Propagation of Ornamental Plants

Vol. 5, № 3, 2005: 156-161

A WOUNDING METHOD AND LIQUID CULTURE IN

PAPHIOPEDILUM DELENATII PROPAGATION

Duong Tan Nhut1*, Phan Thi Thuy Trang1, Nguyen Hong Vu1, Dang Thi Thu Thuy1, Dinh Van Khiem1,
Nguyen Van Binh1 and K. Tran Thanh Van2

1
Dalat Institute of Biology, 116 Xo Viet Nghe Tinh, Dalat, Lam Dong, Vietnam,
*Tel. +84-63-831056, *Fax. +84-63-831028, *E-mail: duongtannhut@yahoo.com
2
Institut de Biotechnologie des Plantes, Université de Paris-Sud,
UMR 0569, F-91405 Orsay Cedex, France.

Abstract
An innovative method for mass shoot propagation of Paphiopedilum delenatii (‘Pink’ slipper orchid), an
endemic and endangered species of Vietnam, was established in this study. Nine-month-old seeds cultured
on Knudson C medium was the optimal condition for in vitro germination. Seeds began swelling and became
greenish in color after 30 to 40 days of culture. Then, rounded bodies, known as protocorm, were observed.
Leaves and roots developed after 90 – 100 days of subculturing on the same medium. The combination of
wounding and liquid culture was shown to be appropriate for highly frequent adventitious shoot formation.
Shoot regeneration rate of 5.2 per wounded seedling could be obtained in MS liquid media supplemented with
1.0 mg l-1 TDZ via this method.

Key words: germination, liquid culture, Paphiopedilum delenatii, TDZ, wounding

INTRODUCTION and plantlet regeneration from shoot tips (Huang 1988)

Paphiopedilum is a terrestrial orchid genus, which
grows from the Himalayas in Southeast Asia to Papua
New Guinea (Teoh 2005). The name of this genus is de-
rived from the Greek word “pedilon”, meaning “sandal”
or “slipper”, giving rise to the common name “slipper
orchid” (Sheehan 2001). Paphiopedilum delenatii,
an endangered species recognized and protected by
the Convention on International Trade of Endangered
Species of Wild Flora and Fauna (CITES), has been a
favorite potted plant for centuries due to its attractive
color and distinctive shapes. However, the increasing
market demand, together with the low multiplication rate
of conventional propagation methods has endangered the
survival of this unique orchid. Consequently, plant cell
tissue culture through which a large numbers of plantlets
can be obtained within a short period has become an ideal
solution for preserving this genus from extinction.
Though Paphiopedilum is a high-value potted plant,
the success of Paphiopedilum micropropagation is rela-
tively limited due to the difficult bacterial and fungal
decontamination of ex vitro-derived explants and the
poor development of explants that survive under in vitro
conditions (Stewart and Button 1975, Huang 1988).
Different methods for Paphiopedilum micropropagion
have been reported, including axillary shoot induction
and shoot multiplication from seedlings (Huang et al.
2001). Recently, plantlets were successfully regenerated
through protocorm-like bodies (PLBs) from totipotent
calli (Lin et al. 2000), and direct shoot bud formation was
obtained from in vitro leaf explants (Chen et al. 2004).
In this paper, the appropriate medium for in vitro
germination of P. delenatii seeds was determined.
Moreover, a practical method for P. delenatii propagation
by wounding the seedlings bases in combination with
liquid culture was established.
MATERIALS AND METHODS
Plant materials
Pollen grains were manually dropped onto the stig-
mata of Paphiopedilum delenatii flowers in the green-
house of the Dalat Institute of Biology. Three-, six-, or
nine-month-old fruits were collected for the germination
experiment. In vitro six-month-old seedlings, 2.5 – 3.0
cm in height, were used as explants for shoot induction
by a wounding method.
Nutrient media
Knudson C (Knudson 1946), basal MS (Murashige
and Skoog 1962), and 1/2 MS (half-strength MS

Received: June 16, 2005 Accepted: August 15, 2005

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 Duong Tan Nhut et al. A wounding method and liquid culture in Paphiopedilum delenatii propagation

Fig. 1. Diagram of the wounding and liquid culture used for shoot regeneration of Paphiopedlium delenatii cultured in vitro
a) Nine-month-old fruit of P. delenatii, b) Seeds spread in vitro, c) Three-month-old seedlings, d) Six-month-old seedlings,
e, f) Nine-month-old seedlings, g) Two-year-old plantlet flowering in greenhouse, m, m1) Wounded plantlets, m1’, m1’’)
Wounded plantlets cultured in liquid medium, m2’, m2’’) Wounded plantlets cultured in solid medium.

medium) media were used for the experiment on the
germination of P. delenatii seeds.
Basal MS medium supplemented with different
concentrations of Thidiazuron (TDZ 0.5, 1.0, or 3.0 mg
l-1) or TDZ (0.25, 0.5, 1.0, or 2.5 mg l-1) in combination
with 0.5 mg l-1 α-naphthaleneacetic acid (NAA), 20 g
l-1 sucrose, and 8 g l-1 agar (for solid media only) were
used for investigating the shoot regeneration on wounded
seedlings.
Basal MS medium supplemented with 0.5 mg l-1
NAA, 2.0 mg l-1 6-benzyladenine (BA), 1 g l-1 activated
charcoal (AC), 20% (v/v) coconut water, 30 g l-1 sucrose,
and 8 g l-1 agar was used as a fresh medium for subcultur-
ing shoots from wounded seedlings.
The rooting medium was a basal MS medium sup-
plemented with 0.5 mg l-1 NAA, 1 g l-1 AC, 10% (v/v)
coconut water, and 25 g l-1 sucrose.
The pH of all media was adjusted to 5.8, prior to the
addition of AC and agar. Seventy ml of solid medium
or 30 ml of liquid medium were added in each 250-ml
Erlenmeyer flask covered by a two-layer polyethylene
cap before autoclaving at 121ºC, 1 atm for 20 minutes.
A 2 x 2 cm piece of filter paper (OSI, France), which
acted as a support keeping the explants from completely
submerging in the medium, was added in the liquid-
containing flask.
Effect of medium and seed age on germination rate
P. delenatii fruit at different ages (Fig. 1a, 2a) were
sterilized by rubbing the entire capsule surfaces in 70%
(v/v) ethanol and dipping in 0.1% (w/v) HgCl2 for 10
min, before rinsing thoroughly with sterile distilled wa-
ter. Seeds were then released by longitudinally splitting
capsules in sterile conditions using a sterilized scalpel,
and evenly spread on nutrient media (Fig. 1b).
Effect of wounding on shoot regeneration in solid
media
Roots of six-month-old in vitro seedlings (Fig. 2b)
were carefully removed and the seedling bases were
pierced three or four times using a sharp needle, whose
diameter was 0.3 mm (Fig. 1m, m1). Three wounded
seedlings were then placed onto one media-containing
flask (Fig. 1m2’, m2’’). Three non-wounded seedlings (per
flask) were placed onto the same media as a control.
Effect of wounding method on shoot regeneration in
liquid media
The experiment was conducted in the same way as
described above, but solid media was replaced by liquid
media (Fig. 1m1’, m1’’). Each explant was placed in the
centre of the piece of filter paper.
Acclimatization
Individual shoots formed at the wounded region of
seedlings were separated before subculturing to fresh
media. Vigorous shoots were then transferred to rooting
medium (Fig. 1e). Rooted plantlets were obtained after
3 months of culture (Fig. 1f), and then acclimatized in
the greenhouse.

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Vol. 5, № 3, 2005: 156-161

A B

C D

E F

Fig. 2. Wounding method and liquid culture effects on Paphiopedilum delenatii propagation. A) Flowers and fruits of P.
delenatii cultured in the greenhouse, B) Six-month-old in vitro seed-derived plantlets, C) Shoots regenerated from wounded
seedlings in liquid medium, D) Shoot cluster regenerated from wounded base of in vitro P. delenatii after 3 months of culture,
E) Shoot cluster developed from wounded base of in vitro P. delenaii on fresh medium after 3 months, F) In vitro rooted
plantlets after 3 months.

Culture conditions circle of sunlight at 27 ± 3oC and 75 – 85% relative hu-

All in vitro cultures were placed at 25 ± 2oC, 70
– 80% relative humidity and a 12-hour photoperiod
at 45 μmol m-2 s-1 photosynthetic photon density flux,
which was provided by fluorescent tubes (Rang Dong,
Vietnam).
Rooted plantlets were planted on styrofoam trays
with a fern substrate and placed under natural light/dark
midity. Plantlets were placed on shade in the greenhouse
and were softly watered every 2 days with tap water.
Statistical analysis
Each treatment was conducted with 12 flasks. Seed
germination rate, shoot survival rate and the number
of shoots per explant were recorded after 3 months of

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 Duong Tan Nhut et al. A wounding method and liquid culture in Paphiopedilum delenatii propagation

culture. Data were analyzed for significance by Duncan’s Table 1. Effect of medium and seed age on germination
multiple range test (Duncan 1995). rate.

RESULTS AND DISCUSSION Media Seed age (months) Germination rate (%)
3 0
Effect of medium and seed age on germination rate
Knudson C 6 8.8
In this experiment, the highest germination rate 9 90.1
(90.1%) and the most vigorous seedlings were obtained MS 9.0
9
on nine-month-old seeds cultured on Knudson C medium 1/2 MS 73.0
(Table 1). After 30 to 40 days of culture, seeds began
swelling and became greenish in color. Then, rounded recorded from wounded seedlings cultured on MS solid
bodies, known as protocorm, were observed. Leaves and medium supplemented with 0.5 mg l-1 TDZ, without
roots developed after 90 – 100 days of subculturing on external auxin. An average of 2.3 green and vigorous
the same medium. It has been reported that immature shoots were obtained on medium containing 0.25 mg
seeds germinate better than mature ones, due to their l-1 TDZ and 0.5 mg l-1 NAA. There is no doubt that the
distended testa cell and metabolically awakened embryos wounding step is a prerequisite for shoot formation
(Linden 1980). Mature seeds, in contrast, are recalcitrant in seedlings. Wounded cells at the damaged site may
to germination primarily due to the change in the quality be responding to stimulating agents (e.g. certain plant
of their food reserves and the dormancy or inhibitory growth regulators (PGRs)) that are present in nutrient
factors that are present inside their cells. However, very media by differentiating into novel organs, such as
young orchid ovules are not appropriate for germina- adventitious shoots. No shoot formation was recorded
tion because they may need time for organogenesis or on non-wounded seedlings in control treatments, sug-
synthesis of nutrients that help embryos recognize the gesting that intact cells of these seedlings were not
stimulating agents, which are present in the medium affected by the stimulators.
and germinate. Young seeds from 6-month-old fruits Explant differentiation depends significantly on me-
developed rather slowly on Knudson C. Seed-derived dium components including minerals, nutrients, sugar,
protocorms were hard to recognize and no seedlings were vitamins, and PGRs. TDZ, a non-purine, cytokinin-like
obtained from 3-month-old seeds in this treatment. These compound has been shown to exhibit stronger effects
results suggest that the appropriate age for collecting P. than any other conventional cytokinin over a wide range
delenatii fruits is 9 months. of species and to be effective for shoot proliferation
Orchid seeds in natural environments have a poor and adventitious shoot organogenesis (Huetteman and
nutrient supply even at maturity. Hence, they require Preece 1993). Тхе mode of action of TDZ may be at-
a suitable stimulus by a fungus which is believed to tributed to its ability to induce cytokinin accumulation
augment the carbohydrate, auxin, and vitamin transport (Victor et al. 1999) and enhance the accumulation and
for their germination in nature (Arditii et al. 1982). translocation of auxin within TDZ-exposed tissues
Knudson (1922) demonstrated that the fungal require- (Murch and Saxena 2001). Therefore, TDZ promotes
ment of orchid seeds can be bypassed during in vitro the induction of shoot formation of wounded seedlings
germination by adding an appropriate carbohydrate in due to its optimal characteristics. TDZ, when supple-
the culture medium. Since then, many nutrient media mented to the medium at an appropriate concentration,
for orchid seed culture have been proposed, includ- enhanced shoot formation. TDZ was applied success-
ing Knudson C, MS and Morel (Morel 1964). In this fully for Paphiopedilum micropropagation in several
study, MS medium seemed inappropriate for germina-
tion of P. delenatii seeds. MS medium may inhibit P. Table 2. Effect of the wounding method on shoot regenera-
delenatii germination due to its high nutrition content. tion in semi-solid medium after three months of culture.
Decreasing the micro- and macro-organic, as well as
the inorganic components in the MS medium may have PGRs (mg l-1)
Shoots per
Survival rate (%)
a positive effect on seed germination (Table 1). The TDZ NAA explant
nutrition-poor Knudson C medium was determined to
be appropriate for germination of P. delenatii seeds in
0.25 0.5 75 2.3 ay
0.5 0.5 75 1.2 c
this study.
1.0 0.5 75 0
2.5 0.5 50 0
Effects of wounding method on shoot regeneration 0.5 - 85 2.1 a
in solid medium 1.0 - 80 1.1 c
As shown in Table 2, no shoot formation was ob- 3.0 - 80 1.6 b
tained in control treatments with non-wounded shoots y
Different letters within a column indicate significant differences
in all kinds of media. The highest survival rate was at α = 0.05 by Duncan’s multiple range test.

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Vol. 5, № 3, 2005: 156-161
previous studies (Huang 1988, Lin et al. 2000, Chen
et al. 2004). In this study, the number of shoots formed
increased with TDZ concentration. Nevertheless, TDZ
at high concentrations may have an inhibitory effect on
shoot formation and reduce the explant survival rate.
In our experiments, this characteristic of TDZ was
obviously shown when it was used in combination
with auxin. The combination of TDZ and auxin (in
this case NAA) promotes the inhibitory effect of TDZ.
Furthermore, high TDZ concentrations also reduced
the explant survival rate. This trend was obvious when
auxin was present and was consistent with the results
from a previous study (Huang et al. 2001) in which TDZ
inhibited shoot proliferation of Paphiopedilum.
Effects of wounding on shoot regeneration in liquid
medium
In semi-solid, auxin-containing media, the number
of newly formed shoots tended to decrease as the con-
centration of TDZ increased. In contrast, the number
of shoots formed in liquid medium increased consider-
ably when TDZ levels were increased (Table 3). These
results suggest that the effects of TDZ on explants
depend significantly on the physical properties of the
medium, and that the physical state of the medium
(liquid or solid) substantially affected shoot formation
in wounded seedlings. Not being completely submerged
in the culture medium, wounded seedlings can uptake
nutrient components and PGRs easier. This stimulates
the differentiation of the affected tissue and results in
higher number of shoots per explant. In liquid media, a
significantly lower adverse effect of TDZ on plant tis-
sue was observed. The survival rate was relatively high
(85%) and did not vary considerably among treatments.
Furthermore, the survival rate was lower when TDZ
was the sole external PGR than when TDZ was used in
combination with auxin. This suggests that the presence
of auxin in liquid media may increase the survival rate.
However, when TDZ was used at a concentration of 1.0
mg l-1, the number of shoots per exlant formed in liquid
Table 3. Effects of wounding method on shoot regeneration
in liquid media after three months of culture.
PGRs (mg l-1)
Survival rate (%) Shoots per explant
TDZ NAA
0.25 0.5 50 1.2 dx
0.5 0.5 85 1.4 d
1.0 0.5 85 2.2 c
2.5 0.5 85 3.0 b
0.5 - 80 3.1 b
1.0 - 75 5.2 a
3.0 - 75 2.9 b
x
Different letters within a column indicate significant differ-
ences at α = 0.05 by Duncan’s multiple range test.
160
media without auxin was 2 times greater than in media
that contained auxin, suggesting that auxin may play
an inhibitory role in liquid media.
CONCLUSION
Knudson C medium and nine-month-old seeds
were found to be appropriate for germination of Pa-
phiopedilum delenatii’s seeds. A wounding method
was demonstrated to be efficient for inducing shoot
regeneration, and highest numbers of shoot were ob-
tained in a liquid medium that contained 1.0 mg l-1 TDZ.
These shoots developed vigorously after 3 months of
subculture on fresh medium. Rooting plantlets could be
obtained after 3 months on rooting medium. Because of
their efficiency, the wounding method and liquid culture
can be applied for large-scale micropropagation of this
endangered species.
Acknowledgment: The authors wish to thank Prof.
Odon Vallét (Université de Sorbonne, France) for his
financial support.
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