out to be fully tetraploid, including the roots
(Table 1).
Summarizing, the absence of endoreduplication in
the above-ground parts enables the analysis of cell lineage in ploidy chimeras of Hosta, viz., the build-up of
the various tissues and organs from the L1, L2, and L3
germ layers.
In addition, the analysis of nuclear DNA content
increases the possibilities for raising new types of
cultivars in this economically important perennial.
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