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Micropropagation of the Thai orchid Grammatophyllum speciosum blume

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DOI: 10.1007/s11240-010-9671-2

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Plant Cell Tiss Organ Cult (2010) 101:143–150
DOI 10.1007/s11240-010-9671-2
ORIGINAL PAPER
Micropropagation of the Thai orchid Grammatophyllum
speciosum blume
Kathawut Sopalun • Kanchit Thammasiri •
Keiko Ishikawa
Received: 15 May 2009 / Accepted: 3 January 2010 / Published online: 9 January 2010
Ó Springer Science+Business Media B.V. 2010
Abstract Protocorm-like bodies (PLBs) were induced
from shoot tips of Grammatophyllum speciosum, a Thai
orchid. The highest frequency of PLBs (93%) were
observed on explants incubated on 1/2-Murashige and
Skoog (MS) liquid medium containing 2% (w/v) sucrose
without any plant growth regulators (PGRs). Tests with
different carbon sources compared to sucrose revealed that
maltose promoted the highest relative growth of G. spec-
iosum PLBs (7-fold increase), while trehalose and sucrose
yielded 5-fold and 4-fold increases, respectively. In 1/2 MS
liquid medium, addition of 15 mg/l of chitosan promoted a
7-fold increase in PLB growth while 25 mg/l promoted a
4-fold increase. However, the relative growth rate in solid
culture was significantly lower than that in liquid culture.
In addition, chitosan supplementation in solid medium
promoted shoot formation but not rooting. Plantlet regen-
eration was induced using a combination of NAA and BA
supplementation in 1/2 MS solid medium with optimum
K. Sopalun
Department of Biotechnology, Faculty of Science, Mahidol
University, Rama VI Road, Phyathai, Bangkok 10400, Thailand
K. Thammasiri (&)
Department of Plant Science, Faculty of Science, Mahidol
University, Rama VI Road, Phyathai, Bangkok 10400, Thailand
e-mail: Kanchitthammasiri@gmail.com
K. Thammasiri
Institute of Molecular Biosciences, Mahidol University,
Putthamonthon, Nakhonpathom 73170, Thailand
K. Ishikawa
Department of Research and Development, Japan Horticultural
Production and Research Institute, Kamishiki, Matsudo, Chiba
270-2221, Japan
induction shoot and root formation at 2.0 mg/l NAA and
1.0 mg/l BA. Using this protocol, approximately 8 months
was required to obtain a hundred plantlets from one shoot
tip. The plantlets showed no changes in ploidy when tested
by flow cytometry.
Keywords Grammatophyllum speciosum 
Micropropagation  Chitosan  Flow cytometry 
Protocorm-like bodies
Introduction
The genus Grammatophyllum contains twelve species.
G. speciosum is the only species of the genus Grammato-
phyllum that is native to Thailand (Pridgeon 2003).
Reaching up to 15 feet in length, it is also known as the
‘‘Tiger orchid’’ or ‘‘Giant orchid’’ and is considered to be
the largest member of the orchid family. It is distributed
from South-East Asia to New Guinea where it can be found
growing on the branches of large trees, but is uncommon in
nature and rarely cultivated. It can remain in bloom for up
to 2 months. In addition to its horticultural attraction for
breeding and commercial purposes, G. speciosum is also
used in Thailand to prepare an herbal elixir against
inflammation.
Since the traditional asexual propagation of orchids is
extremely slow, tissue culture has been adopted as a useful
technique for their micropropagation. Tissue culture was
first introduced and applied to Cymbidium orchids by
Morel in 1960. After that, tissue culture methods, culture
media and various explants for orchid tissue culture were
introduced and developed by several workers. Shoot tip is
the most frequently used for orchid tissue culture (Roy and
Banerjee 2003; Ket et al. 2004; Teixeira da Silva et al.
123
144
2006). Media devised by Vacin and Went (1949) and
Murashige and Skoog (1962) are usually employed for in
vitro orchid culture (Lakshmanan et al. 1995; Ket et al.
2004; Ravindra et al. 2004). Half strength MS medium is
also used successfully for orchid culture (Chen and Chang
2000). Carbon sources are critical components in these
culture media (Islam and Ichihashi 1999) and sucrose is the
most frequently used. However, other sugars used for
orchid culture include glucose, fructose, sorbitol and
maltose (Islam et al. 1998; Islam and Ichihashi 1999).
Another component is often the biodegradable polymer
chitosan, a deacetylated form of chitin that is present in
various organisms, including fungi, crustaceans, insects
and some algae. It has been reported to act as a plant
growth stimulator in some plant species, including orchids
(Nge et al. 2006). For example, supplementation with
1.75% (v/v) chitosan in culture solution enhanced root and
shoot biomass of grapevine plantlets in vitro (Barka et al.
2004). In orchid tissue culture, Nge et al. (2006) reported
that chitosan enhanced growth of Dendrobium phalaen-
opsis PLBs in vitro. Plant growth regulators are also
important for plantlet regeneration in vitro. For example,
NAA and BA are commonly supplemented in orchid cul-
ture media (Tokuhara and Mii 1993; Nayak et al. 2002;
Nasiruddin et al. 2003).
Similar to other orchids, G. speciosum is an inherently
slow-growing plant. However, despite widespread knowl-
edge of its unique characteristics, no established method or
media have been described for its micropropagation and in
vitro culture. This is the first report of an established pro-
tocol for micropropagation of G. speciosum.
Materials and methods
Plant materials and culture media
Shoot tips (2.0 mm) from in vitro seedlings (8–10 cm long)
were excised using a stereo microscope under sterile con-
ditions and cultured on basal solid or in liquid media
including modified Vacin and Went medium (VW) (Vacin
and Went 1949), Murashige and Skoog medium (MS)
(Murashige and Skoog 1962) and half strength MS (1/2 MS).
Each medium contained 2% (w/v) sucrose. Cultures were
maintained under standard conditions at 25 ± 2°C under
white fluorescent light at intensity of 37 lmol m-2 s-1 for
16 h per day. For culture in liquid medium, a shaker was
used at about 110 rpm for 24 h. After 2 months of culture,
survival rate, PLB formation, shoot formation and number
of shoots per explant were recorded.
To study the effects of various carbon sources on
propagation of G. speciosum protocorm-like bodies
(PLBs), 1.0 g of G. speciosum PLBs (0.1–0.2 cm in
123
Plant Cell Tiss Organ Cult (2010) 101:143–150
diameter) were cultured in 1/2 MS liquid medium with 2%
(w/v) of various carbon sources (sucrose, glucose, maltose,
trehalose, sorbitol and mannitol) for 1 month under stan-
dard conditions. Fresh weight of PLBs was recorded after
1-month culture and expressed as relative growth rate
calculated based on the fresh weight of cultured tissue as
follows:
 
Wf  Wi
Relative growth rate ð%Þ ¼  100
Wi
Wf ¼ final weight; Wi ¼ initial weight
To study the effects of chitosan on growth of
G. speciosum PLBs, 1.0 g G. speciosum PLBs (0.1–0.2 cm
in diameter) were cultured in 1/2 MS liquid medium
supplemented with 2% (w/v) of sucrose and various
concentrations of chitosan (0, 5, 10, 15, 20,25 30, 50 or
100 mg/l) at pH 5.7. After 1-month culture, the fresh
weight of PLBs was recorded and expressed as the relative
growth rate as described above. To study its effect on
explant formation, G. speciosum PLBs (0.1–0.2 cm in
diameter) were cultured on 1/2 MS solid medium
supplemented with 2% (w/v) sucrose and various
concentrations of chitosan (0, 5, 10, 15, 20, 25 30, 50 or
100 mg/l) at pH 5.7. Size of whole PLBs, number of new
PLBs, number of shoots per explant, number of roots per
explant and number of leaves per explant were recorded
after 1-month culture.
To study the effects of a-napthaleneacetic acid (NAA)
and benzylaminopurine (BA) on growth and development
of G. speciosum, PLBs of approximately 0.2 cm diameter
were cultured on 1/2 MS solid medium supplemented with
2% (w/v) sucrose and various concentrations of NAA and
BA at pH 5.7. The cultures were incubated under standard
conditions. After 3 months of culture, survival rate, PLB
formation, shoot formation, number of shoots per explant,
root formation and number of roots per explant were
recorded.
Flow cytometry (FCM) analysis
The relative DNA content of each plant tissue sample was
analyzed using a flow cytometer (BD FACSCaliberTM
System, USA). Nuclei from approximately 100 mg fresh-
weight tissue were mechanically isolated by chopping with
a sharp razor blade in 1 ml of nucleus extraction solution
containing 200 mM Tris, 4 mM MgCl2.6H2O, 0.5% (v/v)
Triton X-100, pH 7.5 (Pfossor et al. 1995). The resulting
nuclear suspensions were filtered using 20 lm nylon mesh
and supplemented with 50 lg/ml of propridium iodide (PI,
Sigma, USA) for DNA staining. RNase (50 lg/ml) (Sigma,
USA) was added to prevent staining of double stranded
RNA. Samples were incubated for 10–15 min before
analysis. All flow cytometer assessments were repeated at
Plant Cell Tiss Organ Cult (2010) 101:143–150
least three times for each sample and included a minimum
of 3,000 nuclei per run.
Statistical analysis
All experiments were arranged by completely randomized
design (CRD). Data from in vitro cultures were subjected
to analysis of variance (ANOVA) and means were com-
pared using the least significant difference (LSD) test.
Results and discussion
Establishment of a shoot tip culture to induce formation
of PLBs
After 2 months of G. speciosum shoot tip culture, signifi-
cant differences in survival rate, PLB formation, shoot
formation and number of shoots per explant were recorded
among the three media used (Table1). The results showed
that shoot tips cultured with 1/2 MS liquid or solid media
showed the highest survival rates at 93% and 90%,
respectively. Shoot tips cultured with VW liquid or solid
media exhibited the lowest survival rates at 23% and 30%,
respectively. In addition, shoot tips with VW liquid or solid
media were often bleached after 2 weeks of culture and
died within a month. Most of the surviving shoot tips
cultured in 1/2 MS liquid medium formed PLBs (93%),
while shoot tips cultured on 1/2 MS solid medium formed
16% PLBs and 73% shoots. PLB formation in both 1/2 MS
and full MS liquid media was greater than on solid media
(P \ 0.05). Solid media of all three types tended to result
in shoot formation rather than PLB formation. However,
the number of shoots per explant was low in all media, i.e.,
1.5, 1.7, 2.7, 3.0 and 3.2 shoots per explant in solid VW,
liquid VW, solid MS, liquid MS and 1/2 MS solid media,
respectively. However, there was no shoot formation from
shoot tips cultured in 1/2 MS liquid medium.
Table 1 Effects of three different basal media on survival rate, PLB formation, shoot formation and number of shoots per explant of
G. speciosum shoot tips after 2 months of culture
Media Medium Survival
types rate (%)
VW Liquid 23 ± 3 d
Solid 30 ± 6 d
1/2 MS Liquid 93 ± 3 a
Solid 90 ± 0 ab
MS Liquid 63 ± 3 c
Solid 80 ± 6 ab
Values are presented as means ± standard errors (SE); different letters within a column indicate significant differences at P \ 0.05 according to
analysis of variance (ANOVA). All data were calculated based on the total amount of explants with 10 explants per treatment. The experiment
was replicated three times
145
Three basal media, VW, KC and MS were reported to be
suitable for thin section culture from shoot tip of Aranda
Deborah (Lakshmanan et al. 1995). Tokuhara and Mii
(1993) employed New Dogashima Medium (NDM) con-
taining 0.1 mg/l of NAA and 1 mg/l of BA for induction of
PLB formation, but we found supplementation to be
unnecessary with G. speciosum shoot tips. We have no
explanation for the curious development of bleached shoot
tips after 2 weeks and death within a month in VW liquid
or solid media. There are no previous reports of necrosis of
plant tissue cultured in medium without supplements of
plant growth regulators.
Propagation of PLBs
Effects of various carbon sources on propagation
of PLBs
When various carbon sources were investigated for culture
of G. speciosum PLBs, the medium supplemented with
maltose gave the highest relative growth rate (771%) after
1 month of culture when compared to other carbon sources
(Fig. 1). The relative growth rate was about twice of that
obtained in medium containing sucrose. Medium contain-
ing trehalose also showed a relatively high growth rate
(535%) while medium containing mannitol gave the lowest
growth rate (18%) (Fig. 1).
In orchid culture media, carbon sources are very
important not only as energy sources but also as a osmotic
regulators in induction media (Indrianto et al. 1990).
Although sucrose is the most frequently used sugar for in
vitro culture, other sugars such as glucose, fructose, sor-
bitol and maltose are also used and we found that the rel-
ative growth rates of G. speciosum PLBs were high in 1/2
MS liquid medium containing 2% (w/v) maltose, trehalose
or 2% sucrose. The latter is a disaccharide of fructose and
glucose while maltose and trehalose are stereroisomeric
disaccharides of glucose. Although trehalose is also
PLB Formation Shoot No. of shoots
(%) formation per explant
(%)
17 ± 3 c 7±3d 1.7 ± 0.9 a
13 ± 3 c 17 ± 3 c 1.5 ± 0.3 ab
93 ± 3 a 0 0
16 ± 3 c 73 ± 3 a 3.2 ± 0.2 a
50 ± 0 b 13 ± 3 cd 3.0 ± 0.6 a
17 ± 3 c 63 ± 3 b 2.7 ± 2.5 a
123
146
Fig. 1 Effects of various
carbon sources on relative
growth rate of G. speciosum
PLBs, after cultured 1 month in
1/2 MS liquid medium
containing 2% (w/v) of each
carbon source. Column and bar
represent mean ± standard
error (SE), SE was calculated
from five independent
experiments by one-way
ANOVA. Bar with different
letter differs significantly
(P \ 0.05)
capable of altering plant development, the mechanism by
which this occurs is still unknown (Jheng et al. 2006).
Effects of chitosan on propagation of PLBs
When chitosan was supplemented in 1/2 MS liquid med-
ium containing 2% (w/v) sucrose, the relative growth rate
of G. specioum PLB increased significantly after 1-month
culture (Fig. 2). The highest relative growth rate (756%)
was obtained with 15 mg/l chitosan supplementation.
However, the relative growth rate was significantly reduced
when chitosan concentrations were more than 15 mg/l.
Moreover, supplementation with 100 mg/l chitosan caused
PLB necrosis and release of some browning compounds
into the medium.
When chitosan was supplemented in 1/2 MS solid
medium, 25 mg/l chitosan gave the highest relative growth
rate after 1 month of culture (Fig. 2). PLBs did not survive
on 1/2 MS solid medium supplemented with 100 mg/l of
chitosan. Overall, the relative growth rates of G. speciosum
PLBs on solid media were lower than those obtained using
liquid media at the same concentration of chitosan
supplementation.
Effects of chitosan on development of PLBs
Varying the concentration of chitosan in 1/2 MS solid
medium led to differences in the development of
G. speciosum PLBs (Table 2). At 5, 10, and 15 mg/l,
123
Plant Cell Tiss Organ Cult (2010) 101:143–150
chitosan promoted leaf development and the highest
number of leaves per explant was obtained using 15 mg/l
of chitosan (1.8 leaves per explant). Morever, supplemen-
tation with 5, 10 and 15 mg/l chitosan increased the
number of new PLBs and the number of shoots. However,
chitosan supplementation did not promote rooting.
As previously reported, we confimed with G. speciosum
that chitosan can function as a plant growth stimulator for
orchid production or tissue culture (Uthairatanakij et al.
2007). The optimal amount of 15 mg/l of chitosan sup-
plementation for the highest PLB growth rate and 5 to
15 mg/l supplementation to promote leaf and shoot for-
mation was similar to the results of Nge et al. (2006) who
reported that the optimal concentration of chitosan for
promoting growth of Dendrobium orchid tissue was
also 15 mg/l. Chitosan may play a role in enhancing
growth and development by some signaling pathway to
auxin biosynthesis via a tryptophan-independent pathway
(Uthairatanakij et al. 2007), but as seen here, supplemen-
tation at too high a level such as 100 mg/l can inhibit
growth and kill PLBs.
Effects of a-napthaleneacetic acid (NAA)
and benzylaminopurine (BA)
With respect to PLB and root formation, 100% PLB
formed on 1/2 MS solid medium without any plant growth
regulators and with 0.5 mg/l BA supplementation. The
highest shoot formation was found in media supplemented
Plant Cell Tiss Organ Cult (2010) 101:143–150 147

Fig. 2 Effects of chitosan on

relative growth rate of G.
speciosum PLBs cultured for
1 month in 1/2 MS liquid and
on 1/2 MS solid media
containing 2% (w/v) of sucrose,
supplemented with various
concentrations of chitosan.
Column and bar represent
mean ± standard error (SE), SE
was calculated from five
independent experiments by
one-way ANOVA. Bar with
different letter differs
significantly (P \ 0.05)

Table 2 Effects of chitosan on growth, number of new PLBs per explant, number of shoots per explant, number of roots per explant and number
of leaves per explant of G. speciosum PLBs cultured on 1/2 MS solid medium for 1 month of culture
Chitosan Diameter of No. of new PLBs No. of shoots No. of roots No. of leaves
concentration PLB (mm) per explant per explant per explant per explant
(mg/l)

0 54 ± 2 b 9.2 ± 0.7 b 0.8 ± 0.4 b 0a 0a

5.0 62 ± 4 ab 11.2 ± 1.0 a 3.0 ± 1.0 a 0.1 ± 0.1 a 1.4 ± 0.8 a
10.0 61 ± 3 ab 10.5 ± 0.6 ab 3.1 ± 0.8 a 0.1 ± 0.1 a 0.7 ± 0.3 ab
15.0 69 ± 4 a 11.2 ± 0.7 a 3.1 ± 0.6 a 0a 1.8 ± 0.6 a
20.0 58 ± 7 ab 9.6 ± 1.1 ab 1.1 ± 0.6 b 0a 0a
25.0 58 ± 4 ab 10.7 ± 0.5 ab 0.9 ± 0.3 b 0a 0a
50.0 56 ± 4 ab 8.4 ± 0.8 b 0.6 ± 0.3 b 0a 0a
Values are presented as means ± SE. Different letters within a column indicate significant differences at P \ 0.05 according to analysis of
variance (ANOVA)

with 2.0 mg/l NAA in combination with any concentra-
tions of BA, whereas the highest percentage of root for-
mation was found in medium supplemented with 2.0 mg/l
NAA and 2.0 mg/l BA (Table 3).
There was a trend towards a decrease in PLB survival as
NAA and BA concentrations increased, although concen-
trations of NAA and BA at 0–0.5 mg/l gave 100% survival.
As NAA concentrations increased, there was also a
reduction in PLB formation. Supplementation with BA
alone did not result shoot induction, but supplementation
with 2.0 mg/l NAA induced 40% root formation.
Two main groups of PGRs, cytokinins and auxins, are
commonly used in orchid culture media. Their concentra-
tions significantly affected shoot and root formation. We
showed that NAA alone and BA alone or a combination of
the two can decrease the percentage of PLB formation and
that supplementation with NAA alone increased shoot
formation while supplementation with BA alone induced
root formation in G. speciosum. The combination of NAA
and BA can promote shoot and root formation. However,
the increase of NAA or BA concentration reduced survival
rate. Nayak et al. (1997) found that the combination of
NAA and BA of Acampe praemorsa culture induced PLB
formation but that the induction was very low when com-
pared with the use of BA alone. Seeni and Latha (2000)
reported that the combination 0.8 mg/l of NAA and
2.0 mg/l of BA induced shoot formation from Vanda
coerulea leaves.

123
148 Plant Cell Tiss Organ Cult (2010) 101:143–150

Table 3 Effects of NAA and BA on survival rate, PLB formation, shoot formation, number of shoots per explant, root formation and number of
root per explant of G. speciosum PLBs cultured on 1/2 MS solid media for 3 months
PGRs* Survival rate PLB formation Shoot formation No. of shoots Root formation No. of roots
NAA BA (mg/l) (mg/l) (%) (%) (%) per explant (%) per explant

0 0 100 a 100 a 0 0 0 0
0.5 100 a 100 a 0 0 0 0
1.0 90 ± 3 ab 96 ± 2 a 0 0 14 ± 2 f 0.4 ± 0.2 cd
2.0 82 ± 5 bc 84 ± 2 b 0 0 22 ± 2 e 1.0 ± 0.3 bc
0.5 0 100 a 100 a 14 ± 2 c 1.6 ± 0.2 d 0 0
0.5 100 a 92 ± 2 a 16 ± 4 c 1.6 ± 0.2 d 0 0
1.0 84 ± 4 bc 80 ± 3 b 22 ± 2 c 1.8 ± 0.4 d 22 ± 1 e 1.4 ± 0.2 ab
2.0 76 ± 5 c 64 ± 2 c 14 ± 2 c 1.8 ± 0.4 d 30 ± 3 d 2.2 ± 2.4 a
1.0 0 90 ± 3 ab 54 ± 2 d 32 ± 4 b 5.2 ± 0.4 c 0 0
0.5 90 ± 4 ab 54 ± 2 d 34 ± 2 ab 6.2 ± 0.6 bc 10 ± 0 f 0.4 ± 0.2 cd
1.0 90 ± 4 ab 42 ± 4 e 42 ± 4 ab 7.8 ± 0.4 a 34 ± 0 cd 2.2 ± 0.4 a
2.0 78 ± 6 bc 42 ± 4 e 40 ± 4 ab 6.8 ± 0.4 ab 38 ± 4 bc 2.0 ± 0.4 a
2.0 0 90 ± 3 ab 42 ± 4 e 44 ± 4 a 6.6 ± 0.5 b 40 ± 3 abc 1.8 ± 0.4 ab
0.5 82 ± 3 bc 38 ± 4 e 50 ± 3 a 6.8 ± 0.5 ab 42 ± 4 ab 1.8 ± 0.4 ab
1.0 82 ± 4 bc 24 ± 2 f 48 ± 6 a 6.8 ± 0.4 ab 38 ± 4 bc 2.0 ± 0.3 a
2.0 72 ± 6 e 14 ± 2 g 46 ± 4 a 6.8 ± 0.6 ab 46 ± 2 a 1.6 ± 0.2 ab
*PGRs: Plant growth regulators. Values are presented as means ± SE. Different letters within a column indicate significant differences at
P \ 0.05 according to analysis of variance (ANOVA)

Fig. 3 Establishment of G. speciosum micropropagation; a A shoot
tip was excised under a stereo microscope, b PLBs were induced in
1/2 MS liquid medium (2 months after culture), c PLBs were
subcultured for multiplication (1 month after culture), d PLBs were
cultured on 1/2 MS solid medium supplemented with 0.05% (w/v) of
activated charcoal, 2.0 mg/l NAA and 1.0 mg/l BA, e and f Shoots
and roots were induced (3 months after culture). Bar = 1 mm
Fig. 4 Transferring G. speciosum plantlets to the saranhouse; a
Plantlets (6.0–8.0 cm long) were obtained after 3 months of culture, b
Plantlets were removed from the bottle and washed with tap water, c
Plantlets were transplanted to pots filled with small pieces of coconut
husk d plants grown 6 months in the saranhouse, e plants grown
1 year in the saranhouse and f plants grown 2 s in the saranhouse.
Bar = 1 cm (a to d)

123
Plant Cell Tiss Organ Cult (2010) 101:143–150
Fig. 5 Normal flow cytometry
histogram of G. speciosum
a G. speciosum plant.
b G. speciosum plantlet
regenerated in vitro
In some tests, we found that G. speciosum PLBs cul-
tured on 1/2 MS solid medium containing PGRs for
induction of shoot and root formation tended to release
phenolic compounds into the medium and it was suspected
that these might cause growth inhibition. Since there were
reports that activated charcoal could absorb phenolic
compounds and inhibitory substances and improve plant
growth and elongation (Ket et al. 2004; Ernst 1974), we
added activated charcoal to prevent any inhibitory effects
at the shoot and root induction stage.
Establishment of G. speciosum micropropagation
Based on the results described above, the following
G. speciosum micropropagation procedure was established.
A 2.0 mm shoot tip of an in vitro G. speciosum specimen
was excised using a stereo microscope under sterile con-
ditions (Fig. 3a) and cultured in 1/2 MS liquid medium
containing 2% (w/v) maltose, pH 5.7 to induce PLBs.
Cultures were maintained under standard conditions of
25 ± 2°C under white fluorescent light at an intensity of
37 lmol m-2 s-1 for 16 h per day. After 2 months of
shoot tip culture, PLBs were subcultured onto the same
medium supplemented with 15 mg/l chitosan and cultured
for 1 month to increase the number of PLBs (Fig. 3b and
c). Then PLBs were transferred and cultured on 1/2 MS
solid medium containing 2% (w/v) maltose at pH 5.7
supplemented with 0.05% (w/v) activated charcoal,
2.0 mg/l of NAA and 1.0 mg/l of BA in order to induce
shoot and root formation (Fig. 3d). After 3 months of
culture on 1/2 MS solid medium, complete plantlets were
formed (Fig. 3e and f). Plantlets 6.0–8.0 cm long were
removed from the bottle (Fig. 4a), washed twice with tap
water (Fig. 4b) and transplanted into pots filled with
coconut husks (Fig. 4c). They were acclimatized and
grown in a saranhouse with about 60% shading and 80%
relative humidity. After 3 months, survival was 100% from
100 plantlets. This procedure to obtain 100 plantlets from
one shoot tip took approximately 8 months. Figure 4e and
f, respectively, show 1 and 2-year-old G. speciosum plants
grown in the saranhouse.
149
Ploidy stability of G. speciosum planlets grown in vitro
Results of ploidy analysis by flow cytometry of G. spec-
iosum plants developed from in vitro shoot tip culture
revealed that histograms obtained from G. speciosum
plantlets grown in vitro showed the same pattern as those
from G. speciosum plants grown in the saranhouse (Fig. 5).
Thus, there was no change in ploidy level in plants prop-
agated using the devised protocol.
In summary, we have successfully established a protocol
for micropropagation of G. speciosum, the largest orchid in
the world. The procedure to obtain about a hundred
plantlets from one shoot tip requires approximately
8 months. This achievement will benefit G. speciosum
conservation and promote its sustainable use as an orna-
mental and medicinal plant.
Acknowledgments This work was supported by a grant from The
Royal Golden Jubilee Ph.D. Program, Thailand Research Fund,
Thailand. We thank Professor T.W. Flegel for reviewing the manu-
script. We are also grateful to Prof. Masahiro Mii, Chiba University,
Dr. Pranee Fucharoen, Dr. M.L. Saovaros Svasti and Mr. Teerasakdi
Chaiya, Thalassemia Research Center, Institute of Molecular Bio-
sciences, Mahidol University, for kindly providing help in flow
cytometry analysis and to the Japan Horticultural Production and
Research Institute, Mastudo, Chiba for training.
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