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Breeding and Cytogenetic Characterizations of New Hexaploid Drosera Strains Colchicine-Induced from Triploid Hybrid of D. rotundifolia and D. spatulata


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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 solutions 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 colchicineinduced 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.
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© 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:
DOI: 10.1508/cytologia.81.263
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
Plant materials
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.
Chromosome observation
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
hexaploid strains.
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.
Concentration of
colchicine (%)
Duration period
Total number of
explant s
Survival rate
after 60 d (%)
Number of
Number of
Frequency of
mixoploid (%)
Frequency of
hexaploid (%)
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).
Chromosome counting
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.
Species Accession
number (s)
number (2n)
Kar yoty pe
Present d atab
2C (pg) DNA amount of
2C (Mbp)
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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... The procedure of colchicine treatment was employed following the protocol of Tungkajiwangkoon et al. (2016). Adventitious buds were produced from young leaves by tissue culture in 1/2 MS liquid medium supplemented with 1.0% sucrose after transferring. ...
... Charoenwattana (2014) used colchicine to produce tetraploids from plantlet in vitro of the diploid D. spatulata (2n= 20), and obtained the chromosome-doubled strains treated with lower concentrations (6 or 12 mg L 1 ) for longer period (7 d). In contrast, Tungkajiwangkoon et al. (2016) created the colchicine-induced hexaploids from adventitious buds generated on leaf explants in vitro of the triploid hybrid of D. rotundifolia (2n=20) and the tetraploid D. spatulata (2n= 40), by treating with high concentrations (50 or 100 mg L 1 ) for short periods (1 d or 3 d). The concentration, duration, and explant type for the mitosis inhibitor treatment is a key to success for polyploidy induction (Kazi et al. 2015, Eng and Ho 2019), due to toxic effects of the treatment (Trojak-Goluch and Skomra 2013). ...
... Even though we could simply speculate the monomodal forming in D. anglica by adding or subtracting the similar 2C values of D. linearis and D. rotundifolia, the fact of bimodal karyotype formation in hybrid without any exception made us difficult to conclude same sizes of chromosomes or genomes between D. linearis and D. rotundifolia. A genome size reduction after speciation has been discussed in some plant species with amphiploid origin (Ozkan et al. 2003), including in Drosera (Tungkajiwangkoon et al. 2016). Tungkajiwangkoon et al. (2016) compared the genome sizes between wild amphidiploidal species D. tokaiensis and the artificial hexaploid created by chromosome doubling of triploid hybrid between D. rotundifolia and D. spatulata Labill. ...
Artificial tetraploid and octaploid strains were induced from the wild species of Drosera rotundifolia (2n=2x=20) and D. anglica (2n=4x=40), respectively. The optimal condition of colchicine-treatments for polyploid inductions was determined first. A flow cytometry (FCM) analysis showed that the highest mixoploid score of D. rotundifolia was 20% in the treatment of 0.3% for 2 days (d), or 0.5% for 3 d, while the highest mixoploid score of D. anglica was 20% in the treatment of 0.5% for 2 d. Next, to remove chimeric cells, adventitious bud inductions were carried out using the FCM-selected individuals in both species. One strain from a total of 360 colchicine-treated leaf explants in each species had pure chromosome-double numbers of 2n=40 (tetraploid) in D. rotundifolia and 2n=80 (octaploid) in D. anglica. In both species, the guard cell sizes of the chromosome-doubled strains were larger than those of the wild types. The leaves of the chromosome-doubled strains of D. rotundifolia were larger than those of the wild diploid D. rotundifolia, while the leaves of the chromosome-doubled strains of D. anglica were smaller than those of the wild tetraploid D. anglica.
... C-values of 11 species in the Droseraceae have previously been recorded (Rothfels and Heimburger 1968, Greilhuber 2008, Shirakawa et al. 2011, Veselý et al. 2012, Jensen et al. 2015, Tungkajiwangkoon et al. 2016. In this study, C-value data of D. anglica, D. burmannii and D. communis were measured for the first time. ...
... A similar value of 1.76 pg was obtained from flow cytometer measuring data by Shirakawa et al. (2011). On the other hand, Greilhuber (2008) and Tungkajiwangkoon et al. (2016) determined a 2C-value of 2.73 pg for this species, which is the same as our present data. Greilhuber (2008) mentioned that C-values in the Droseraceae published by Rothfels and Heimburger (1968) using an improved Feulgen method are likely underestimated due to tannin error, since secondary metabolites made flow cytometric determinations as well as Feulgen measurements problematic for genome size estimation. ...
2C-values of nine species of the genus Drosera, and two monotypic genera Aldrovanda and Dionaea were estimated to provide an overview of the genome diversity and chromosome differentiation in the Droseraceae. The measured DNA contents of all species used in this study ranged over nine-fold from 2C=0.63 pg in D. burmannii to 5.67 pg in D. anglica. In the genus Drosera, even though the polyploid species were excluded, the difference of the 2C DNA contents among diploid species was still high, ranging 4.3-fold from 0.66 pg in D. spatulata to 2.85 pg in D. intermedia. In subgenus Drosera, especially the polyploidal group, two chromosome types were identified by means of their size; this therefore made it possible to discriminate two groups of the genomes: one group was of a smaller genome size (S genome group) consisting of a total of 10 small-sized chromosomes (x=10s), and the other group was of a larger genome size (M genome group) consisting of a total of 10 middle-sized chromosomes (x=10 m). The Cx-value of the S genome group was less than 0.4 pg (ca. 400 Mbp). On the other hand, the Cx-value of the M genome group showed a range of 1.3–1.5 pg (1270–1470 Mbp). Moreover, the 2C DNA content of the hexaploid species D. tokaiensis (2n=6x=20 m+40s, 2C=3.57 pg), which originated from naturally occurring interspecific hybridization event between D. rotundifolia (2n=2x=20 m, 2C=2.73 pg) and the tetraploid D. spatulata (2n=4x=40s, 2C=1.38 pg), was less (86.9%) than the sum of their putative parental species.
... Although 1Cx of 495 Mbp was estimated both in the diploid and tetraploid B. gigantea with the highest DNA amount in all species, 1Cx of the tetraploid B. filifolia was 1.3 times larger than that of diploid, and contrariwise 1Cx-values of B. guehoi and B. rorida were smaller than those of other species, being supported in our chromosome observation (Fig. 1). The speciation accompanied by genome size-changing both increase and decrease in DNA amounts have been known in plants (e.g., Johnston et al. 2005, Tungkajiwangkoon et al. 2016. As a consequence, the genus Byblis has a wide range of genome sizes, seeming to arise through not only natural polyploidization events after a chromosome insertion/deletion but also genome size changes keeping chromosome uniformity as a monomodal karyotype. ...
Chromosome numbers and 2C-values of six Byblis species were determined to investigate the chromosome differentiation and genome diversity in this genus. Somatic chromosome numbers of the B. liniflora complex showed the diploid and the tetraploid numbers of 2n=16 and 32. In contrast, the chromosome number of B. gigantea, which belongs to the B. gigantea complex, displayed only the tetraploid numbers of 2n=36. The 2C DNA contents across all species studied were over a three-fold range, from the lowest value of 2C=0.64 pg in B. rorida to the highest value of 1.93 pg in B. gigantea. Since two cytotypes with different ploidy levels were seen in B. filifolia, the 2C contents of 0.70 pg and 1.81 pg were measured in the diploid (2n=2x=16) and the tetraploid (2n=4x=32), respectively. Haploid genome sizes of the tetraploid B. filifolia were, thus, 1.3 times larger than that of the diploid. The lowest haploid value of 1Cx=271 Mbp was shown in B. guehoi.
... The verification of the identity of individual species using DNA molecular analysis is a crucial step for the genetically related individual Drosera species that have similar morphological characters [Tungkajiwangkoon et al. 2016] that may be slightly altered from in vitro cultivation [Batagin et al. 2009, Trejgell et al. 2012]. In our case, this type of molecular verification of individual carnivorous species precedes their further detailed characterisation in respect of molecular processes involving the insect prey digestion. ...
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Drosera L. is a genus of carnivorous plants that comprises approximately 250 species, although this number is probably not complete. Some of these taxa exhibit only small differences in morphological traits that can be partly influenced if the taxa are propagated in vitro. Here, we focus on the verification of putative clones of Drosera spathulata Labill., Drosera rotundifolia L. and Drosera binata var. Dichotoma species cultivated in vitro using molecular markers covering the internal transcribed spacer (ITS) region of 45S ribosomal DNA (rDNA). Following the polymerase chain reaction (PCR) amplification of ~360-bp DNA fragments and sequencing, the sequences were aligned with corresponding sequences in the National Center for Biotechnology Information (NCBI) database. In addition, each of tested PCR amplicons had a specific restriction profile that predominantly enables the differentiation of D. rotundifolia and D. spathulata; the shape of the leaves does not have to be a clear morphologically distinguishable trait.
The chromosome number and karyotype analysis of ten taxa of genus Tanacetum found native to Turkey were examined in detail. Somatic chromosome numbers were determined as follows: 2n=18 in T. balsamitoides Sch.Bip., T. parthenifolium Sch.Bip., T. zahlbruckneri (Náb.) Grierson, T. densum (Labill.) Sch.Bip. ssp. laxum Grierson, T. densum (Labill.) Sch.Bip. ssp. amani Heywood, T. argenteum (Lam.) Willd. ssp. argenteum, T. tomentellum (Boiss.) Grierson, 2n=36 in T. nitens (Boiss. & Noë) Grierson, T. polycephalum Sch.Bip. ssp. argyrophyllum (K.Koch) Podlech, T. abrotanifolium (L.) Druce. It was observed that chromosomes of Tanacetum taxa, of which karyotype analyses were examined, have generally median (m), submedian (sm), and rarely median point (M), subterminal (st) centromeres. In T. parthenifolium, T. nitens, T. abrotanifolium and T. argenteum ssp. argenteum; one pair of satellite chromosomes was observed.
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The aim of this study was to develop a protocol for production of polyploid M1-generation plants of Viola × wittrockiana Gams. Two variants of colchicine treatment were compared for their efficiency. Early detection of novel ploidy levels was achieved by screening of stomata size, leaf index value (leaf blade length/width), and other morphological characteristics of the M1-generation. Secondary screening for novel ploidy levels was performed by flow cytometry (FCM). Hexadecaploid, aneuploid, and mixoploid plants were successfully identified by FCM.
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Background: Ploidy manipulation is considered an efficient method to increase production potential of medicinally important compounds. Dracocephalum kotschyi Boiss. is an endangered medicinal plant of Iran. Various concentrations of colchicine (0.05, 0.10, 0.20, and 0.50% w/v) were applied to shoot apical meristems of D. kotschyi seedlings in two and four-leaf stages to induce tetraploidy. Results: According to the results, 0.5% (w/v) of colchicine can be effective for polyploidy induction in D. kotschyi. Putative tetraploids were selected by morphological and microscopic characteristics and their ploidy level was confirmed by flow cytometry analysis and chromosome counting. The chromosome number of original diploid plant was confirmed to be 2n = 2× = 20 whereas that of the tetraploid plant was 2n = 4× = 40. Tetraploid and mixoploid plants showed different morphological, physiological and microscopic characteristics from those of diploid counterparts. The total content of flavonoids was increased from 1583.28 in diploids to 1890.07 (μg/g DW) in stable tetraploids. Conclusion: High-Performance Liquid Chromatography with Diode-Array Detection (HPLC-DAD) confirmed over accumulation of methoxylated hydroxyflavones in solid tetraploid plants of D. kotschyi.
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Polyploidy represents a useful tool for increasing marketability of floriculture crops. The efficacy of 250 μMcolchicine [0.01% (w/v)] as a means of inducing polyploidy in six South African Watsonia species (W. borbonica subsp. ardernei, W. humilis, W. laccata, W. lepida, W. pulchra, and W. vanderspuyiae), as determined through highresolution flow cytometry, is reported. Exposure to colchicine during imbibition and as 24-, 48-, or 72-h pulse treatments for in vitro germinated seeds resulted in seedlings with increased ploidy, reaching a maximum of 60% induction after the 72-h pulse treatment. The greatest proportions of induced individuals from both the pre- and post-germination exposure treatments were of mixed ploidy. These mixoploids were induced in five species. Non-chimeric tetra- and octaploids were produced in low frequencies only for W. vanderspuyiae during radicle-pulse exposure of 24 and 48 h. Increasing colchicines exposure at radicle emergence manifested as aberrant phenotypic expression and was typified by a reduction in leaf length and rooting capacity in vitro coupled with overall slowed growth. In vitro regeneration and multiplication is easily achievable for the genus and should allow for the capture and refinement of desirable polyploidy tissues.
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Australian acacias are widely planted as exotics and in some cases as invasive. Impact may be reduced if sterile triploid planting stock can be developed. This article reports the first step in such a breeding programme, the production of a population of tetraploid lines for inter-breeding with diploids. Three methods of polyploid induction with colchicine were compared. A conversion rate of 8.9 per cent was obtained by applying 1.5 per cent colchicine to the shoot apical meristem of seedlings. A 7 per cent conversion rate was obtained by germination of scarified seed on filter paper saturated with 0.02 per cent colchicine for 16 h and this method is recommended on logistical and safety grounds. Poor results were obtained when scarified seed were submerged in aqueous solutions of colchicine. Flow cytometry is the preferred method for ploidy determination, sampling after vegetative phase change on a minimum of two opposing phyllodes per plant. Visual classification was inaccurate due to the confounding effects of growth-retarding properties of colchicine. Size and distribution of stomata can also be used but is more time consuming than flow cytometry. At 26Â months, tetraploid plants had heavier, thicker, wider and more cupped shaped phyllodes than diploids and the bark: stem diameter ratio was greater.
The Drosera chromosomes studied were not localized-centromeric but diffused-centromeric. Among the Drosera taxa studied, only D. filiformis (2n=20) had 20 weak CMA-negative orDAPI-positive large bands more than 2.97 μm2 area each at the same site of 20 chromosomes. Thus, its hybrid with D. intermedia (=Drosera × hybrida, 2n=20) had ten CMA weakly-negative orDAPI-positive bands more than 3.00 μm2 each in the same site of ten chromosomes which could be of the gamete of D. filiformis. Drosera petiolaris (2n=14) displayed the meiotic chromosome configuration of six circular bivalents with four distinct chromatids held each other with end-to-end association and two univalents at metaphase I, while D. rotundifolia (2n=20) displayed ten ring-shaped bivalents, that were quite similar to the bivalents of the localized-centromeric chromosomes. These differences in CMA-positive and DAPI-negative bands, IBAS-area, and meiotic chromosome behavior might be correlated with chromosome differentiation and speciation in the species of Drosera of the Northern and the Southern Hemispheres.
Using sequential fluorescent staining method and fluorescence in situ hybridization (FISH) technique, karyomorphological and molecular cytogenetic investigations of two centromere differentiating Drosera species, D. arcturi and D. regia were carried out. Drosera arcturi had chromosome number of 2n = 58, while D. regia had chromosome number of 2n = 34. Many chromosome bands stained with CMA positive and DAPI positive (CMA+DAPI+) were the most common in both species. CMA positive and DAPI negative (CMA+DAPI–) sites were shown in two chromosomes of both species. Four sites stained with CMA+DAPI–appeared on both sides of the constrictions of two larger chromosomes in D. arcturi, while two CMA+DAPI–sites appeared at terminal positions of two chromosomes in D. regia. Two-color FISH of 5S and 45S rDNAs showed two regions with major 45S rDNA signals in the both species, and four sites with clear 5S rDNA signals in D. arcturi. Drosera arcturi did not show any primary constriction in all chromosomes, except for two larger chromosomes. In contrast, D. regia had localized-centromeric position or well-differentiated primary constrictions in most metaphase chromosomes.