Effect of Colchicine Tablets on Morphology of Torenia fournieri

2013 International Transaction Journal of Engineering, Management, & Applied Sciences & Technologies.

International Transaction Journal of Engineering,
Management, & Applied Sciences & Technologies
http://TuEngr.com

Effect of Colchicine Tablets on
Morphology of Torenia fournieri
a

Sasiree Boonbongkarn , Thunya Taychasinpitak
a
b
Shermarl Wongchaochant , and Shinji Kikuchi

a*

,

a

Department of Horticulture, Faculty of Agriculture, Kasetsart University, Jatujak, Bangkok 10900.
THAILAND
b
Graduate School of Horticulture, Chiba University, Chiba, JAPAN
ARTICLEINFO

ABSTRACT

Article history:
Received 02 August 2013
Received in revised form
06 September 2013
Accepted 10 September 2013
Available online
12 September 2013
Keywords:
Polyploid induction;
Cromosome;
Flow cytometry;
Morphological characteristics;
Cytological characteristics.

The effects of colchicine tablets on Torenia fournieri were
studied. Leaves were cut and soaked in different concentrations of
colchicine solution: 0, 5, 10, 15 and 20 ppm for 0, 1, 2 and 3 days.
The survival rate decreased when colchicine concentration and
treatment duration were increased. The stomata length was found to
be greater in the putative polyploids. Flow cytometric analysis
demonstrated that the nuclear DNA of putative polyploid Torenia
plants was doubled relative to that of control diploid plants, and
microscopy results confirmed that the chromosome number of the
tetraploid plants was 2n = 4x = 36. The highest frequency of
tetraploid induction was 6.67% at 15 ppm of colchicine solution
soaked for 3 days. Morphological characteristics of tetraploid and
diploid plants were compared. The results showed that growth of
tetraploid plants were less than diploid plants. Tetraploid plants also
had larger leaves and flower sizes when compared with diploid plants.
2013 INT TRANS J ENG MANAG SCI TECH.

.

1. Introduction
Torenia spp. are dicotyledons, belonging to the class Magnoliopsida, order Scrophulariales
and family Scrophulariaceae, known as the ‘figwort family.’ The Scrophulariaceae comprise
306 genera and approximately 5850 species native to South East Asia, Africa and Madagascar.
Yamazaki (1985) reported a total of 50 Torenia species, 20 species from Cambodia, Laos and
*Corresponding author (T. Taychasinpitak) Tel: +66-2-579-0308 Fax: +66-2-579-1951. E-mail
address: agrtyt@ku.ac.th.
2013 International Transaction Journal of Engineering,
Management, & Applied Sciences & Technologies. Volume 4 No.4
ISSN 2228-9860
eISSN 1906-9642. Online Available at http://TuEngr.com/V04/299-309.pdf

299

Vietnam, and 19 species from Thailand. Other reports indicate 40 species of Torenia (Fischer
2004; Spencer 2006). Torenia spp. have an extended history of cultivation and safe use as an
ornamental plant and are usually found in nurseries, home gardens and landscaping situations.
Torenia have been grown as a front border plant especially in lightly shaded areas and are
popularly grown in hanging baskets or as trailing specimens in patio planters (Starman, 2005).
Flowers of Torenia fournieri are reportedly edible and can be used as salad material (Shindu et
al., 2008). Torenia fournieri (2n = 18) has also been used to study the location and movement
of chromosomes and their centromeres in the early stages of embryogenesis in interspecific
hybrid plants (Kikuchi et al., 2005).

Torenia is generally a diploid plant. The basic

chromosome number varies with species. Tetraploids can be induced by colchicine treatment
of young seedlings and have relatively large flowers, offering potential for developing better
horticultural varieties. However, these tetraploids exhibit significant reduction in pollen
viability, seed setting and unequal distribution of chromosomes at anaphase when compared to
their diploid progenitors (Tandon & Bhutani, 1965).
Inducing polyploidy is an effective means for the generation of innovative germplasm
resources suitable for selective breeding. In general, polyploid plants have larger leaves and
flowers, and autopolyploids tend to be less fertile.

Previously unsuitable habitats can

sometimes be better tolerated by polyploids, compared to the original diploids (Hancock,
1997). Polyploids are superior to diploids in terms of a higher genetic adaptability and
improved levels of resistance or tolerance to environmental stresses (Shao et al., 2003).
Colchicine treatment is the classical method to induce doubling of chromosome number.
Jensen (1974) reported the techniques used for chromosome doubling of barley haploid with
colchicine. Preliminary morphological screening for putative polyploids stated that stomata
length was the accurate indicator of polyploidy level in many plants. Wright (1976) also
showed that stomatal measurement was a quick way to determine whether or most of the leaves
on a branch were polyploidy.
The research objectives were to study the optimum concentration and duration of
colchicine solution exposure for polyploid induction in Torenia fournieri and to study the effect
of colchicine solution from colchicine tablets on morphological and cytological characteristics
and chromosome number variation of Torenia fournieri.

2. Materials and Methods
Purple-flowered

300

native

Thai

Torenia

(Torenia

fournieri)

with

a

semi-erect,

S. Boonbongkarn, T. Taychasinpitak, S. Wongchaochant, and S. Kikuchi

semi-recumbent habit was the subject for this research. Polyploidy was induced using
colchicine from medicinal tablets for treating gout manufactured in Switzerland and purchased
at a pharmacy in Thailand. Each tablet contains 0.6 mg colchicine along with lactose,
magnesium stearate and starch.

2.1 Polyploidy Induction
Leaves were cut from native Torenia fournieri and for the different treatments, the petioles
were soaked in 0, 5, 10, 15 and 20 ppm colchicine solution for 0, 1, 2 and 3 days (30 leaves per
treatment), after which they were placed upright in peat moss for rooting. Once roots were
established, the leaf cuttings were transferred to 4-inch pots containing a mixture of sand, rice
husk charcoal, coconut coir, coconut husk chips, and manure

1:1:1:1:1/2. Slow-release

fertilizer of formula 14-14-14 was added at the rate of 5 g per pot, and liquid fertilizer of
formula 21-21-21 was also provided once a week at the concentration of 30 g per 20 L of water.
The survival rate was recorded weekly.

2.2 Selection of Putative Polyploids
When the plants had grown for 90 days, putative polyploids were selected based on
morphological characteristics, such as slower growth, darker green leaves, thicker, larger and
rougher leaves, or larger flowers. The stomata size was examined by examining impressions of
the abaxial sides of leaves under a light microscope.

2.3 Flow Cytometry
One young leaf (mature but newly emerged) of Torenia fournieri for each specimen to be
tested was chopped in a Petri dish with 500 µl of Partec CyStain (a one-step extraction and
DAPI stain solution) and filtered through a 30 µm filter before being analyzed in a Partec PAII
flow cytometer.

2.4 Karyotype by DAPI staining
Chromosome preparation with flower bud and the use of FISH for meiotic chromosomes
were performed according to the procedures described in Kikuchi et al., 2008. The slides were
mounted with Vectashield (Vector) containing 5 mg/mL 4’, 6-diamidino-2-phenylindole
(DAPI) for staining of chromosomes. All images were captured with an Olympus BX61
fluorescence microscope equipped with a cooled-CCD camera (Photometrics CoolSNAP
fx:Roper Scientific) and processed using the Meta imaging series 5.0 software (Universal
Imaging Corporation).
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2.5 Comparison of Morphological and Cytological Characteristics of
Diploid and Tetraploid Torenia
Stem cuttings (about 7 cm long) were taken from 5 plants each of the diploid and
confirmed tetraploid native Torenia and placed in peat moss for rooting. After the stem cuttings
were established, they were transferred to 4-inch pots containing a mixture of sand, rice husk
charcoal, coconut coir, coconut husk chips, and manure 1:1:1:1:1/2. Slow-release fertilizer of
formula 14-14-14 was added at the rate of 5 g per pot, and liquid fertilizer of formula 21-21-21
was also provided once a week at the concentration of 30 g per 20 L of water. The experiment
was a Completely Randomized Design (CRD) with 2 treatments (diploid and tetraploid) and 5
replicates. Growth characteristics were recorded, comprising plant height, spread, number of
branches, stem thickness, leaf width, length and thickness and flower characteristics such as
flower width and length and petal thickness.

2.6 Statistical Analysis
Statistical differences among data were tested to 95% level confidence using Duncan’s
New Multiple Range Test (DMRT).

3. Results and Discussion
3.1 Survival Rate Following Colchicine Treatment
After leaves were cut from native Torenia fournieri and the petioles/leaf stalks were
soaked in 0, 5, 10, 15 or 20 ppm colchicine solution for 0, 1, 2 or 3 days, and then placed in peat
moss for rooting, the survival rate was recorded after 90 days. The control group had the highest
mean survival rate, and the survival rate dropped with increasing concentration of colchicine
and increasing exposure time. The survival rate dropped to zero for the 20 ppm concentration
and 3-day exposure time (Table 1). Report the survival rates of colchicine-treated shoots were
lower, with the extent of the reduction depending on the colchicine concentration and treatment
duration (Jiranapapan et al. 2011). This is consistent with the findings of Espino and Vazquez
(1979), who reported that high concentrations of colchicine could cause tissue necrosis when
parts of leaves of African violet were soaked in different concentrations of colchicine solution.
This is because colchicine does not only have an effect on cell division but spreads throughout
the cell, interfering with cellular mechanisms and causing toxicity at high concentrations
(Dermen, 1940). Colchicine apparently affects the viscosity of the cytoplasm so the cell cannot
function normally (Cook and Loudon, 1952).

302


3.2 Selection of Polyploids and Stomata Guard Cell Size
When the leaf cuttings had grown for 90 days, putative polyploids were selected based on
changes in morphological characteristics, such as slower growth, darker green leaves, thicker,
larger and rougher leaves, or larger flowers. When the plants were 120 days old the stomata
size was examined to select those plants with larger stomata than the control. The mean
stomata guard cell length of the putative polyploid plants was 31.43±1.59 µm, which was
longer than the mean of the control group (22.77±0.48 µm) (Table 6 and Figure 2, A and B).
This was in accordance with the findings of Ye et al. (2010) who applied different
concentrations of colchicine for different durations to the shoot tips of 3 varieties of crape
myrtle sprouts during cotyledon stage (‘Zi Wei,’ ‘Hong Wei’ and ‘Yin Wei’ varieties). They
found that the morphological and cytological characteristics of large leaf size, greater leaf
thickness, darker green leaves, larger stomata, lesser stomata density on the abaxial leaf
surface, and more chloroplasts in the guard cells were all indicators that could predict the
presence of tetraploids in the large colchicine-treated population. In another study on
polyploidy induction in Torenia fournieri, colchicine solution at concentrations of 0.05, 0.10,
0.20% were applied to seed-grown seedlings with 4-6 leaves for a duration of 10 hours.
Tetraploids were obtained from the 0.20% colchicine treatment, and they had characteristics
that were deemed valuable for breed development, such as larger flowers, larger leaves, larger
anthers and larger stomata (Tandon and Bhutani, 1965).
Table 1: Survival rate of native Torenia fournieri leaf cuttings 90 days after planting (control
and colchicine treatments)

3.3 DNA Volume Analysis by Flow Cytometry
Flow cytometry was used to measure the DNA volume of putative polyploidy plants that
were selected based on morphological characteristics, such as slower growth, darker green
leaves, thicker and larger leaves and flowers, and larger stomata size. Flow cytometry was
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carried out with a Partec PA II flow cytometer, and the histograms showed that the ploidy level
of some of the colchicine-treated plants had increased from 2n, as in the control, to 4n or
tetraploid (Figure 4). Flow cytometry is now routinely used for ploidy analysis and is regarded
as the most accurate tool for ploidy determination (Loureiro et al., 2005). Advantages of using
flow cytometry to estimate ploidy level include the ease of plant sample preparation, requiring
just a few milligrams of tissue, and multiple samples can be analysed in one working day
(Dolezel, 1997).

3.4 Chromosome Counting
Cytological analysis revealed that the diploid native Torenia in the control group had the
expected chromosome number of 2n = 2x = 18 (Figure 3 A) and the 7 tetraploid plants from the
colchicine treatment had twice as many chromosomes 2n = 4x = 36 (Figure 3B). Previously
reported screened the 14 regenerated plants with morphological characteristics that could
indicate polyploidy In addition, we confirmed that the 14 regenerated plants carried 34
chromosomes Thus, our treatment produced 14 amphidiploid plants from the 600 detached
leaves treated with colchicine (Jiranapapan et al. 2011). However colchicine is an antimitotic
mutagen that has the property of disrupting normal cell division during mitosis by reacting with
microtubule proteins and preventing the formation of spindle fibers, so chromosomes can not
migrate to opposite poles of the cell during anaphase, resulting in tetraploids cells (Elliott,
1958).
Table 2: Frequency of polyploids induced in native Torenia fournieri 90 days after colchicine
treatment
Colchicine
concentration
(ppm)
5

10

15

20

Duration
(days)
1
2
3
1
2
3
1
2
3
1
2
3

Total plants
treated
(plants)
30
30
30
30
30
30
30
30
30
30
30
30

Tetraploids
(plants)
2
1
0
0
1
0
1
0
2
0
0
0

Tetraploid
induction rate
(%)
6.67
3.33
0
0
3.33
0
3.33
0
6.67
0
0
0

3.5 Frequency of Polyploidy
Data on the number of tetraploid plants induced by colchicine treatments at the different
concentrations and different exposure times tested is shown in Table 2. In this research, a total
of 7 tetraploid plants were formed. The most effective treatments were 5 ppm colchicine for 1
304


day and 15 ppm for 3 days, both of which yielded 2 tetraploids, or an induction rate of 6.67%
(Table 2). The highest frequency (8%) of polyploidy induction was seen in the treatment with
15 µmol mol-1 colchicine for 2 days. (Jiranapapan et al. 2011).

Figure 1 Comparison of diploid (left) and induced tetraploid Torenia fournieri (right):
(A) leaves; (B) branches; and (C) flowers.

3.6 Morphological Comparison of Diploid and Tetraploid Native Torenia
When stem cuttings were made from 5 plants each of the diploid and confirmed tetraploid
native Torenia, the growth parameters of plant height, plant width, number of branches, stem
thickness, leaf width, length and thickness were compared. After 90 days the mean height of
the tetraploids plants was less than the diploids, but the mean plant width, leaf length and leaf
thickness of the tetraploids were all greater than the diploids to a statistically significant degree.
There was no statistically significant difference in mean number of branches, leaf width or stem
thickness between the diploid and tetraploid plants (Table 3 and Figure 1A).
Table 3: Comparison of growth parameters of 90-day-old stem cuttings of diploid and
tetraploid Torenia
Mean height

Mean spread

Mean number of branches

Mean stem thickness

Diploid

(cm)
22.40 ± 2.08a

(cm)
29.30 ± 2.17a

(branches)
11.40 ± 3.05

(cm)
1.69 ± 0.11

Tetraploid

13.30 ± 2.16b

20.80 ± 2.84b

13.00 ± 4.8

1.80 ± 0.14

Ploidy

*
*
t-test
Note ns means not statically significant
* means statistically different to the confidence level of 95%
by Duncan ’s New Multiple Range Test (DMRT)

ns

ns

Visual comparison showed that the leaves of tetraploids plants from stem cuttings were
darker green, larger and thicker than the diploid group (Table 4 and Figure 1B). As for the
floral characteristics, at 90 days the flowers on tetraploids were wider than flowers on diploids
to a statistically significant degree. There was no statistically significant difference in mean
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305

flower length or petal thickness between the diploid and tetraploid plants (Table 5 and Figure
3C). This is the consistent with the results of Zhang et al (2010), who induced polyploidy in
the diploid (2n = 24) Cucumis melo pure line ‘M01-3’ through the application of colchicine.
They reported that the tetraploid melon plants were noticeably taller, with larger leaves and
flowers as well as thicker stems. Tetraploids can be induced by colchicine treatment of young
seedlings and have relatively large flowers offering potential for developing better horticultural
varieties. However these tetraploids exhibit significant reduction in pollen viability, seed
setting and unequal distribution of chromosomes at anaphase when compared to their diploid
progenitors (Tandon & Bhutani 1965).
Table 4 Comparison of mean leaf sizes of 90-day-old stem cuttings of
diploid and tetraploid Torenia fournieri.
Ploidy

Mean leaf
width
(cm)

Mean leaf
length
(cm)

Mean leaf
thickness
(mm)

Diploid

3.77 ± 0.15b

0.31 ± 0.05b

0.31 ± 0.04

Tetraploid

4.10 ± 0.13a

0.44 ± 0.04a

0.48 ± 0.11

t-test
*
*

ns

Table 5 Comparison of mean flower sizes of 90-day-old stem cuttings of
diploid and tetraploid Torenia fournieri


Figure 2: Comparison of stomata of diploid Torenia fournieri (A) and
tetraploid Torenia fournieri (B) (bar= 20 µm)
306


Table 6: Stomata size of Torenia fournieri plants that were selected as putative polyploids from
colchicine treatment, 120 days after treatment.
Plant
Control
Selected plant (colchicine concentration –
days – number)
5-1-1
5-1-2
5-2-1
10-2-1
15-1-1
15-3-1
15-3-2

Mean stomata guard cell length ± SD
(µm)
22.77 ± 0.48

28.34 ± 3.82
27.22 ± 0.95
28.61 ± 2.68
29.17 ± 3.01
30.81 ± 0.84
31.43 ± 1.59
31.11 ± 1.27

Figure 3: Comparison of the chromosome number of diploid Torenia fournieri (2n=18) (A)
and an induced tetraploid Torenia fournieri (2n=36) (B) (bar =10µm)

Figure 4: Flow cytometry histograms showing the DNA content of diploid (A) and tetraploid
(B) Torenia fournieri

4. Conclusion
The concentration and duration of colchicine treatment affected the survival rate of native
Torenia fournieri.

Survival rate decreased with increasing colchicine concentration and

exposure time. Flow cytometry analysis demonstrated that the amount of DNA in the tetraploid
plants had doubled. The treatments of 5 ppm colchicine for 1 day and 15 ppm colchicine for 3
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307

days were both found to be effective for inducing polyploidy in Torenia fournieri, because both
treatments resulted in 2 tetraploids, which were confirmed to have the doubled chromosome
number of 2n=4x=36. A comparison of the morphological and cytological characteristics of
diploid and tetraploids Torenia fournieri showed that the tetraploids grew more slowly and
were shorter than the diploids, but had greater spread, darker green leaves, larger and thicker
leaves, and larger flowers. The stomata size of the tetraploids was also larger than the diploids.

5. References
Cook, J.W. and L.D. Loudon. 1952. Colchicine. The Alkaloid Chemistry and Physiology.
2: 261-329.
Dermen, H. 1940. Colchicine polyploidy and technique. The Bot. Rev. 6: 599-635.
Dolezel, J., 1997. Applications of flow cytometry for the study of plant genomes. Journal of
Applied Genetics. 38:285–302.
Elliott, F.C. 1958. Plant Breeding and Cytogenetics. McGraw-Hill, Inc, New York.
Espino F. J. and A. M. Vazquez. 1981. Chromosome numbers of Saintpaulia zonantha
plantlets regenerated from leaves cultured in vitro with caffeine and colchicine.
Euphytica. 30: 847-853.
Fischer, E. (2004). The Families and Genera of Vascular Plants.
Kadereit, ed. Springer-Verlag, New York. pp 333-391.

Volume VII, JW

Hancock, J.F., 1997. The colchicine story. Hortscience 32: 1011–1012.
Jensen, C.J. 1974. Chromosome doubling techniques in haploids, pp. 153-190. In K.L. Kasha
(ed.). Haploids in higher plants-Advances and potential. University of Guelph,
Canada.
Jiranapapan, J., Kikuchi, S., Manochai, B., Taychasinpitak, T., Tanaka, H., Tsujimoto, H.
(2011). A simple method chromosome doubling suing colchicine in Torenia
(Linderniaceae), and the behavior of meiotic chromosome in amphidiploids.
Chromosome Science 14: 29-32.
Kikuchi, S., Kishii, M., Shimizu, M., Tsujimoto, H. (2005). Centromere-specific repetitive
sequences from Torenia, a model plant for interspecific fertilization and whole-mount
FISH of its interspecific hybrid embryos. Cytogenetic and Genome Research 109:
228-235.
Kikuchi, S., Matsui, K., Tanaka, H., Ohuishi, O., Tsujimoto, H. (2008). Chromosome
evolution among seven Fagopyrum species revealed by flouresence in situ
hybridization (FISH) probed with aDNAs. Chromosome Science. 11: 37-43.
Loureiro, J., Pinto, G., Lopes, T., Dolezˇel, J., Santos, C., 2005. Assessment of ploidy stability
of somatic embryogenesis process in Quercus suber L. using flow cytometry. Planta.
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Yamazaki, T. (1985). A Revision of the Genera Limnophila and Torenia from Indochina. J
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Thunya TAYCHASINPITAK is an Associate Professor in Department of Horticulture, Faculty of
Agriculture, Kasetsart University, Bangkhen, Bangkok, THAILAND. He is teaching and researching in
floriculture and floriculture crop improvement.

Dr. Shermarl WONGCHAOCHANT is a lecturer in the Department of Horticulture, Faculty of Agriculture,
Kasetsart University, Bangkhen, Bangkok, THAILAND. She earned an B.S. (Agriculture)(HONS.) from
Chiang Mai University, Thailand, an M.A. and a Ph.D. (Plant Biotechnology) from Osaka Prefecture University,
Japan. She is teaching and researching in floriculture crop improvement and plant molecular genetics.
Dr. Shinji KIKUCHI is an Assistant Professor in Graduate School of Horticulture, Chiba University, Chiba,
JAPAN. He is teaching and researching in Chromosome Science and Plant Breeding

Sasiree BOONBONGKARN is a graduate student in Department of Horticulture, Faculty of
Agriculture, Kasetsart University, Bangkhen, Bangkok, THAILAND. Her research encompasses plant
breeding technology.

Peer Review: This article has been internationally peer-reviewed and accepted for publication
according to the guidelines given at the journal’s website.

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Effect of Colchicine Tablets on Morphology of Torenia fournieri

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