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Not Sci Biol, 2015, 7(2):192-197
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Not Sci Biol, 2015, 7(2):192-197. DOI: 10.15835/nsb.7.2.9546
Organic Additives Improves the in Vitro Growth
of Native Orchid Vanda helvola Blume
Devina DAVID1, Roslina JAWAN2, Hartinie MARBAWI2, Jualang Azlan GANSAU2*
1
University Malaysia Sabah, Faculty of Sustainable Agriculture, Locked Bag No.3, 90509 Sandakan Sabah, Malaysia; ddevina@ums.edu.my
2
University Malaysia Sabah, Faculty of Science and Natural Resources, UMS Road, 88450 Kota Kinabalu Sabah,
Malaysia; roslinaj@ums.edu.my; hartinie@ums.edu.my; azlanajg@ums.edu.my (*corresponding author)
Abstract
In vitro seed germination has been proven to be the most efficient technique to propagate orchid. The application of this aseptic
technique has contributed to conservation of many endangered orchid species. In this study, undehisced capsules of Vanda helvola Blume
were collected from Orchid Conservation Centre in Lagud Sebrang Agriculture Park, after 120 days from hand pollination and aseptically
cultured on three types of basal media such as Murashige and Skoog (MS), Knudson C (KC) and Vacin and Went (VW). After 90 days of
culture, 66.40 ± 4.14% of seeds successfully germinated on KC medium. The effect of organic additives such as tomato juice, coconut water,
peptone and yeast extract at different level of concentrations in KC basal medium were also tested on seed germination and seedling
development of this native orchid. After 90 days of culture, over 90% of seeds were tremendously germinated on KC medium
supplemented with 10% or 15% (v/v) of tomato juice. The incorporation of peptone at 0.1% (w/v) in KC basal media promoted rapid
development of protocorm to seedling. Seedlings on this treatment produced an average of three leaves and two roots after 90 days of
culture and were successfully acclimatized.
Keywords: basal media, conservation, in vitro seed germination, protocorm, Orchidaceae
Introduction
Orchidaceae belongs to the largest family of flowering plants in
the world, with an estimation of 800 genera and 20,000 species
around the world. It was estimated that 2,500-3,000 of orchid
species are found in Borneo (Chan et al., 1994). Vanda helvola
Blume is an epiphytic orchid, with a vast distribution in Borneo, but
mostly found at Mt Kinabalu and Tambunan areas. The flower has
a coppery red mountain form and it flowered at regular intervals
throughout the year.
The illegal activity on orchid collection from the natural habitat
has resulted in nearly extinction for many native orchids including V.
helvola. Since this orchid was listed in Appendices II of the
regulations formulated by the Committee for International Trade in
Endangered Species Wild Fauna and Flora (CITES 2014), an
effective strategy becomes essential to conserve and multiple this
orchid species, for conservation as well as for horticultural purposes.
Plant tissue culture techniques have been proven as an
important tool for orchid micropropagation, as well as to conserve
many rare or endangered orchid species (Harrison and Arditti,
1978; Vij et al., 1994; Shimora & Koda, 2004; Zeng et al., 2012).
As for Vanda sp. the in vitro seed germination protocol has been
widely used for mass propagation of superior varieties such as V.
joaquim (Rao and Advhani, 1964), Vanda hybrids (Mathews and
Rao, 1980; Johnson and Kane, 2007), V. tessellata (Roy and
Banerjee, 2002), V. teres (Sinha and Roy, 2004), V. coerulea Grif ex.
Lindl (Roy et al., 2011) and V. dearei (Jualang et al., 2014).
The incorporation of organic additives such as potato extract,
tomato juice, banana, coconut water and peptone into the growth
medium has influenced the protocorm and seedling development
of some epiphytic orchids as reported previously in V. roxburgii
(Islam et al., 2011), Cymbidium penduculum (Saranjeet and
Buthani, 2012), Phalaenopsis hybrids (Shekarriz et al., 2014) and
V. dearei (Jualang et al., 2014).
Therefore, the current study was carried out to develop and
establish an efficient protocol for in vitro propagation, by studying
the effect of basal media and organic additives on in vitro seed
germination and seedling development of V. helvola.
Materials and methods
Capsule source and sterilization
Undehisced capsules of Vanda helvola Blume were collected
from Orchid Conservation Centre in Lagud Sebrang Agriculture
Park, after 120 days from hand-pollination. Capsules of V. helvola
were gently scrubbed with soap solution then rinsed and left under
running tap water for 30 min. The capsules were surface sterilized
by immersion in a 30% (v/v) of Clorox® solution added with two
drops of Tween 20 and agitated for another 20 min. The capsules
were then rinsed five times with sterilized distilled water, followed
by dipping in 95% (v/v) ethanol for 10 s and passed briefly through
flame.
Received: 06 Mar 2015. Received in revised form: 16 June 2015. Accepted: 16 June 2015. Published online: 21 June 2015.
David D et al. / Not Sci Biol, 2015, 7(2):192-197
193
Fig. 1. (a) Flowering plant of Vanda helvola Blume; (b) Orchid capsule
Effect of basal media on seed germination
Three basal media were assayed for their effectiveness in
promoting seeds germination of V. helvola. Seeds were aseptically
inoculated on Murashige and Skoog (MS) (Murashige and Skoog,
1965), Knudson C (KC) (Knudson, 1946) and Vacin and Went
(VW) (Vacin and Went, 1949) basal media and cultures were
maintained at 25 ± 2 °C under continuous light.
Effect of organic additives on seed germination
The effect of organic additives on seed germination was tested
using KC as basal medium. The medium was individually
supplemented with tomato juice or coconut water at 10, 15 and
20% (v/v) and peptone or yeast extract at 0.1, 0.2 and 0.3% (w/v),
respectively. Tomato juice was prepared by cutting the freshly ripe
tomato into smaller pieces (without skin and seed) and grounded
in a mixer. Coconut water was obtained from young and tender
fruit purchased from local market. The coconut water was filtered
prior using. Peptone and yeast extract were obtained from BactoTM
(Beckon, France). Basal medium devoid additive served as control
and all treatments were maintained at 25 ± 2 °C under continuous
light.
Data Collection
For seed germination study, observation and data collection
were carried out up to 90 days of culture with an interval of 10
days. The percentage of germinated seeds was calculated by
dividing the number of germinated seeds by the total number of
cultured seeds (Shimora and Koda, 2004).
For seedling development study, the percentage of leaf and
root formation was recorded. The length of leaf (third leaf) and the
longest root were measured in millimetre (mm) every month.
Statistical analysis
Experiments were carried out in a completely randomized
design (CRD). Every treatment in all experiments consisted of 10
replicates. Data were analyzed by SPSS (Statistical Package for
Social Science) software and subjected to analysis of variance
(ANOVA). The mean values were compared using Duncan’s
Multiple Range Test (DMRT) at p < 0.05 significance level.
Results and discussions
Effect of organic additives on seedlings development
To see the effectiveness of organic additives on seedlings
development, 60 days-old of protocorm derived from in vitro
germination was used as explant in this study. A total of 15
protocorms were selected and cultured on every petri dish
containing KC basal media supplemented with different organic
additives such as tomato juice or coconut water at 10, 20 or 40%
(v/v); peptone or yeast extract at 0.1, 0.2 and 0.4% (w/v). All
treatments were maintained at 25 ± 2 °C under continuous light.
Capsule and seed characterization
Flower of V. helvola varies from dull pale yellow – brown to
coppery red with dull yellow (Fig. 1a). Pollination of orchid
flowers was done manually and the green capsules harvested after
120 days from hand pollination. Weight and length of capsule
were determined at 12.56 g and 9.5 cm respectively (Fig. 1b).
Seeds of V. helvola were minute, dust-like, ranging from 200 µm
to 300 µm in length. Seed at this age was yellowish and the testa
(seed coat) was transparent. The characterization of capsule and
seed of V. helvola were comparatively smaller than V. dearei as
previously reported by Jualang et al. (2014).
Acclimatization of V. helvola seedlings
Seedlings of V. helvola at 2.5 cm height and above, with welldeveloped rhizomes and shoots were taken out from flasks and
washed thoroughly under tap water to remove traces of agar-gelled
medium. Seedlings were treated with 0.5% (w/v) fungicide for 15
min. They were then planted in pots containing brick pieces and
coconut husks (1:2) and mulched with moss (Sphagnum sp.). The
brick pieces and coconut husk were autoclaved-sterilized prior to
potting, while Sphagnum mosses were treated with 0.5% (w/v)
fungicide for 15 min. The plants were initially covered with a
polythene sheet for one month to maintain high humidity and
were irrigated twice a week with tap water.
Effect of basal media on in vitro seed germination
Among the three types of basal media, KC medium was
found superior for germination of V. helvola seeds, compared to
MS and VW media. Seeds sown on KC medium swollen within
28 days, developed into protocorms stage by day 60. Observation
after 90 days of culture revealed that the germination percentage
was significantly high in KC basal medium, with 66.40 ± 4.14%
(Table 1). It was followed by MS medium with 18.90 ± 4.01%
and VW medium yielded the lowest germination percentage at
9.70 ± 1.89%.
The in vitro seed germination of V. helvola was obviously
affected by the type of basal medium. According to Arditti and
David D et al. / Not Sci Biol, 2015, 7(2):192-197
194
Table 1. Effect of basal media on germination of Vanda helvola seeds after 30, 60 and 90 days of culture
y
Germination percentage (% ± SD) by days
Germination response
(day)
30
60
90
KC
28
12.10 ± 2.07a
33.70 ± 1.56a
66.40 ± 4.14a
MS
29
1.90 ± 0.74b
10.30 ± 1.57b
18.90 ± 4.01b
VW
35
0.30 ± 0.67c
6.90 ± 1.72c
9.70 ± 1.89c
y
Data are means of 10 replicates. Mean followed by the same letter (column) did not differ significantly at p < 0.05 according to Duncan multiple range tests. SD:
Standard deviation; Basal media KC (Knudson C, 1946); MS (Murashige & Skoog,1962); VW (Vacin & Went, 1949)
Basal media
Table 2. Effect of organic additives on Vanda helvola seeds germination, cultured on KC basal medium
y
Germination percentage (% ± S.D)
Germination
response (day)
30 days
60 days
90 days
0
28
12.10 ± 2.08e
33.70 ± 1.57e
66.40 ± 4.14e
10
23
42.80 ± 2.53c
81.60 ± 1.35a
91.20 ± 1.55a
Tomato juice
15
23
44.30 ± 3.53c
83.90 ± 2.47a
92.30 ± 1.42a
(%, v/v)
b
a
20
21
47.90 ± 4.31
83.10 ± 3.18
80.60 ± 1.90b
10
38
0h
9.20 ± 3.08g
19.80 ± 2.49j
Coconut water
h
fg
15
38
0
11.70 ± 3.56
24.20 ± 4.02i
(%, v/v)
20
35
0h
13.80 ±3.12f
26.50 ± 4.74i
g
c
0.1
28
3.40 ± 1.78
47.30 ± 5.12
73.20 ± 2.97d
Peptone
0.2
28
8.20 ± 1.48f
49.50 ± 4.77d
74.90 ± 2.08cd
(%, w/v)
0.3
28
7.10 ± 1.10f
52.50 ± 2.99d
76.10 ± 2.02c
0.1
15
44.80 ± 4.08c
74.20 ± 2.44b
62.10 ± 3.03f
Yeast extract
0.2
15
39.10 ± 4.89d
73.90 ± 1.72b
58.10 ± 3.14g
(%, w/v)
0.3
14
53.50 ± 4.09a
73.10 ± 3.62b
49.20 ± 3.01h
y
Data are means of 10 replicates. Mean followed by the same letter (column) did not differ significantly at p < 0.05 according to Duncan’s Multiple Range Tests. SD
– Standard deviation.
Organic
additive
Control
Concentration
Fig. 2. Seedling development stages of Vanda helvola. (a) Seeds 10 days of culture. (b) Embryo enlarged after 20 days of culture. (c) Germinated seeds
after 30 days of culture. (d) Protocorm at 40 days of culture. (e) Initiation of shoot from protocorm. (f) Protocorm with two leaves. (g) Established
seedling with two leaves and two roots. (h) Seedling treated with 40% (v/v) tomato juice. (i) Acclimatized seedlings of Vanda helvola at Sabah
Agriculture Park. Bar (a – h) 1.0 mm; (i): 5 cm
David D et al. / Not Sci Biol, 2015, 7(2):192-197
195
Table 3. Effect of organic additives on Vanda helvola seedlings development, after 90 days of culture on KC basal medium
y
94.70 ± 4.92b
No. of leaf per
responsive
explant
(± SD)
2.30 ± 0.48cd
y
Organic additives
(Concentration)
Protocorms with
leaves (% ± SD)
Control
y
Leafm
Rootn
y
Length
(mm ± SD)
y
Width
(mm ± SD)
y
Length
(mm ± SD)
89.40 ± 2.12b
No. of root per
responded
explant
(± SD)
1.65 ± 0.47d
6.00 ± 1.15d
2.21 ± 0.25cd
6.00 ± 1.15d
y
Protocorms with
roots
(% ± SD)
Tomato juice (%, v/v)
10
20
40
97.30 ± 2.21ab
94.90 ± 3.07b
70.60 ± 4.65d
1.90 ± 0.74de
2.58 ± 0.50bc
1.71 ± 0.41ef
97.90 ± 1.85a
96.00 ± 2.91a
76.90 ± 5.47c
2.25 ± 0.72bc
2.42 ± 0.62b
3.14 ± 0.51a
8.20 ± 1.14c
9.30 ± 0.95b
6.12 ± 0.48d
2.56 ± 0.21ab
2.61 ± 0.23ab
2.79 ± 0.47a
8.26 ± 1.09b
9.48 ± 1.48a
9.58 ± 0.96a
Coconut water (%,v/v)
10
20
40
84.20 ± 4.73c
80.80 ± 6.56c
58.60 ± 6.80e
2.19 ± 0.54cd
2.05 ± 0.55de
1.29 ± 0.44fg
77.00 ± 4.27c
40.90 ± 3.45d
0f
1.82 ± 0.39d
1.06 ± 0.41e
0g
4.94 ± 0.60e
4.61 ± 0.85e
2.60 ± 0.52f
2.13 ± 0.32cde
2.16 ± 0.33cde
1.96 ± 0.25de
1.8 ± 0.46e
1.8 ± 0.41e
0f
Peptone (%, w/v)
0.1
0.2
0.4
99.50 ± 1.08a
94.20 ± 4.66b
32.30 ± 4.08f
3.10 ± 0.74a
2.91 ± 0.30ab
2.21 ± 0.34cd
98.70 ± 1.34a
88.50 ± 6.19b
14.40 ± 3.13e
1.97 ± 0.41d
1.80 ± 0.63d
0.60 ± 0.19f
10.97 ± 0.75a
10.84 ± 0.69a
2.85 ± 0.51f
2.37 ± 0.16bc
2.37 ± 0.20bc
2.04 ± 0.22de
6.86 ± 0.54c
6.58 ± 0.61c
1.26 ± 0.24e
0.1
83.70 ± 3.33c
2.28 ± 0.52cd
0f
0g
5.98 ± 0.55d
2.11 ± 0.10de
0f
d
g
f
g
e
e
0.2
80.90 ± 4.09
1.54 ± 0.28
0
0
5.51 ± 0.60
1.95 ± 0.18
0f
0.4
0g
0h
0f
0g
0g
0f
0f
y
Data are means of 10 replicates. Mean followed by the same letter did not differ significantly at p < 0.05 according to Duncan’s Multiple Range Tests. SD: Standard
Deviation; m the length of leaf was recorded from the third leaf of seedling ; n the length of root was recorded from the longest root formed
Yeast extract (%, w/v)
Ernst (1993), germination was considered difficult, as specific
nutritional and environmental conditions are needed for in vitro
germination of orchid seeds. Previous studies have revealed that
seed germination of orchid depends on nitrogen source
contained in the germination medium (Roy and Banerjee, 2002;
Kauth et al., 2006; Stewart and Kane, 2006). In the present
study, the total nitrogen content in MS medium (60.01 mM) is
considerably higher than in KC (16.04 mM) and VW (8.97
mM). It was also observed that seeds of V. helvola prefer to
germinate better in KC medium. This might be due to high
water requirement for seed germination, so the elimination of
mineral salt is advantageous (Rasmussen, 1995) or the genotype
dependent (Johnson and Kane, 2007) of V. helvola as an
epiphytic orchid. The efficacy of the KC medium for seed
germination was also demonstrated earlier in Blue Vanda (Seeni
and Latha, 2000) and V. dearei (Jualang et al., 2014).
culture (Table 2). Even though seeds were germinated earlier in
this treatment, the yield however decreased to 49.20 ± 3.01% as
some of the protocorms turn to yellowish and later died after 90
days of culture. The supplementation of 10 or 15% (v/v) tomato
juice on KC basal media had significantly increased the
germination percentage to 91.20 ± 1.55% and 92.30 ± 1.42%
respectively after 90 days of culture. Meanwhile, coconut water at
10, 15 or 20% (v/v) gave the lowest percentage of germination
after 90 days of culture. The incorporation of suitable organic
additives obviously increased the germination rate of V. helvola
compared to KC medium alone. The development from seed to
protocorm was fast and the size of protocorm was larger on
medium containing tomato juice compared to other treatments.
This result was supported earlier by Rao and Avadhani (1964)
who concluded that the addition of tomato juice had a
significant influence on seed germination and protocorm
differentiation on Vanda Joaquim.
Effect of organic additives on in vitro seed germination
The process of seed germination started when the embryo
began to swell after 10 days of inoculation on KC medium
containing 10% (v/v) of tomato juice (Fig. 2a). Colours of
embryos varied from milk-white at the beginning of germination
to bright green from 10 to 20 days of culture. Embryos were
then enlarged by two times and occupied the whole seed coat
(Fig. 2b). Within 23 days of culture, the embryos were finally
discharged from the seed coat, moment when it was considered
that the seeds germinated (Fig. 2c). According to Harrison and
Arditti (1978) and Johnson & Kane (2007), a seed was
considered germinated after the embryo discharged from the
seed coat. The germinated seeds were then enlarged further to
form a cone-shaped known as protocorm by approximately 40
days (Fig 2d). Some of the protocorms formed papillae and they
remained at this stage up to 65 days. It was also observed that the
protocorms were spherical and green.
The addition on KC basal medium of organic additives such
as tomato juice, coconut water, peptone or yeast extract gave the
earliest respond for seed germination on medium containing
0.3% (w/v) of yeast extract which germinated within 14 days of
Effect of organic additives on seedling development
Protocorms of V. helvola responded variably when cultured
on different organic additives for the first month of culture. The
seedlings development process began with the formation of tiny
leaf primordial at the apex of protocorm (Fig. 2e). First leaf
initiated after 40 days of culture, followed by a formation of
second leaf one month after that (Fig. 2f). The formation of first
root started after 60 days of culture and it was followed either by
the formation of a second root or the third leaf (Fig. 2g). After 90
days of culture, a seedling could produce up to three leaves and
two roots.
The effect of organic additives on seedling development is
presented in Table 3. The results revealed that the addition of
organic additives had significantly enhanced the development of
seedlings compared to control medium. It was observed that
99.50 ± 1.08% of protocorms treated in KC medium containing
0.1% (w/v) peptone successfully produced 3.10 ± 0.74 leaves
after 90 days of culture with an average length of leaves at 10.97 ±
0.75 mm per responsive explant. The nutrients requirements for
V. helvola growth varied from seed to seedling stage. From the
David D et al. / Not Sci Biol, 2015, 7(2):192-197
196
current findings, the addition of peptone at 0.1% (w/v) was
beneficial in promoting the leaf and root formation of V. helvola.
Previously, Nhut et al. (2008) described that peptone has been
used as a source of carbon and nitrogen in plant tissue culture.
The characteristic of peptone being water soluble protein
hydrolase with very high amino acid content might also
contribute to culture growth (Saranjeet and Bhuthani, 2012).
The beneficial effect of peptone incorporated into basal media to
improve the growth rate of V. tessellata was also reported earlier
(Roy and Banerjee, 2002), or to increase uniformity and seedling
development of C. tuberosus (Kauth et al., 2006) and in vitro
multiplication of Phalaenopsis hybrid (Shekarriz et al., 2014).
The addition of tomato juice at 40% (v/v) promoted the
highest number of roots. Explants cultured on this treatment
produced 3.14 ± 0.51 roots, with average length of 9.58 ± 0.96
mm after 90 days of culture. Seedlings treated with tomato juice
at concentrations of 10, 20 or 40% (v/v) however, were observed
slightly yellowish compared to other treatments (Fig. 2h). This
might be contributed by the bright red carotenoid pigment in
tomato. In previous study by Rao and Avadhani (1964), tomato
juice improves the rate of cell division, maximum vacuolation
and early differentiation of the orchid embryos. Rao and
Avadhani (1964) also added that seedlings treated with tomato
juice could produce high number of roots 3 to 4 times longer,
thin walled and branched. Several studies also demonstrated the
effect of tomato juice on growth, however the finding gave low
growth rate on Phalaenopsis violacea (Gnasekaran et al., 2012)
and Dendrobium hybrids (Nambiar et al., 2012).
Supplementation of coconut water or yeast extract was not
significant on promoting the V. helvola seedlings development
when compared to control medium, which is devoid of any
additive. Even though coconut water is commonly used in
orchid micropropagation, this finding however is in contrast
with previous results that coconut water was found beneficial on
seed germination of Vanda coerulea x Ascocenrtum auranticum
(Kishor et al., 2006) and Geodorum sp. (Sheelavantmath et al.,
2000). The incorporation of yeast extract was also not suitable
for V. helvola development even though in previous study by
Jawan et al. (2010) it was suggested that 0.2% (w/v) of yeast
extract significantly increased the shoot formation of Vanda
dearie, an endemic orchid of Borneo.
Acclimatization of V. helvola seedlings
The mean survival rate of V. helvola seedlings was 55.67%
after six months of acclimatization with size reached up to 4.72 ±
0.22 cm of height and 7.00 ± 0.71 of leaf produced per explant.
The seedlings were then shifted to former habitat at Orchid
Conservation Centre in Lagud Sebrang Agriculture Park for
further growth (Fig. 2i). The acclimatization of some orchids has
been successfully demonstrated in V. coerula planted on charcoal
chips and broken tiles (2:1) (Seeni and Latha, 2000; Malabadi et
al., 2004); Dendrobium tosaense planted on moss (Sphagnum sp.)
(Lo et al., 2004) and Ascocenda ‘Kangla’ seedlings planted on
brick:charcoal (2:1) and mulched with moss (Sphagnum sp.)
(Kishor et al., 2006).
Conclusions
An efficient protocol for in vitro seed germination and
seedlings development of V. helvola was achieved using selected
organic additives at suitable concentration. After 90 days of culture,
over 90% of seeds germinated on KC medium supplemented with
10% or 15% (v/v) of tomato juice. The incorporation of peptone
at 0.1% (w/v) in KC basal media promoted rapid development of
protocorm to seedling. Seedlings on this treatment produced an
average of three leaves and two roots after 90 days of culture and
were successfully acclimatized. This protocol has a tremendous
potential as a conservation tool to protect the local orchid species in
Borneo.
Acknowledgements
We would like to acknowledge Mr. Jain Linton and the
team from Lagud Sebrang Agriculture Park for providing the
orchid capsules for this research.
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