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Enhancement of direct somatic embryogenesis and plantlet growth from leaf

explants of Phalaenopsis by adjusting culture period and explant length

Article  in  Acta Physiologiae Plantarum · July 2010

DOI: 10.1007/s11738-009-0438-5

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Acta Physiol Plant (2010) 32:621–627
DOI 10.1007/s11738-009-0438-5

ORIGINAL PAPER

Enhancement of direct somatic embryogenesis and plantlet

growth from leaf explants of Phalaenopsis by adjusting
culture period and explant length
Wee-Peng Gow • Jen-Tsung Chen • Wei-Chin Chang

Received: 11 June 2009 / Revised: 22 November 2009 / Accepted: 3 December 2009 / Published online: 22 December 2009
Ó Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2009

Abstract Two Phalaenopsis orchids, Phalaenopsis am- Abbreviations

abilis and Phalaenopsis ‘Nebula’, were used to test the
effects of induction period (30, 45 and 60 days), subculture
period (30, 45 and 60 days), and explant length (1, 1.5 and
2 cm) on direct somatic embryogenesis from different
regions (leaf tip, adaxial side, abaxial side and cut end) of
leaf explants from in vitro grown seedlings. The results
showed that the cut end had a highest competence to form
embryos than the other regions of the leaf explants from
both orchids. In addition, the suitable culture conditions
were 60 days for induction period in darkness, 45 days for
subculture period in light and 1 cm for explant length.
Besides, the combinations of N6-benzyladenine (BA) and
naphthaleneacetic acid were tested on their effects on
plantlet conversion and further development of leaf-
derived embryo. It was found that 0.5 mg/l of BA showed
the highest response on plantlet conversion rate and the
lowest browning rate of explants. In this communication,
the embryo structures and development were proved by
scanning electron microscopy.
Keywords Culture condition
Embryogenic
competence
Embryo development
Moth orchid
Communicated by S. Werbrouck.
W.-P. Gow
W.-C. Chang
Institute of Plant and Microbial Biology, Academia Sinica,
Taipei, Taiwan, ROC
J.-T. Chen (&)
Institute of Biotechnology, National University of Kaohsiung,
Kaohsiung 811, Taiwan, ROC
e-mail: jentsung@nuk.edu.tw
BA N6-benzyladenine
NAA Naphthaleneacetic acid
TDZ Thidiazuron,1-phenyl-3-(1,2,3-thiadiazol-5-yl)-
urea
Introduction
Phalaenopsis (Orchidaceae), commonly known as moth
orchids, are distributed throughout Southeast Asia with a
few species extending from Taiwan and Sikkim to
Australia and the Pacific (Teob 1989). The production of
potted Phalaenopsis has high economical value in
international flower markets. The conventional in vitro
culture protocols have been well developed in this genus
and usually for the propagating purpose (Tanaka et al.
1975; Arditti and Ernst 1993; Tokuhara and Masahiro
1993; Ernst 1994; Chen and Piluek 1995; Duan et al.
1996; Ishii et al. 1998; Islam and Ichihashi 1999; Chen
et al. 2000; Young et al. 2000; Tokuhara and Mii 2001;
Park et al. 2002). These methods mainly used regener-
ation system via protocorm-like body or shoot formation.
In our previous reports, protocols for induction of direct
somatic embryogenesis in Phalaenopsis orchids and
factors affecting embryo production were investigated
(Kuo et al. 2005; Chen and Chang 2006; Gow et al.
2008; Gow et al. 2009). In this communication, further
we test the effects of induction period, subculture period
and explant length on direct somatic embryogenesis of
two Phalaenopsis species, and provide useful data to
improve the protocol for regenerating Phalaenopsis and
other genus of orchids.

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Materials and methods Effect of explant length on direct embryo formation

Plants and culture conditions Three explant lengths (1, 1.5 and 2 cm) were used to test
their effects on direct embryo formation from different leaf
Two Phalaenopsis orchids, Phalaenopsis amabilis Shi- locations of P. amabilis on a medium containing 3 mg/l of
madzu var. formosa with large white flowers (one of the TDZ. Five replicates (dishes) each with four leaf explants
most beautiful native orchid species in Taiwan) and were performed for each treatment.
Phalaenopsis ‘Nebula’ (one of the most popular com-
mercial cultivar), were used as donor plants in this study. Effects of BA and NAA on development of leaf-derived
In vitro grown plants of P. amabilis were purchased from embryos
Taiwan Sugar Corporation (TSC), Taiwan. While the
seedlings of Phalaenopsis ‘Nebula’ were collected by in Two-month-old leaf-derived embryos of P. amabilis were
vitro seed germination on a 1/2-strength modified Mu- used to test combinations of BA (0, 0.5 and 1 mg/l) and
rashige and Skoog (1/2 MS) medium (Murashige and NAA (0, 0.5 and 1 mg/l) on plantlet conversion and
Skoog 1962) containing half-strength macro- and micro- development. Four replicates (dishes) each with five
elements (mg/l) supplemented with myo-inositol (100), embryos were performed for each treatment.
niacin (0.5), pyridoxine HCl (0.5), thiamine HCl (0.1),
glycine (2.0), peptone (1,000), NaH2PO4 (170), sucrose
Data collection and statistic analysis
(20,000) and Gelrite (2,200). Plant growth regulators were
added according to the experimental design and the pH of
The percentage of explants forming somatic embryos was
the media was adjusted to 5.2 with 1 M KOH or HCl prior
recorded for entire explants and in different locations of the
to autoclaving for 15 min at 121°C. The plants were
explants. The number of embryos formed from each
subcultured every 2 months on a hormone-free 1/2 MS
responding explant was counted under a stereomicroscope
medium in 250 ml flasks which were placed under a 16-h
(SZH, Olympus, Tokyo, Japan) at the protocorm stage. The
photoperiod at irradiance of 28–36 lmol m-2 s-1 (day-
percentage of plantlet conversion was counted as the final
light fluorescent tubes FL-30D/29, 40 W, China Electric
numbers of survival plantlets divided by the initial numbers
Co., Taipei, Taiwan) and temperature of 26 ± 1°C. The
of incubated leaf-derived embryos. Data expressed as
seedlings with 3–5 leaves and 2–4 roots were used as
percentage were transformed using arc sine prior to anal-
donor plants.
ysis of variance (ANOVA) and converted back to the ori-
ginal scale (Compton, 1994). Treatment means were
Effect of induction period on direct embryo formation compared by following Duncan’s Multiple Range Test
(Duncan 1955).
Leaf tip segments (about 1 cm in length) of first two
leaves taken from the donor plants were used to induce
Scanning electron microscopic observation
direct somatic embryogenesis as the protocol described
by Chen and Chang (2006). Three induction periods (30,
Samples for scanning electron microscope (SEM) were
45 and 60 days) to test their effects on the amount of
fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer
direct embryo formation from different locations (LT,
(pH 7.0) for 4 h at 4°C, dehydrated in ethanol (Dawns
leaf tips; Ad, adaxial sides; Ab, abaxial sides; and CE,
1971), critical point dryer (HCP-2, Hitachi, Tokyo, Japan),
cut ends) on leaf explants. Five replicates (dishes) each
and coated with gold in an ion coater (IB-2, Giko Engi-
with four leaf explants were performed for each
neering Co., Tokyo, Japan). SEM (DSM-950, Carl Zeiss,
treatment.
Jena, Germany) was used to examine and photograph the
specimen.
Effect of subculture period on direct somatic
embryogenesis
Results and discussion
Three subculture periods (30, 45 and 60 days) were used to
test their effects on the amount of direct embryo formation Embryo formation
from different leaf locations of P. amabilis on 3 mg/l of
TDZ- or 3 mg/l of BA-containing medium. Five replicates Somatic embryos formed mainly from leaf surfaces of the
(dishes) each with four leaf explants were performed for adaxial side and the cut end of both cultivars after 60 days
each treatment. of culture (Fig. 1a and b). These embryos showed whitish

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Acta Physiol Plant (2010) 32:621–627 623

Fig. 1 Plant regeneration via

direct somatic embryogenesis
from leaf explants of
P. amabilis and Phalaenopsis
‘Nebula’. (The bar in upper left
denotes fitting for all panels).
a Direct somatic embryogenesis
from a leaf explant of
P. amabilis after 2 months of
culture (bar = 2.5 mm).
b Direct somatic embryogenesis
from a leaf explant of
Phalaenopsis ‘Nebula’ after 2
months of culture
(bar = 1.5 mm). c Several
mature leaf-derived embryos of
P. amabilis (bar = 1 mm).
d Plantlets of P. amabilis
(bar = 8.5 mm)

to pale yellow in colour together with pale green parent
explants due to the darkness during the 60 days of induc-
tion period (Fig.1a and b). After 60 days of induction,
embryos were then transferred to light for further growth.
These embryos enlarged, turned green and were converted
into plantlets successfully on a growth regulator-free 1/2
MS medium (Fig. 1c, d).
Effect of induction period on direct embryo formation
In both the cultivars, 60 days was the most suitable
induction period for direct embryo formation with 50 and
80% of the leaf explants forming an average of 8.2 and 3.5
embryos per responding explant, respectively (Table 1). In
P. amabilis, the cut end gave the best of embryogenic
frequency (40%) than the leaf tip (20%), the adaxial side
(10%) and the abaxial sides (0%) after 60 days of induction
on 3 mg/l of TDZ-containing medium (Table 1). In Pha-
laenopsis ‘Nebula’, the order of embryogenic responses
was different with P. amabilis that the cut end (80%) was
the best, and then the adaxial side (30%), the leaf tip (5%)
and the abaxial side (5%) (Table 1). Although 60 days is
considered the best for embryo induction on TDZ-con-
taining medium, the cultures usually started to become
brown after 60 days of culture without transferring them
into a new medium for embryo development. Working with
direct somatic embryogenesis in orchids, the leaf tip and
the cut end had the most competence to form embryos from
leaf explants when cultured on TDZ-containing medium in
Oncidium and Dendrobium, respectively (Chen and Chang
2001; Chung et al. 2005; Chung et al. 2007), while the cut
end gave the best embryogenic response than other leaf
regions in this study (Table 1). Leaf explants of P. amabilis
had higher percentage of embryo-bearing explant than
Phalaenopsis ‘Nebula’, but had lower mean number of
embryos per explant (Table 1).
Effect of subculture period on direct somatic
embryogenesis
Based on our knowledge, the leaf cultures of Phalaenopsis
became brown easily and then necrotic when the subculture

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Table 1 Effect of induction period on direct somatic embryogenesis from leaf explants of P. amabilis and Phalaenopsis ‘Nebula’
Induction Percentage of Percentage of embryogenesis on different explant locations (%) No. of embryos per
period (days) embryogenesis on responding explant
the entire explant (%) CE Ad Ab LT

P. amabilis
30 35 a 30 a 0a 0a 0b 1.3
45 40 a 40 a 5a 0a 0b 3.1
60 50 a 40 a 10 a 0a 20 a 8.2
Phalaenopsis ‘Nebula’
30 45 b 45 a 15 a 0a 0a 2
45 60 ab 60 a 15 a 0a 5a 2.9
60 80 a 80 a 30 a 5a 5a 3.5
Data were scored after 30, 45 and 60 days of culture, respectively. The amounts of direct embryo formation were counted at entire explants or at
different explant locations (LT, leaf tips; Ad, adaxial sides; Ab, abaxial sides; and CE, cut ends). Data expressed as percentage were transformed
using arc sine prior to ANOVA and converted back to the original scale (Compton, 1994). Five replicates (dishes) each with four leaf explants
were performed for each treatment. Means with same letters within columns are not significantly different at p \ 0.05 (Duncan, 1955)

Table 2 Effect of subculture period on direct somatic embryogenesis from leaf explants of P. amabilis on 3 mg/l of TDZ- or 3 mg/l of BA-
containing medium
Growth Subculture Percentage of Percentage of Percentage of embryogenesis No. of embryos per
regulator period (days) browning (%) embryogenesis on on different explant locations (%) responding explant
the entire explant (%)
CE Ad Ab LT

TDZ (3 mg/l) 30 25 ab 30 a 20 a 15 a 5a 0b 6.2

45 5b 60 a 45 a 20 a 0a 25 a 6.7
60 45 a 50 a 40 a 20 a 0a 0b 3.7
BA (3 mg/l) 30 10 b 30 a 25 a 15 a 0a 0b 5.2
45 20 ab 40 a 35 a 5a 0a 10 ab 6.8
60 15 ab 25 a 15 a 10 a 0a 0b 3.4
Data were scored after 60 days of culture. The amounts of direct embryo formation were counted at entire explants or at different explant
locations (LT, leaf tips; Ad, adaxial sides; Ab, abaxial sides; and CE, cut ends). Data expressed as percentage were transformed using arc sine
prior to ANOVA and converted back to the original scale (Compton, 1994). Five replicates (dishes) each with four leaf explants were performed
for each treatment. Means with the same letters within columns are not significantly different at p \ 0.05 (Duncan, 1955)

period is not suitable. As the result showed in Table 2, 45
days of subculture gave the best embryogenic response and
the lowest percentage of explant browning on TDZ-con-
taining medium. However, at 3 mg/l of BA, the 45 days of
treatment showed the best embryogenic response but also
the highest percentage of explant browning. In all of the
treatments, cut ends still had the highest ability to form
embryos (Table 2).
Effect of explant length on direct embryo formation
Obviously, 1 cm long explants had the best embryogenic
responses than 1.5 and 2 cm long explants after 2 months of
culture on 1/2 MS medium supplemented with 3 mg/l of
TDZ. Leaf explants longer than 1 cm turn brown easily and
had extremely low embryogenic response. Therefore, older
explants longer than 1 cm were not suitable for embryo
induction in P. amabilis.
Effects of BA and NAA on development of leaf-derived
embryos

The leaf explants taken from older donor plants had longer When cultured on hormone-free medium, 60% of leaf-
length, and these leaves were cut into three segments for derived embryos could be converted into plantlets. How-
testing to their competences to form embryos (Table 3). ever, the percentage of browning was high (Table 4).

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Acta Physiol Plant (2010) 32:621–627 625

Table 3 Effect of explant length on direct somatic embryogenesis from leaf explants of P. amabilis
Explant Percentage of Percentage of Percentage of embryogenesis at different explant locations (%) No. of embryos per
length (cm) browning (%) embryogenesis on responding explant
the entire explant (%) CE Ad Ab LT

1 30 a 30 a 20 a 25 a 5a 5a 13.5
1.5 60 a 0b 0b 0b 0a 0a 0
2 15 a 0b 0b 0b 0a 0a 0
Data were scored after 60 days of culture. The amounts of direct embryo formation were counted at entire explants or at different explant
locations (LT, leaf tips; Ad, adaxial sides; Ab, abaxial sides; and CE, cut ends). Data expressed as percentage were transformed using arc sine
prior to ANOVA and converted back to the original scale for demonstration in table (Compton, 1994). Five replicates (dishes) each with four leaf
explants were performed for each treatment. Means with the same letters within columns are not significantly different at p \ 0.05 (Duncan,
1955)

Table 4 Effects of BA and NAA on plantlet conversion of leaf-derived embryos of P. amabilis

BA (mg/l) NAA (mg/l) Plantlet conversion (%) Percentage of browning (%)
45 days 60 days 45 days 60 days

0 0 60 b 60 bcd 40 ab 40 abc
0.5 0 90 a 90 a 10 c 10 d
1 0 80 ab 80 ab 20 bc 20 cd
0 0.5 65 b 45 d 45 a 55 a
0 1 75 b 55 cd 25 ab 45 ab
0.5 0.5 60 b 60 bcd 40 ab 40 abc
0.5 1 75 b 70 bc 25 ab 30 bc
1 0.5 75 b 75 bc 25 ab 25 bc
Data were scored after 45 and 60 days of culture. The percentage of plantlet conversion was counted as the final numbers of survival plantlets
divided by the initial numbers of incubated leaf-derived embryos. Data expressed as percentage were transformed using arc sine prior to ANOVA
and converted back to the original scale (Compton, 1994). Four replicates (dishes) each with five embryos were performed for each treatment.
Means with same letters within columns are not significantly different at p \ 0.05 (Duncan, 1955)

BA promoted embryo development and at 0.5 mg/l gave
the highest rate of plantlet conversion (90%) and the lowest
browning rate (Table 4). On the contrary, NAA inhibited
embryo development and at 0.5 and 1 mg/l both decreased
plantlet conversion rate and gave a higher percentage of
browning explant (Table 4). Besides, BA reduced the
inhibitory effects of NAA on embryo development (Table 4).
SEM observation
It reveals that somatic embryos formed directly from the
surfaces of leaf explants of both cultivars in an asynchro-
nous way (Fig. 2a and b). In other words, different devel-
opmental stages of embryos were obtained on the same
cultured expant (Fig. 2a and b). In addition, theses embryos
clustered on the leaf surface and often with an average of
5–10 embryos per group. Obviously, the basal regions of
embryos showed a partial connection to each others and to
the parent explant (Fig. 2d). After transferring the embryos
into the growth medium, the size of embryo gradually
enlarged and sheath leaves developed (Fig. 2c). At this
stage, the entire embryos were enclosed by two of the
sheath leaves (Fig. 2c). With a low frequency, both sides of
leaf explants were competent to form embryos (Fig. 2e). At
maturity, the sheath leaves unfold and the shoots further
developed (Fig. 2f).
In summary, the suitable factors for induction of direct
somatic embryogenesis from Phalaenopsis leaf explants
were (1) 60 days in darkness for induction period; (2) 45
days for subculture period; (3) the leaf explant length
should be less than 1 cm; and (4) 0.5 mg/l of BA for
plantlet conversion.

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626 Acta Physiol Plant (2010) 32:621–627

Fig. 2 SEM observation on

direct somatic embryogenesis
from leaf explants of
Phalaenopsis. (The bar in upper
left denotes fitting for all
panels). a Direct somatic
embryogenesis from a leaf
explant of P. amabilis
(bar = 1.5 mm). b Direct
somatic embryogenesis from a
leaf explant of Phalaenopsis
‘Nebula’ (bar = 1 mm).
c Several mature leaf-derived
embryos of P. amabilis
(bar = 250 lm). d Clusters of
embryos of Phalaenopsis
‘Nebula’ (bar = 1.5 mm).
e Mature embryos from both
sides of a leaf of Phalaenopsis
‘Nebula’ (bar = 1.5 mm).
f A mature leaf-derived embryo
with sheath leaves (0.5 mm)

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