© 2016 The Japan Mendel Societ y Cytologia 81(3): 263–269
Breeding and Cytogenetic Characterizations of
New Hexaploid Drosera Strains Colchicine-Induced
from Triploid Hybrid of D. rotundifolia and D. spatulata
Santhita Tungkajiwangkoon1, Junichi Shirakawa2,
Masako Azumatani3 and Yoshikazu Hoshi4*
1 Graduate School of Bioscience, Tokai University, Kawayo, Minamiaso-mura, Aso-gun,
Kumamoto 869–1404, Japan
2 Department of Bioscience, School of Agriculture, Tokai University, Kawayo, Minamiaso-mura, Aso-gun,
Kumamoto 869–1404, Japan
3 Graduate School of Agriculture, Tokai University, Kawayo, Minamiaso-mura, Aso-gun,
Kumamoto 869–1404, Japan
4 Department of Plant Science, School of Agriculture, Tokai University, Kawayo, Minamiaso-mura, Aso-gun,
Kumamoto 869–1404, Japan
Received April 8, 2016; accepted April 27, 2016
Summary An in vitro technique was connected with application to produce colchicine-induced hexaploid from
the artiﬁcial-crossing triploid hybrid of Drosera rotundifolia and D. spatulata. The colchicine-treated plants
of artiﬁcial-crossing triploid hybrid showed morphologically mutated characteristics. Three hexaploid strains
by chromosome doubling were produced after screening of clones treated with 0.05 and 0.1% colchicine solu-
tions for one or three days. Each of the strains had 2n=60 with 20 middle size and 40 small size chromosomes
(2n=6x=20M+ 40S). The stomata guard cell sizes of colchicine-induced hexaploid and the wild hexaploid
species D. tokaiensis were larger than that of the artiﬁcial-crossing triploid hybrid. Flow cytometry analysis
showed that 2C DNA-values of D. rotundifolia (2n=2x=20M), D. spatulata (2n=4x=40S) and D. tokaiensis
(2n=6x=20M+ 40S) were 2.73, 1.41 and 3.74 pg, respectively. In contrast, the artiﬁcial-crossing triploid hybrid
(2n=3x=10M+ 20S) and colchicine-induced hexaploid (2n=6x=20M +40S) were 2.31 and 4.41 pg, respectively.
The genome size of the artiﬁcial-crossing triploid hybrid (2.31 pg) was nearly half the size of the colchicine-
induced hexaploid (4.41 pg). Compared to the colchicine-induced hexaploid, the genome size of D. tokaiensis
was unexpectedly small, even though they contained the same genome constitutions.
Key words Colchicine, Drosera, Hexaploid, Genome size estimation, Triploid hybrid.
Carnivorous plants are extraordinary plants because
of their highly specialized morphological characters
for insect trapping (Rivadavia et al. 2003, Hoshi et
al. 2010). The family Droseraceae, which is one of
the representative carnivorous plants, consists of three
genera: Aldrovanda, Dionaea and Drosera (Rivadavia
et al. 2003, Shirakawa et al. 2011). In contrast to the two
monotypic genera Aldrovanda and Dionaea, the Drosera
consists of more than 150 species with extensive genetic
diversity and has useful compounds, which are natural
sources of medication (Banasiuk et al. 2012). It has
been reported that some Drosera species have revealed
anti-inﬂammatory and antibacterial effects. Moreover, a
traditional medicine Drosera Herba, which is comprised
mostly of D. rotundifolia, has been used for the treat-
ment of pertussis or whooping cough (Fukushima et al.
Three Japanese Drosera species are useful mod-
els to study genome organization for the trapping
system to capture insects (Dixon et al. 1980, Zhang
et al. 2010), inﬂammatory effects (Fukushima et al.
2009) and complexity of genetic diversity with chro-
mosome evolution (Shirakawa et al. 2011). Geographi-
ca lly, D. rotundifolia is distributed across the northern
hemisphere, and D. spatulata is distributed mostly in
Southeast Asia including parts of Japan and Australia.
D. tokaiensis is found only in Japan. D. rotundifolia
is the diploid species with middle size chromosomes
(2n=2x=20M), while D. spatulata is the tetraploid
species with small size chromosomes (2n=4x=40S). In
contrast, D. tokaiensis is the amphidiploidal hexaploid
species (2n=6x= 20M+40S) with a hybrid origin
between D. spatulata and D. rotundifolia (Hoshi et al.
Triploid hybrids from D. spatulata and D. rotundi-
folia have been produced by artiﬁcial crossing and are
sterile. Natural triploid hybrids occur occasionally in the
ﬁeld and are usually sterile (Hoyo and Tsuyuzaki 2013).
The fertility of these triploid hybrids can be rescued by
* Corresponding author, e-mail: firstname.lastname@example.org
264 S. Tungkajiwangkoon et al. Cy to log ia 81(3)
chromosome doubling using colchicine (Campos et al.
2009). The induction of polyploids is a valuable tool for
breeding in several plants and discovering mutation in
ornamental crops (Thompson et al. 2010).
Chromosome counting is a direct method of analysis
to determine the ploidy level of plants. On the other
hand, a measurement of guard cell size is a convenient
tool for plant morphology analysis. These methods
make the investigation difﬁcult due to time-consuming
work when dealing with a huge number of individuals is
necessary (Campos et al. 2009). In contrast, ﬂow cytom-
etry analysis is a principal method with high-throughput
analysis for nuclear DNA contents (Cousin et al. 2009).
Although a fast, stable, and easy estimation of ploidy
level has been established in many plants (Ajalin et
al. 2002), accurate determinations of new strains with
chromosome doubling require chromosome counting
methods because detections of chromosomal aberrations
or small number of chimeric-cells per plant are difﬁcult
using ﬂow cytometry. Thus, three methods are necessary
for screening and establishing pure polyploid strains.
Polyploid shows the importance of genetic diversity and
plant evolution (Zahedi et al. 2014).
The aim of the present study is to characterize
the hexaploid strains induced by colchicine treatment
from the artiﬁcial-crossing triploid hybrid strain of
D. rotundifolia and D. spatulata. We initially exam-
ined the effect of colchicine on morphological charac-
teristics, and then screened and identiﬁed the ploidy
level by chromosome counting, stomatal guard cells
measurement, and ﬂow cytometry analysis. Moreover,
established strains of colchicine-induced hexaploids
were compared with the donor triploid hybrid, wild type
species D. tokaiensis, and their parental species.
Materials and methods
The plant materials used were D. rotundifolia L.
(accession No. 010816-sera1), D. spatulata Labill.
(accession No. Jpn Ha4x-6), artiﬁcial-crossing triploid
hybrid (accession No. 10 RS-01) of D. rotundifolia D.
spatulata, and D. tokaiensis (Komiya & C. Shibata)
T. Nakamura & Ueda (accession No. Jpn Ha6x-9).
For the internal standards in measurement of genome
size, Oryza sativa L. Hinohikari and Miscanthus
sinensis Andersson (Sashiki-strain, kindly given by
Kyushu Okinawa Agricultural Research Center, Nation-
al Agriculture and Food Research Organization) were
used. All of the plants used were cultivated in the Labo-
ratory of Plant Environment Science, Department of
Plant Science, School of Agriculture, Tokai University.
Production of new plantlets from leaf explants
Leaf materials of the triploid hybrid (10 RS-01) were
obtained from in vitro tissue culture clones. To induce
the plantlets, young leaves of the triploid hybrids were
cut and cultured on 1/4 B5 liquid medium supplemented
with 2.0% sucrose after transferring.
Hexaploid induction from the triploid hybrid (10 RS-01)
The cultured leaf explants, which produced new
bud, shoots and plantlets, were used and soaked in 0%,
0.01%, 0.05%, 0.1% and 0.5% colchicine solutions for
one, two, and three days (20 plantlets per treatment). Af-
ter that they were cultured on half-strength Murashige–
Skoogs basal medium (Murashige and Skoog 1962)
supplemented with 3.0% sucrose and 0.2% gellan gum at
25C in continuous light condition.
Root tips of treated plants were pretreated with 0.05%
colchicine at 18C for 2 h. Then they were ﬁxed in 45%
acetic acid for 30 min on ice. The root tips were hydro-
lyzed in a mixture of 1 M HCl and 45% acetic acid (2 : 1)
at 60C for 7 s. Root meristems were cut and squashed
in 45% acetic acid. The preparations were air-dried at
room temperature. After removing coverslips, they were
stained with 5 µg mL1 DAPI solution in McIlvaines buf-
fer. The chromosomes stained with DAPI were observed
under a ﬂuorescence microscope with a U ﬁlter. More
than 100 metaphase cells were observed in each strain to
check for chromosomal aber rations and chimeric-cells in
Observation of leaf stomata guard cells
Guard cell lengths and widths of the colchicine-
induced hexaploids were measured on 30 guard cells per
plant species. Guard cell sizes were estimated from the
lower epidermis in the middle parts of the leaves. Guard
cell length and width measurements were carried out us-
ing Image J software (ver. 1.48v).
Flow cytometry (FCM) analysis
Some in vitro leaves of strains of Drosera used in this
study were chopped in propidium iodide (PI) chopping
buffer containing 1% Triton X-100 (v/v), 140 mM 2-mer-
captoethanol, 0.5 M Na2SO3, 0.5 M Tris–HCl (pH 7.5),
0.2 mM MgCl2 and 20 mg mL1 polyvinylpyr rolidone
and 1 mg mL1 PI. Afterward, the samples were ﬁltered
through a 48 µm nylon mesh, and then the samples were
incubated for 5 min and stained with PI chopping buffer.
The DNA content was measured by using FCM (Guava
EasyCyte™ 12 HT Flow cytometer). Oryza sativa
(1C= 0.5 pg calculated from Bennett and Smith 1976)
and Miscanthus sinensis (1C= 2.65 pg calculated from
Nishiwaki et al. 2011) were used to calculate genome
size in absolute units.
2016 Characterization of Colchicine-Induced Hexaploid Drosera 265
Results and discussion
Survival rate after colchicine treatment
A few treatments caused phytotoxic effects. The sur-
vival rate was recorded after 60 d. The control group
had the highest survival rate. The results showed that
as the colchicine concentration increased, the survival
rate decreased with exposure time under the same condi-
tion (Table 1). The highest lethality was observed in the
treatments with 0.5% colchicine solution for three days.
Toxic effects of colchicine were correlated with concen-
tration and duration. It was described in a previous re-
port that long soaking periods in a higher dose of colchi-
cine solution negatively inﬂuenced the vigor and rooting
of the plantlets (Trojak-Goluch and Skomra 2013).
Changes in morphological characteristics
Mutation characteristics of the artiﬁcial-crossing trip-
loid hybrid are shown in Fig. 1. Colchicine treatment
with various concentrations for 60 d resulted in some
leaves changing from a simple to heart-shape when
treated with 0.1% colchicine for three days (Fig. 1A).
Rarely, in the same treatment condition, the separation
at the basal part of the leaf blade was observed (Fig. 1B).
Treatment with 0.1% colchicine for one day resulted in
some of the leaves changing into a bell-shape (Fig. 1C).
Colchicine treatment affects not only chromosome
doubling, but also morphological changes such as leaf
shape. The phenomena of leaf changes, including size,
margin, double leaves and forked apexs of leaves, were
previously described (Obute et al. 2007). The possible
causes of leaf-morphological changes might be due to
chromosomal instability, reduction of auxin level, activ-
ity of enzymatic change, and variations in ascorbic acid
dose (Obute et al. 2007).
Frequency of polyploid
Using FCM, mixoploid plants could be detected after
colchicine treatment. The highest mixoploid score was
20% in 0.05 and 0.5% treatment for one day. These
mixoploids might be a mericlinal chimera, having cells
with different chromosome numbers in one meristem,
or they may be a periclinal chimera, having the same
ploidy cells in parallel layers in one plant (Harbard et
al. 2012). We isolated the pure synthetic hexaploid from
several mixoploid clones and established three colchi-
Table 1. Survival rate and frequency of poly ploidization of arti ﬁcial-crossing tr iploid hybrid 60 d after colchicine treatment.
Total number of
after 60 d (%)
0 1 20 95 0 0 0 0
2 20 90 0 0 0 0
3 20 85 0 0 0 0
0.01 1 20 85 0 0 0 0
2 20 55 0 0 0 0
3 20 75 0 0 0 0
0.05 1 20 70 4 0 20 0
2 20 85 1 0 50
3 20 85 1 1 5 5
0.1 1 20 85 1 1 5 5
2 20 85 1 0 50
3 20 75 3 0 15 0
0.5 1 20 55 4 0 20 0
2 20 70 1 0 50
3 20 20 0 1 0 5
Fig. 1. Morphological characteristic changes 60 d after colchicine treatment. Heart-shaped leaf (A), double-headed (two leaf
bla de) leaf (B) and bell-shaped leaf (C) were observed in the 0.1% colchicine treatment for one day (C) and three days
(A and B). Arrows ind icate ty pical leaf. Bar=1 cm.
266 S. Tungkajiwangkoon et al. Cy to log ia 81(3)
cine-induced hexaploid strains (Tables 1, 2).
Cytological analysis revealed that the artiﬁcial-cross-
ing triploid hybrid possessed a chromosome number of
2n=30 (2n=3x=10M+20S) (Fig. 2a). Of all 300 explants,
only three hexaploid strains, No. 63-Santhita-01-1-3,
No. 243-Santhita-005-3-3, and No. 283-Santhita-05-3-3,
were induced under treatment of 0.1% colchicine for
one day, 0.05% for three days, and 0.5% for three days,
respectively. These strains possessed chromosome num-
ber of 2n=60 (2n=6x= 20M+40S) (Fig. 2b) without any
Comparison of leaf morphology
Ninety days later after colchicine treatment, the col-
chicine-induced hexaploids were morphologically com-
pared with the artiﬁcial-crossing triploid hybrid and
D. tokaiensis. The leaves of the triploid hybrid tend to
be rounded, while the leaves of the colchicine-induced
hexaploids and wild hexaploid species D. tokaiensis took
similar shapes, which were intermediate between round
and spatulate shapes (Fig. 3). On the other hand, the leaf
blade length of the colchicine-induced hexaploids was
shorter than that of D. tokaiensis. Moreover, the leaves
of the colchicine-induced hexaploids were darker, thick-
er and wider than those of the artiﬁcial-crossing triploid
hybrid. Aditionally, the neighboring tentacles of the
colchicine-induced hexaploids were thicker than those
of the artiﬁcial-crossing triploid hybrid (Fig. 3). Usually,
polyploid has higher chloroplast number than diploid,
and thus the total of photosynthetic cells per unit leaf
area consistently reduced with rising ploidy from diploid
to hexaploid (Murti et al. 2012). Polyploid plants typi-
cally tend to have darker green leaves, thicker stems, and
an expanded width/length ratio of leaves (Nonaka et al.
2011). Thus, the general trend of morphological changes
caused by polyploidization was seen in genus Drosera.
Evaluation of stomata guard cell size
The effect of colchicine treatment on stomata guard
cell length and width was investigated among the
artiﬁcial-crossing triploid hybrid, colchicine-induced
hexaploid and wild hexaploid species D. tokaiensis. The
stomata guard cell sizes of colchicine-induced hexaploid
and the wild hexaploid species D. tokaiensis were larger
than that of the artiﬁcial-crossing triploid hybrid (Fig.
4, Table 3). Interestingly, the stomata guard cell width
of the colchicine-induced hexaploid was signiﬁcantly
wider than that of D. tokaiensis, while the stomata guard
cell length of D. tokaiensis was signiﬁcantly longer than
that of the colchicine-induced hexaploid (Fig. 4, Table 3).
Our previous paper demonstrated that the hexaploid D.
tokaiensis had amphidiploidal hybrid origin between D.
spatulata and D. rotundifolia (Hoshi et al. 2010). Since
the artiﬁcial-crossing triploid hybrid was made from D.
spatulata and D. rotundifolia, the genome constitution
of the colchicine-induced hexaploid is the same as D.
Table 2. Chromosome nu mber and genome size of three Japanese Drosera species a nd two new bred strains.
Kar yoty pe
Present d atab
2C (pg) DNA amount of
D. rotundiﬂoria 010816 Sera-1 20 2x20M 2.73 2745
D. spatulata Jpn Ha4x-6 40 4x40S 1.41 1549
D. tokaiensis Jpn Ha6x-9 60 6x20M+ 40S 3.74 3784
Triploid hybrid 10 RS-01 30 3x10M +20S 2.31 2265
Induced hexaploid 63, 243 and 283 60 6x20M +40S 4.41 4324
a M: middle size chromosome, S: small size ch romosome (see Hoshi and Kondo 1998). bGenome size was calculated from the equivalence
1 pg=980 Mbp (Bennett et al. 2000, see Dolezel et al. 2003 for the calculation).
Fig. 2. Fluorescence staining of mitotic-metaphase chromosomes with DAPI. A. Artiﬁcial- crossing triploid hybrid (2n=3x=
10M+20S). B. Colchicine-i nduced hexaploid (2n=6x=20M +40S). Bar = 5 µm.
2016 Characterization of Colchicine-Induced Hexaploid Drosera 267
tokaiensis. Thus, the present results of guard cell sizes,
including leaf shape, showed unexpected morphological
differences between colchicine-induced and wild type
hexaploids, suggesting existence of variance within the
hexaploid genome constitution between them. Indeed,
a wild habitat where hybrids occur spontaneously is
considered to have environmental selection gradients
of genetic diversity and morphological composition of
populations (Hamilton and Aitken 2013).
2C DNA-value estimation by FCM
The present study showed that the 2C DNA value of
the Japanese strain of D. rotundifolia (2n=2x=20M)
was 2.73 pg (Fig. 5, Table 2). This result strongly sup-
ported the previous data of Greilhuber (2008). In
contrast, the 2C DNA value of Japanese D. spatulata
(2n=4x=40S) was 1.41 pg (Fig. 5, Table 2). The 2C
DNA value of the artiﬁcial-crossing triploid hybrid
(2n=3x=10M+20S) between D. rotundifolia and D.
spatulata was 2.31 pg, while that of the colchicine-
induced hexaploid (2n=6x=20M+ 40S) made from the
artiﬁcial-crossing triploid hybrid by chromosome dou-
bling treatment was 4.41 pg (Fig. 6, Table 2). Thus, the
observed 2C DNA value of the artiﬁcial-crossing triploid
(2.31 pg) was close to the expected hybrid 2C DNA value
(2.07 pg), calculated by a sum of haploid genomes of D.
rotundifolia (2.73 pg) and D. spatulata (1.41 pg). The
genome size of the artiﬁcial-crossing triploid hybrid
Fig. 3. Leaves of 60- d (A– C) and 90-d (D–F) old artiﬁcial-crossing triploid hybrid (2n=3x= 10M+ 20S) (A and D), colchicine-
induced hexaploid (2n=6x=20M+ 40S) (B and E), and D. tokaiensis (2n=6x=20M+ 40S) (C and F). Bar=5 mm.
Fig. 4. Comparison of the stomata guard cell size of triploid hybrid (2n=3x=30) (A), artiﬁcial hexaploid (2n=6x=60) (B) and D.
tokaiensis (2n=6x=60) (D). Bar= 20 µm.
Table 3. Effect of colchicine concentration and durat ion period on
stomata guard cell size.
Mean stomata guard cell
Length SD (µm) Width SD ( µm)
D. tokaiensis 44.42 6.91c22.474.65a
Triploid hybrid 23.214.75a21.313.29a
Induced hexaploid 30.464.17b30.872.56b
a–cDifferent letters within the same colum ns indicate statistically
signiﬁcance difference usi ng the Duncans test at 95% (p<0.05).
268 S. Tungkajiwangkoon et al. Cy to log ia 81(3)
(2.31 pg) was nearly half size of the colchicine-induced
hexaploid (4.41 pg). Thus, these genome-size estimations
indicated an additive cytogenetic effect of DNA contents
from parental species and triploid donor plant. In allo-
polyploid, these doubling of homologous chromosome
segment or gene were established by different donor taxa
in the polyploidy mechanism. The combination of two
different species with related genomes should expect the
genomic additivity related to both parental species (Liu
and Wendel 2002).
There are some interesting reports that the speciation
accompanied with polyploid formation provides both
increase and decrease of DNA amounts in plants (e.g.,
Johnston et al. 2005). The phenomenon of polyploidy
is due to the interspeciﬁc allopolyploidal-hybridization
(Renny-Byﬁeld et al. 2012). It has been proposed that
this formation might cause reproducible, rapid and direc-
tional changes to their parental sub-genomes (Feldman
and Levy 2009). Compared to colchicine-induced hexa-
ploid (4.41 pg), the genome size of D. tokaiensis (3.74 pg)
was unexpectedly small (Figs. 5, 6, Table 2), even
though they contained the same genomic constitutions.
Non-additive change and DNA loss in genome size seem
to be due to the genomic stress after allopolyploidal hy-
bridization, which has already been discussed in various
taxa such as Tr i ticu m , Gossypium, and Brassica (Ozkan
et al. 2003). As is the case with genome upsizing, the
downsizing effect might be also associated with transpo-
son elements (TEs) (Fedoroff 2012). The TEs including
retrotransposons contain many repeats in the genome.
The mechanism of TEs could efﬁciently increase or per-
haps decrease in genome size (Bennetzen et al. 2004). In
the case of DNA loss, unequal intrastrand homologous
recombination between these repeats might affect the net
loss of native DNA and could reduce a certain amount
of the repeated DNAs (Devos et al. 2002). In Arabidop-
sis, the genome had shared a surge of retrotransposon
ampliﬁcation in a short time (Devos et al. 2002). It has
been expected that genome size decreases through re-
combinant mechanisms. One of the possible mechanisms
is an existence of solo LTRs, which is a single long
terminal repeat sequence making large-size delete pos-
sible in the genome (Zhou and Cahan 2012). Recently,
solo LTRs were also found in vascular plant species that
contain many retrotransposons (SanMiguel and Vitte
2009). Unequal homologous recombination between two
LTRs happened in Oryza sativa (Vitte and Panaud 2003)
and was similarly reported in Arabidopsis (Devos et al.
2002). However, solo LTRs are rarely found in Zea mays
(Vitte and Panaud 2003). These reports suggested that
solo LTR recombination or a similar mechanism might
occur in Drosera genome.
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