Agric. sci. dev., Vol(3), No (10), October, 2014. pp.

317-322

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Agriculture Science Developments

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Copyright 2014. All rights reserved for TI Journals.

Coconut Water and Peptone Improve Seed Germination and Protocorm

Like Body Formation of Hybrid Phalaenopsis
Parisa Shekarriz *
Department of Horticultural Sciences, Karaj Branch, Islamic Azad University, Karaj, Iran.

Mohsen Kafi
Department of Horticultural Sciences, Faculty of Agriculture Science & Engineering, University of Tehran, Karaj, Iran.

Shirin Dianati Deilamy

Department of Horticultural Sciences, College of Abouraihan, University of Tehran, Tehran, Iran.

Masoud Mirmasoumi
Department of Plant Sciences, Faculty of Biology, College of Sciences, University of Tehran, Tehran, Iran.
*Corresponding author: parisashekarriz@gmail.com

Keywords

Abstract

Phalaenopsis
Protocorm like body
Coconut water
Peptone
Media culture

Phalaenopsis is one of the most popular orchids in the market and it is easy to flowering. Orchid seed is
rather difficult to germination rather than other angiospermic seeds, because of lack endosperm, radical and
leaf rudiments. Seed germination and protocorm like body (PBL) formation of Phalaenopsis hybrid
Manchester was performed. This study was conducted to determine the effects of coconut water (CW),
peptone and optimal media culture. Seeds from sterilized capsules cultured on five medium (modified
FAST, modified KC, modified MS, modified MS and modified VW) containing 0 and 15% (v/v) and 0
and 2 gl-1 peptone. The highest percentage of survival percentage on modified KC 2 gl-1 peptone without
CW was observed. The highest germination percentage was observed in 15% (v/v), 2 gl-1 peptone and
modified VW medium. This show no significant difference with modified FAST medium containing 15%
(v/v) and 2 gl-1 peptone, modified KC medium containing 15% (v/v) without peptone and modified FAST
medium containing 2 gl-1 peptone without CW and the highest PBL number formation was in modified
MS medium with peptone and CW with mean value 3.5. Overall, this study showed that Phalaenopsis seeds
cultured on a MS medium containing CW and peptone can be used for clone propagation.

1.

Introduction

The Orchidaceae is one of the largest families of flowering plant which is economically valuable in the floriculture industry as cut flowers and
potted plants [1]. Phalaenopsis hybrids are the most popular orchid genus which is result of intensive breeding of plants of increasing market
value as cut flowers and even more as potted plants [2]. Orchid seeds are very unique than the seeds of other flowering plants. They are very
minute, dust like and produced in large numbers within a capsule or pod but lacks storage tissue [3]. Orchids grow in nature through seeds but in
absence of mycorrhizal association they do not germinate in adequate numbers, so it remains as a vulnerable species [4]. Most of the
commercially importance orchids have been propagated by regenerating protocorm-like bodies (PLBs) [5]. In the first tissue culture study on a
new system, it is essential to prepare a perfect medium to fulfill specific requirements of that tissue and nutritional requirements for optimal
growth of a tissue in vitro may vary with the species [6].
There are some studies on the most suitable culture media for sowing orchids such as Cymbidium with Murashige and Skoog [MS, 7] medium
[8], Dendrobium with MS [9], Phalaenopsis with XER [10] medium [11], Teixeira Cymbidium (TC) medium and Dactylorhiza fuchsia with
Knudson C (KC) medium [12; 13].
Growth rate of the tissues can be increased by the addition of organic supplements and plant extracts [14]. Many different organic additives like
coconut water, banana pulp, peptone, tomato juice, slap honey, date palm syrup, corn extract, papaya extract and beef extract influenced medium
culture and provide undefined mixture of organic nutrients and growth factors [15; 16]. Coconut milk is the extract of white, solid endosperm of
matured coconut after grinding and squeezing [17]. This compound is often used when no other composition of known defined components
produces the desire growth or development [18].Coconut water (CW) is the colorless liquid endosperm of green coconuts (Cocos nucifera)
which its liquid endosperm contains a number of amino acids, organic acids, nucleic acids, several vitamins, sugars and sugar alcohols, plant
hormones (auxins, cytokinins), minerals and other unidentified substances, none of which alone is totally responsible for growth promoting
qualities [17]. Peptones are enzymic or acid hydrolyzates of plant or animal tissue prepared under rigidly specified production condition [19].
This study was carried out to investigate the possibility of inducing PLBs from the seeds explants of the Phalaenopsis hybrid Manchester.

2.

Materials and Methods

Capsules of popular Phalaenopsis hybrid namely Manchester were harvested 110 days post self-pollination when they were dark green and
not-cracked. They were disinfected with dish soap for 30 min, and then top water, followed by sodium hypochloride solution (commercial
bleach, 50% v/v) for 30 min afterward they rinsed in sterile distilled water (3-4 time). Finally they submerged in absolute ethanol and then
immediately flamed. The capsules were cut longitudinally into 3 sections with a sterile surgical blade and the seeds per carpel were poured in a
sterile vial containing 5 mgl-1 liquid medium and placed on vortex and shake. Three drops (10 ml-1) of liquid medium with seeds with sampler
micropipette placed on petri dishes containing five types medium: modified FAST medium [20], modified Knudson C (KC) medium, modified
MS, modified MS and modified and modified Vacin and Went (VW) medium [21]. The composition of all culture media used is described in

Parisa Shekarriz *, Mohsen Kafi , Shirin Dianati Deilamy , Masoud Mirmasoumi

318

Agriculture Science Developments Vol(3), No (10), October, 2014.

Table 1. The pH was adjusted to 5.5 prior to autoclaving. These mediums were supplemented with different concentrations of coconut water
(CW) 0 and 15% (v/v) and peptone 0 and 2 gl-1 in various combinations. All cultures were maintained at 251 C under photoperiod 16 h and
light intensity 2500 lux. There were 3 replicates per treatment an, arranged in a completely randomized design (CRD). Survival percentage,
germination percentage and protocorm like body formation were recorded. All data were analyzed using the SAS software (version 9) according
to analysis of variance (ANOVA). Percent data were arcsine transformed before performing ANOVA. The means compared by Duncans
multiple range test (DMRT) at the level of P 0.05.
Table 1. Composition of culture media used as treatment for PBL formation in Phalaenopsis orchid Manchester cultivar.

Components
Ca(NO3)24H2O
NH4NO3
KNO3
Calcium gluconate
KH2PO4
KCl
CaCl22H2O
MgSO47H2O
(NH4)2SO4
Na2Fe-EDTA
Na2MoO42H2O
CoCl26H2O
ZnSO47H2O
MnSO47H2O
MnSO44H2O
MnSO4H2O
CuSo45H2O
H3BO3
NiCl2
KI
Ascorbic acid
Nicotinic acid
Thiamine
Pyridoxine
Glycine
Inositol
Sucrose
Fructose
GelriteTM

3.

modified FAST
80
160
390
80
160
80
16
1
0.1
0.03
1
0.3
0.01
50
10
5
0.5
2
12
53000
3000

Culture media (mgl-1)

modified KC modified MS modified MS
695
500
33000
16500
3800
1900
250
3400
1700
250
8800
4400
250
500
35
37
37
50
50
5
5
1720
1720
4460
2230
5.68
5
5
1240
1240
166
166
100
100
100
100
100
100
400
400
20000
10000
20000
30000
30000
2000
2000
2000
2000
2500

modified VW
613
22.5
202
272
71.6
122
500
21.3
5.68
226
20000
2000

Results

Using this sterilization method 100% sterile seeds and culture were obtained. Seeds germinated on all media culture.
3.1 Survival percentage
CW and peptone treatment significantly affected survival percentage but there was no significant difference between five medium cultures.
According to Table 2. Between two CW concentrations, survival percentage in control was more rather than 15% (v/v). In peptone treatment, 2
gl-1 showed more survival percentage than media without peptone (Table 2). It is shown organic additive is effective in survival of Phalaenopsis
orchid Manchester cultivar.
Table 2. Survival percentage, germination percentage and protocorm like body (PBL) number which influenced by coconut water, peptone and culture media.

Treatment
CW
0
15
Peptone
0
2
Media culture
modified FAST
modified KC
modified MS
modified MS
modified VW

Survival (%)

Germination (%)

PBL formation

40.5 a
28.5 b

41.3 b
47.6 a

1.5 a
1.6 a

27.4 b
41.1 a

42.4 a
46.6 a

1.4 b
1.8 a

35.1 a
35.5 a
37.4 a
39.8 a
38.1 a

62.4 a
54.8 a
31.2 b
27.9 b
60.0 a

1.1 b
1.2 b
1.7 a
2.0 a
1.8 a

In per column, for the same variable, means with different letters were significantly different at the 5% level as determined by Duncans multiple range test
(DMRT).

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Coconut water and peptone improve seed germination and protocorm like body formation of hybrid Phalaenopsis
Agriculture Science Developments Vol(3), No (10), October, 2014.

Survival (%)

A significant interaction effects between CW and peptone were detected in survival percentage. We observed the highest survival percentage in
media without CW and 2 gl-1 peptone (Figure 1). Also interaction effect of CW and medium culture showed the highest survival percentage with
15% (v/v) CW and modified MS however, this was not statistically significant from modified KC, modified MS and modified VW medium
without CW. The lowest survival percentage was obtained in 15% (v/v) and modified MS medium (Figure 2). Significant interaction between
CW, peptone and media culture showed that the highest survival percentage was in modified KC medium with 2 gl-1 peptone and without CW
with mean value 90% (Table 3).

60
b

40

20

0 peptone

2
0
15
Coconut concentration (v/v)

Survival (%)

Figure 1. Interaction effect between coconut water and peptone on survival percentage

60
50
40
30
20
10
0

b b

a
b

bc

0 CW
c

FAST

KC

MS

15
1/2MS

VW

media culture
Figure 2. Interaction effect between coconut water and media culture on survival percentage.

Table 3. Effect of coconut water, peptone and media culture on studied characteristic.

CW (v/v)
0
0
0
0
0
0
0
0
0
0
15
15
15
15
15
15
15
15
15
15

Treatment
Peptone (gl-1) Media culture
0
FAST
0
KC
0
MS
0
MS
0
VW
2
FAST
2
KC
2
MS
2
MS
2
VW
0
FAST
0
KC
0
MS
0
MS
0
VW
2
FAST
2
KC
2
MS
2
MS
2
VW

Survival (%)

Germination (%)

PBL formation

0g
2.9 g
36.5 cde
47.4 bcd
31.4 def
50.0 bcd
90.0 a
58.4 bcd
12.3fg
65.2 bcd
35.3 de
30.0 def
9.1 fg
29.7 def
39.8 cde
19.3 efg
9.6 fg
0g
67.1 bcd
19.3 efg

46 cd
53.7 bc
20.3 e
23.6 e
57.1 bc
67.2 ab
44.3 cd
46.7 cd
26.6 e
51.4 c
50.0 c
67.3 ab
28.8 e
33.2 de
58.1 bc
74.5 ab
54.4 bc
30.5 e
29.8 e
74.5 a

0j
0.2 ij
1.0 fghi
0.9 ghi
2.5 b
2.0 bcde
2.1 bcd
1.9 bcdef
2.2 bc
1.7 bcdefg
0.7 hij
1.2 defgh
2.3 b
1.8 bcdef
1.0 fghi
1.4 cdefgh
1.2 efgh
1.0 fghi
3.5 a
1.4 cdefgh

3.2 Seed germination

The effect of CW, peptone and media culture on seed germination percentage are shown in Table 2. CW, peptone and media culture were
significantly influenced germination percentage. Germination in presence of CW was better than control. Germination increased when modified
FAST, modified KC and modified VW medium were used for seed germination in comparison to modified MS and modified MS medium
(Table 2). Interaction effect between peptone and media culture showed that 2 gl-1 peptone and modified FAST medium produced the highest
germination percentage and the lowest germination percentage was obtained with modified MS and modified MS without peptone (Figure 3).

Parisa Shekarriz *, Mohsen Kafi , Shirin Dianati Deilamy , Masoud Mirmasoumi

320

Agriculture Science Developments Vol(3), No (10), October, 2014.

Interaction effect between CW, peptone and media culture significantly affected germination percentage and the highest germination percentage
was observed in 15% (v/v), 2 gl-1 peptone and modified VW medium. This show no significant difference with modified FAST medium
containing 15% (v/v) and 2 gl-1 peptone, modified KC medium containing 15% (v/v) without peptone and modified FAST medium containing 2
gl-1 peptone without CW (Table 3).

Germination (%)

80

b
60

bc

cd

40

f ef

0 peptone

20

0
FAST

KC

MS

1/2MS

VW

Media culture
Figure 3. Interaction effect between peptone and media culture on germination percentage.

PBL number

3.3 PBL formation

Peptone and media culture significantly affected PBL formation in Phalaenopsis hybrid Manchester but CW did not show significant effect on
PBL formation. The highest PBL number was showed with 2 gl-1 peptone in compare to control (0 gl-1). Between media culture, modified MS,
modified MS and modified VW medium showed the highest PBL formation (Table 2). Interaction effect between CW and peptone showed the
highest PBL formation in treatment with peptone and with or without CW (Figure 4). Also interaction effect between CW and media culture
showed that the highest mean was obtained in modified VW medium without CW and modified MS and MS medium with 15% (v/v) CW
(Figure 5). significant Interaction effect between peptone and media culture showed that the lowest PBL formation in modified FAST and
modified KC medium both without peptone and other treatment showed the highest mean and there were no significant difference between them
(Figure 6). Interaction effect between CW, peptone and media culture showed the highest PBL formation in modified MS medium with
peptone and CW with mean value 3.5 and the lowest PBL formation observed in modified FAST medium without peptone and CW (Table 3).

3
2
1

0 peptone

2
0

15

Coconut water (v/v)

PBL number

Figure 4. Interaction effect between coconut water and peptone on PBL number.

3
2.5
2
1.5
1
0.5
0

a
ab
c

bc

bc
c

0 CW
15

FAST

KC

MS
1/2MS
media culture

VW

Figure 5. Interaction effect between coconut water and media culture on PBL number.

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Coconut water and peptone improve seed germination and protocorm like body formation of hybrid Phalaenopsis
Agriculture Science Developments Vol(3), No (10), October, 2014.

PBL number

2.5
2

a a
a

a
a

1.5
b

1
0.5

0 peptone

0
FAST

KC

MS
1/2MS
media culture

VW

Figure 6. Interaction effect between peptone and media culture on PBL number.

4.

Discussion

Many tissue culture methods have been developed for Phalaenopsis mass propagation. The germination of the orchid seed is rather difficult in
comparisons to the germination of other angiospermic seeds, because they lack endosperm, radical and leaf rudiments [22].
In this study seed germination and PBL formation of orchid Phalaenopsis using CW and peptone in different media was examined. Peptone was
effective on PBL formation of Phalaenopsis hybrid Manchester. Peptone has been used as the source of carbon and nitrogen for plant tissue
culture [23] and some reports have shown a positive effect on the growth of explants, such as embryo production in Oncidium [24]. A special
role of peptone as factor to enhance the growth and development of plant tissues was suggested [25]. Kanjilal and Datta [26] reported that
peptone (2 g-1) was effective in promoting the survival percentage of explants a terrestrial orchid Geodorum densiflorum (Lam) Schltr. but had
no effect on PLB production.
Our result about CW showed that CW in medium decreased seed survival percentage but increased seed germination. Tawaro et al. [27] results
were in agreement with our results. They showed that seed germination of Cymbidium findlaysonianum was higher in VW medium with 15%
CW. Baque et al. [28] have shown that addition of coconut water with Hyponex medium can enhance growth attributes of Calanthe hybrids
(BukduseongHyesung and ChunkwangHyesung) up to 50 ml-1 but higher concentration of CW (100 ml-1) decreased all the growth and
morphological features. Gnasekaran et al. [15] indicated that addition of coconut water nut to the culture medium promoted proliferation of
PLBs in Vanda Kasems Delight orchid. In our study coconut water showed no significant effect on PBL formation and it was in contrast with
them.
Basal medium is a key factor to the success of a tissue culture protocol. In comparison between five media culture, we demonstrated that
modified VW medium was the best medium and showed the highest germination percentage and PBL formation. When we compared all
treatment applied in this experiment, modified MS medium with CW and peptone showed the highest PBL formation but it showed low
germination percentage. Modified FAST medium and modified KC medium showed the worst response. Park et al. [29] showed that among
MS, VW, KC, LM [30] and Hyponex media for PLB induction from leaf explants of some Phalaenopsis hybrids and obtained optimal results
with the explants cultured on MS medium. vila-Daz et al. [31] reported that in seed culture of orchid Laelia speciosa, among the different
media, MS and MS media with sucrose provided the best response rather than on KC medium. Our results were in consistent with them.
Teixeira da Silva [32] VW medium and similar media with weak concentrations of macro and micro nutrients, were necessary for PLB induction
and formation while higher levels of these nutrients were required for callus formation, namely MS, MS and TC-2.

5.

Conclusion

We have performed in vitro clonal propagation of Phalaenopsis hybrid Manchester and we conclude that modified MS medium and
modified VW medium are well suited as compare to FAST, KC and MS medium. Additionally, for survival of seed on medium peptone is
required and for optimal seed germination and PBL formation, medium should supplemented with 2 gl-1 peptone and 15% (v/v) coconut water.
This study has developed a simple, economical and reliable protocol to propagate PLBs of Phalaenopsis hybrid Manchester. The regeneration
system was efficient, and could provide large amounts of plantlets in a short time and is to be considered in transgenic work and a starting work
for production of synthetic seeds.

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