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org Thai Journal of Agricultural Science 2013, 46(1): 59-63

Polyploid Induction by Colchicine Treatments and Plant

Regeneration of Dendrobium chrysotoxum

P. Atichart

Department of Biology, Faculty of Science, Mahasarakham University

Kantarawichai District, Mahasarakham 44150, Thailand

Corresponding author. Email: [email protected]

Abstract
The purpose of this research was to investigate the effect of colchicines concentration and
duration time to polyploid induction and plant regeneration of Dendrobium chrysotoxum L. The
method was conducted by inclusion of colchicine into semi-solid VW medium. Protocorm like
bodies (PLBs) of diploid D. chrysotoxum were treated with 0, 0.01, 0.02, 0.03, 0.04, and 0.05%
colchicines (w/v) for 1, 2, 3, 4 and 5 days. The most effective treatment was 0.04% colchicine for
1 day which resulted in about 84% surviving PLBs and with 47 % of tetraploid orchids, as
measured by flow cytometry. The treated PLBs were cultured on the same medium supplemented
with 0, 0.5 and 1 mg L-1 NAA and 0, 0.5 and 1 mg L-1 BA for plant regeneration. Treated PLBs
with 0.01% and 0.02% colchicines, the highest number of proliferated shoot (2.36 per explants and
2.44 per explant respectively) was obtained from 1 mg L-1 NAA and 0.5 mg L-1 BA. In the
treatment with 0.03% and 0.04% colchicines, the highest number of proliferated shoot (3.40 per
explants and 4.35 per explants respectively) was obtained from the culture media supplemented
only with 0.5 mg L-1 BA and 1 mg L-1 BA .

Keywords: colchicine, protocorm, flow cytometry, orchid

Introduction be used as a means to create and select new and

better breeds for further use. In order to produce
Dendrobium is one of the most important types polyploidy plants, the chemical colchicine is widely
of commercial orchids used for cut flowers and used because of its effectiveness and availability
potted plants, flower sizes of some varieties are but it is toxic to cells. Polyploids were induced
small such as D. chrysotoxum. It is a cluster of successfully from in vitro plants of diploid by
bright yellow, fifty cent size and honey scented. treating with cochicine in different concentrations
Chromosome doubling has been used in breeding in Miscanthus sinensis (Petersen et al., 2003), oil
program for improved new characteristics of orchid palm (Madon et al., 2005), sesame (Mensah et al.,
flower. Colchicine treatment has become a common 2007), Ginger (Sakhanokho et al., 2009), Basil
tool use for polyploid induction in many plants. (Omidbaigi et al., 2010) and cocoyam (Oumar et
Economic crops such as wheat, oats, cotton, coffee, al., 2011), different explant materials were treated
apples, roses, and bananas are polyploid (Luckett, with colchicine to induce chromosome doubling.
1989; Thao et al., 2003; Sundov et al., 2005). The Ploidy levels could be easily determined by flow
process can occur naturally or through human cytometry (Costich et al., 1993; Allum et al., 2007;
manipulation. In general, polyploid plants exhibit Sarathum et al., 2011; Oumar et al., 2011. The
superior phenotypes to those of diploids such as objectives for this experiment were to determine the
stronger stems, and thicker and larger leaves, effective concentrations of colchicine, the
flowers, fruits, and seeds. Polyploid induction can appropriate duration time in the polyploid induction
60 P. Atichart Thai Journal of Agricultural Science

and plant regeneration from treated PLBs of D. plants were used for flow cytometric measurement.
chrysotoxum. Approximately 1 g of each leaf was chopped with a
sharp razor blade in a 55-mm plastic Petri dish
Materials and Methods containing hypotonic buffer Cy stain RUV ploidy
(one step DAPI staining solution) and then filtered
Plant Material through a 30 µm celltrics disposable filter. The
PLBs of D. chrysotoxum were used as explant samples were analyzed with Partec PAII.
sources derived from cultured of D. chrysotoxum
seed on Vacin and Went (1949) medium (VW) Statistical Analysis
containing 100 mg L-1 myo-inositol, 1 mg L-1 Mean values for each duration and
thiamine, 1 mg L-1 nicotinic acid, 1 mg L-1 concentration of colchicine treatment and plant
pyridoxine and 4 mg mg L-1 glycine, 20 g l mg L-1 regeneration were tested in five replications and
sucrose, 15% coconut water and 0.8% agar at pH subjected to factorial in Completely Randomized
5.4. The explants were cultured under 16 h Design (CRD) analysis of variance and compared
photoperiod (light intensity 40 µmole m-2 s-1) at by Duncan’s new multiple range test (DMRT) at
25±2oC for 4 weeks. After 4 weeks of cultured P<0.05.
seeds were formed Protocorm like bodies (PLBs).
Results
Colchicine Treatment
PLBs were treated with 0, 0.01, 0.02, 0.03, Survival Rate
0.04, and 0.05% colchicine for 1, 2, 3, 4 and 5 days. There are statistically significant differences
After the treatment, they were washed with steriled (p<0.05) between the average survival rates over all
distilled water and then cultured on VW medium duration and concentration of colchicines treatment,
containing 100 mg L-1 myo-inositol, 1 mg L-1 declining from 100% in the colchicines free control
thiamine, 1 mg L-1 nicotinic acid, 1 mg L-1 to 44% at the highest concentration of 0.05%
pyridoxine, 4 mg mg L-1 glycine, 15% coconut treated for 3 days. The interaction between
water, 20 g L-1 sucrose 8 g L-1 agar, pH 5.4. Each concentration and the duration of the colchicine
treatment was replicated 5 times and 50 protocorms treatments found that higher concentration and
were cultured per each replication. After culturing longer duration reduced survival of explants
for 12 weeks, the survival plantlets was examined. (Figure 1).

Plant Regeneration Plant Regeneration

Treated PLBs of D. chrysotoxum with Regeneration of treated PLBs after treated with
colchicines were cultured on VW medium for 3 colchicines on modified VW medium supplemented
month they could not regenerated therefore treated with 0, 0.5 and 1 mg L-1 NAA and 0, 0.5 and 1 mg
PLBs would transfer to VW medium supplemented L-1 BA. The mean number of proliferated shoot
with 0, 0.5 and 1 mg L-1 -naphthalene acetic acid were evaluated after 20 weeks of cultures. The
(NAA) and 0, 0.5 and 1 mg L-1 Benzyl adenine result showed significantly (p<0.05), treated PLBs
(BA) for plant regeneration. The explants were with 0.01% and 0.02% colchicines, the highest
cultured under 16 h photoperiod (light intensity 40 number of proliferated shoot (2.36±0.4 per explants
µmole m-2 s-1) at 25±2oC. The observation was and 2.44±1.3 per explant respectively) was
taken at regular intervals of one week up to the 20 obtained from 1 mg L-1 NAA and 0.5 mg L-1 BA.
weeks and the obtained result was recorded. But the treated PLBs with 0.03% and 0.04%
colchicines, the highest number of proliferated
Ploidy Level Determination shoot (3.40±1.26 per explants and 4.35±1.45 per
After 20 weeks, the proliferated shoots were explant respectively) was obtained from the culture
subjected to test their ploidy levels by flow media supplement only with 0.5 mg L-1 BA and 1
cytometry. Young leaves of colchicine treated mg L-1 BA, respectively (Table 1).
Vol. 46, No. 1, 2013 Polyploid induction of D. chrysotoxum 61

Determination of Ploidy Level by Flow

Cytometric Analysis
In diploid control plants, used as an external
standard, 100% of the nuclei were in G1 phase of
the cell cycle, peaking at a relative nuclear DNA
content of 200 (chanel number) indicated that the
diploid (Figure 3A), whereas the tetraploid
remaining nuclei appeared at a relative nuclear
Figure 1 The effect of in vitro colchicine treatment on the DNA content of 400 (chanel number), in late S or
survival rate of D. chrysotoxum PLBs.
G2 phase of the cell cycle (Figure 3B), and
mixoploid remaining nuclei appeared at a relative
Table 1 Effect of different concentration of - nuclear DNA content of 200 and 400 (chanel
naphthalene acetic acid (NAA) and Benzyl adenine (BA) number) (Figure 3C)The ploidy level of the treated
on plant regeneration of D. chrysotoxum treated plantlets was affected by the concentration of
protocorm with colchicines. colchicine and the duration of treatment. When
Treatment Growth Mean number of plantlets were treated with 0.04% colchicine,
concentration regulator regenerated shoots plantlets exhibit increases in ploidy level. At the
(%) (mg L-1) (mean±S.E) same concentration of colchicines (0.04%) but
NAA BA when the time of exposure was increased to 2 days,
0.01 0 0 1.72 ± 0.2bc
30% tetraploids and 46% mixoploids (2x+4x) were
0 0.5 1.20 ± 0.4abc
0 1 0.40 ± 0.5 a found among tested plantlets (Table 2).
0.5 0 0.00 ± 0.3 a
0.5 0.5 0.60 ± 0.1ab Discussion
0.5 1 0.76 ± 0.3 ab
1 0 2.36 ± 0.7
1 0.5 2.36 ± 0.4 c In vitro induction of polyploid in D.
1 1 0.20 ± 0.2 a chrysotoxum proved successful when treated PLBs
0.02 0 0 2.30 ± 1.35bc with colchicines concentration at 0.04% for 1 and 2
0 0.5 0.70 ± 0.4ab days, Tetraploid and mixoploid were found. Several
0 1 3.13 ± 1.267c reports used colchicine as antimitotic substances. It
0.5 0 0.40 ± 0.4a
0.5 0.5 0.80 ± 0.6ab binds to cell protein tubulin and arrests mitosis in
0.5 1 1.30 ± 0.3ab metaphase due to failure of spindle formation. It
1 0 1.46 ± 1.23 abc causes depolymerisation and disappearance of the
1 0.5 2.44 ± 1.35bc fibrillar microtubules in granulocytes and other
1 1 0.00 ± 0.23 ab
motile cells, inhibiting their migration as well as
0.03 0 0 2.26 ± 1.23bc
0 0.5 3.40 ± 1.26 ab metabolic and phagocytic activity (Sundov et al.,
0 1 0.50 ± 0.4a 2005) In many plant species colchicine causes side
0.5 0 1.52 ± 0.6ab effects such as sterility, abnormal growth and
0.5 0.5 1.52 ± 0.6 ab morphology, chromosome losses or rearrangements
0.5 1 2.10 ± 1.34 bc
1 0 0.80 ± 0.5 ab
and gene mutation (Luckett, 1989). Using high
1 0.5 0.90 ± 0.4 ab colchicines concentration at 0.1, 0.15 and 0.2% for
1 1 1.44 ± 1.15 ab longer than 24 h, chrolophyll contents were
0.04 0 0 0.86 ± 0.5 a decreased and plantets were died after treated. The
0 0.5 3.56 ± 1.52b survival of the explants after colchicine treatments
0 1 4.35 ± 1.45 b
0.5 0 1.70 ± 1.23a depend on the concentration and duration of the
0.5 0.5 0.80 ± 0.5 a treatment. In general, higher coccentration and
0.5 1 0.86 ± 0.5a longer duration reduced survival of plants (Thao et
1 0 1.40 ± 1.25a al., 2003; Atichart and Bunnag, 2007).
1 0.5 1.00 ± 0.9 a
1 1 0.40 ± 0.3 a
62 P. Atichart Thai Journal of Agricultural Science

Treated PLBs of D. chrysotoxum with

colchicines in various concentration and duration
could not regenerated to plantlet. Used of growth
regulators may help them to shoot proliferated. The
A B
type and concentration of growth regulators are an
initial consideration for micropropagation of orchid
species (Genkov and Ivanova, 1995). Addition of
cytokinin to the medium increase the number of
shoots which demonstrates the significance of
exogenous cytokinin to enhance the multiple
shoots. BA influences shoot proliferation by
stimulating quick cell divisions to induce large
C D number of multiple shoots (Yakimova et al., 2000;
Roy and Banerjee, 2002; Ronzhina, 2003; Hameed
Figure 2 Plant regeneration of D. chrysotoxum treated et al., 2006). Results are also according to Asghar
protocorm with co% cultured on 0.03% cultured on VW et al. (20011) who reported that axillary buds of
lchicines (A), 0.01% cultured on VW supplement with 1 mg orchid Dendrobium nobile var. Emma white were
L-1 NAA and 0.5 mg L-1 BA (B), VW supplement with 1 mg
L-1 NAA and 0.5 mg L-1 BA (C), and 0.02 supplement with 0.5 proliferated by using phytotechnology medium
mg L-1 BA 0.04% cultured on VW supplement with 1 mg L-1 (O753) supplemented with benzylaminopurine
BA (D).
(BAP) and kinetin (Kin) as well as coconut water
(CW) and Roy and Banerjee (2002) who reported
A that BAP enhances the shoot multiplication more
Number of nuclei

2n actively than Kin. BAP provided smaller lengths of

proliferated shoots in contrast to shoots number.
Being a strong cytokinin, it depresses shoot length
by an increase in number of axillary buds (Hameed
et al., 2006).
The following conclusions regarding the
efficiency of polyploidy induction, base on the in
B vitro application of colchicines to D. chrysotoxum,
Number of nuclei

plantlets of this study will multiply and observation

about plant growth, plant morphology such as the
4n number of leaves and flower per plant, plant size
and flower size and determine chlorophyll content
for the next experiment.

Acknowledgments
2n
C
Number of nuclei

This research was supported by Mahasarakham

University and 2553 research fund form National
Research Council of Thailand.
4n

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Vol. 46, No. 1, 2013 Polyploid induction of D. chrysotoxum 63

Table 2 Ploidy level was analyzed two months after colchicines treatment.
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