Indian Journal of Biotechnology

Vol 6, April 2007, pp. 256-261

Effects of different culture media on seed germination and subsequent

in vitro development of protocorms of Hygrochilus parishii
(Veith & Rchb.f.) Pfitz (Orchidaceae)
R Shadang, Padmanabh Dwivedi1*, S N Hegde and N Ahmed
State Forest Research Institute, Post Box No. 159, Itanagar 791 111, India
1
Department of Botany, Rajiv Gandhi University, Itanagar 791 111, India
Received 19 September 2005; revised 24 May 2006; accepted 20 July 2006
Seeds of Hygrochilus parishii (Veith & Rchb.f.) Pfitz were inoculated on eight different culture media (basal) MS,
MKC, V&W, I&Y and half strength of respective media. Seeds germinated within 15-20 d almost on all media with varying
percentage and developed into greenish protocorm after 80-100 d. Best seed germination was observed in strength MKC
medium. Healthy protocorms were observed within 100 d in V&W media. Sub-cultured protocorms in all the above media
supplemented with NAA, BAP, 2,4-D and IAA in a range of 0.5-2.0 mg L-1, both individually and in combination, and
additives like banana pulp (BP, 10%) and coconut milk (CM, 15%) produced well developed callus. Multiplication of
protocorms was best on strength MS + NAA (2.0 mg L-1). Formation of root and leaf was early in V&W medium having
CM (15%) and BP (10%) but without sucrose.

Keywords: culture media, Hygrochilus parishii, orchid, protocorms, seed germination

IPC Code: Int. Cl.8 A01H4/00

Introduction
North-Eastern part of India is home to a number of
rare and endemic orchid species. Some of these
species are disappearing due to extensive collection
and habitat destruction by anthropogenic activities.
Hygrochilus parishii (Veith & Rchb.f.) Pfitz, an
epiphytic plant is one of those species that is endemic
and has become rare1. Due to its handsome
multicoloured and long lasting flower quality, the
species has received wide attention of floriculturists
and orchid lovers. As measure to conserve this
species, experiments on seed germination and
subsequent protocorms and seedling growth have
been carried out in vitro. Though most of the orchid
species produce millions of seeds in a single
capsule2,3, in natural conditions less than 5%
germinate due to specific nutritional requirement4,5.
Asymbiotic in vitro germination of orchid seeds has
been reported by several workers6-8. In the present
communication, an attempt has been made for in vitro
seed germination and subsequent development of
________
*Author for correspondence:
Tel: 91-360-2277572; Fax: 91-360-2277317
E-mail: pdwivedi25@rediffmail.com

protocorms and seedling growth from green

undihisced mature capsules of H. parishii to produce
a large number of plants.
Materials and Methods
Eight different basal culture media - MKC
(Modified Knudson C)9, MS (Murashige &
Skoog)10, V&W (Vacin & Went)11 and I&Y
(Ichihashi & Yamashita)12 and their half strengths
were used for seed germination and protocorm
development. pH of the media were adjusted to 5.6
before autoclaving. The growth and development of
protocorms and subsequent seedlings were tested on
all the above media supplemented with filtersterilized NAA, BAP, IAA, 2,4-D (0.5-2.0 mg L-1),
either singly or in combination. The media were also
enriched with additives like coconut milk (CM) and
banana pulp (BP).
Undihisced 8-month-old green capsules were
washed thrice in running tap water followed by
application of a detergent solution with brush. The
capsules were then dipped in 70% ethanol for 5 min
and washed thoroughly with sterilized distilled water.
The capsules were flamed before opening by a sterile
blade. Seeds were then distributed directly onto the

SHADANG et al: PROTOCORM DEVELOPMENT OF H.PARISHII IN VITRO

culture media. The inoculated flasks were kept at

room temperature (22'-2OC) under 2000-2500 lux
light with 14 h photoperiod.
The number of days required for germination and
protocorm development were recorded. Protocorms
thus obtained were inoculated onto basal media used
for seed germination with different combinations of
NAA, BAP, IAA, 2,4-D and additives like CM and
BP. Growth response in all the cultures was recorded.

Results and Discussion

The seeds of H. parishii inoculated in eight basal
nutrient media germinated after 15-20 d and
developed into greenish protocorms within 100 d of
culture. Seed germination was 90% in MKC, followed
by 80% in MS medium. On V&W medium though
only 60% germination occurred, the protocorms
showed better growth with early leaf ( 1 ~ 2in numbers)
and roots (1-2 in numbers) formation 4-5 months after
culture. 50-60% germination was recorded in I&Y,
34 MKC and ?h MS, and 20-40% in ?hV&W and 34 I
and Y (Table 1).
The promotory effect of growth regulators in in
vitro culture has been worked out by a number of
worker^'^-'^. In the present study, BAP (2.0 mg L-') in
MS media showed significant response in shoot and
root development (Table 2). Higher concentration of
NAA (2.0 mg L-I) in 34 MS, V&W media favoured
protocorm multiplication (Figs 1 & 2) but 2,4-D
(2.0 mg L-') in W K C initiated callus in protocorms
(Fig. 3). IAA (1.0 mg L-') in MS initiated shoot bud
formation after 60 d of sub-culture (Fig. 4). All
protocorms developed shoot buds in both full and ?h
strength MS media supplemented with BAP (2.0 mg
L-') and NAA (2.0 mg L-I) (Figs 5&6). With BAP
(1.0 mg L-') + NAA (2.0 mg L-') in 95 strength MKC
protocorm
was observed'
Excellent callus was observed in MKC, MS or %MS
media in combination of BAP (1.0 mg L-') + 2,4-D
(2.0 mg L-') + IAA (1.0 mg L-I). BP, CM and potato
extract have been successfull used in place of
sucrose in Sarcanthine orchids17. In our experiments,
incorporation of CM (15%) or BP (10%) instead of
sucrose in MKC, MS, 34 strength MS and V&W
media initiated direct differentiation of shoots and
roots from protocorms. In other media the response
was very poor (Table 2).
Seed germination frequency was lower in
34 strength than in full strength of culture media.
In case of protocorms, higher concentration of BAP

Figs 1-7 - 1&2. Multiplication of protocorms on Yz strength MS

md V&W supplemented with NAA (2.0 mg ~ 1 ) 3.; Callus in H
MKC+ 2,4-D (2.0 mg L-1); 4. shoot bud initiation in MS+IAA
(1.0 mg L-j); 5 & 6. Protocorms developed shoot & buds in MS &
Yz strength MS supplemented with BAP (2.0 mg L-I) and NAA
(1.0 mg L-I); and 7. Seedling growth in V&W +CM (15%)+BP
(10%).

(2.0 mg LA') initiated bud in majority of culture

media. 2,4-D (2.0 mg L-') caused callusing of
protocorms and NAA (2.0 mg L-') proved effective
for protocorm multiplication. Combination of BAP
(1.0 mg L-I), 2,4-D (2.0 mg L-I) and IAA (1.0 mg L-')
favoured formation of shoot buds. However, CM
'
(15%) and BP (10%) when added in place of sucrose

'

INDIAN J BIOTECHNOL, APRIL 2007

258

Table 1 Response of seed germination in Hygrochilus parishii.

S..No Media

Time taken for Number of days taken for

Observation
%
seed germination formation of protocorm- Germination
(d)
like bodies (Plbs)

MKC

16

80

90-100

Inoculated seeds swell after 16 d, followed by germination,

Plbs found after 80 d, Plbs small in size.

MKC

16

90

50

Inoculated seeds swell after 16 d, followed by germination,

40% of Plbs turned brownish and died after 90 d, Plbs
were small in size.

MS

16

90

80

Inoculated seeds swell after 16 d, followed by germination,

Plbs found after 90 d, Plbs large in size.

MS

16

90

50-60

Inoculated seeds swell after 16 d, followed by germination,

few of the protocorms turned brownish and died after 90 d.

V&W

20

100

50-60

Inoculated seeds swell after 20 d, followed by germination,

protocorms formed with 1-2 leaf, 2-5 mm long, 1-2
root 5-10 mm long, after 5 months.

V&W

20

100

40

Inoculated seeds swell after 20 d, followed by germination,

protocorms formed with 1-2 leaf, 2-5 mm long, 1-2
root 5-10 mm long, after 4 months.

I&Y

16

70

50

Inoculated seeds swell after 16 d, followed by germination,

Plbs found after 80 d.

I &Y

16

80

20

Inoculated seeds swell after 16 d, followed by germination,

white protocorms formed after 90 d only, few turned
green.

Time taken for Plbs: n = 120, df = 7, F = 18.367, p< 0.001 [p = 0.000]

Time taken for germination: n = 120, df = 7, F = 3.702, p< 0.005 [0.001]
Result: n = 120, df = 7, F = 18.367, p < 0.001 [p = 0.000]
Table 2 Response of protocorms grown on various culture media supplemented with different growth regulators
Culture
Media
MKC

Growth regulators (mg L-1)

% Response

BAP

NAA

2,4-D

IAA

CM

BP

Callus Plbs multiplication Shoot buds after 2 month

after 40 d
after 40 d
of Plbs inoculation

1.5

20

30

50

2.0
-

1.5
2.0
-

1.0
1.5
0.5
1.0
2.0
-

0.5
1.0
2.0
-

15%

10%

40
50
50
-

0.5
0.5
1.5
2.0
1.0
1.5
2.0
1.0
-

1.0
1.5
2.0
1.5
2.0
1.0
-

20
10
20
50
20
30
50
10
30
10
50
60
30
30
40
60

60

1.0
1.5
2.0
-

30
80
-

30
60
60
60
60
30
70
30
40
50
80

SHADANG et al: PROTOCORM DEVELOPMENT OF H. PARISHII IN VITRO

259

Table 2 Response of protocorms grown on various culture media supplemented with different growth regulators Contd.
Culture
Media
MKC

MS

MS

Growth regulators (mg L-1)

% Response

BAP

NAA

2,4-D

IAA

CM

BP

1.0

20

1.5
2.0

1.5
2.0

1.0
1.5
2.0
2.0
0.5
1.0
1.5
2.0
1.0
1.0
1.5
2.0
1.0
-

50
70

20
50
-

15%
15%
15%

10%
10%
10%

50
20
-

10
10
50

20
50
20
60
50
60
60
70
10
10
30
60
50
50
50
50
80
50
50
60
50
40
30
80
30
60
70

2.0
1.0
2.0
0.5
1.0
1.5
2.0
2.0
1.0
0.5
1.0
2.0
1.0
1.5
2.0
0.5
1.5
2.0
1.0
2.0
1.0
-

1.0
1.5
2.0
1.0
2.0
1.5
2.0
1.5
0.5
1.0
2.0
-

2.0
1.0
1.5
1.5
2.0
1.0
2.0
1.5
2.0
1.0
2.0
-

Callus Plbs multiplication Shoot buds after 2 month

after 40 d
after 40 d
of Plbs inoculation

60
50
60
30
70
70
50
40
70
-

40
50
20
20
30
20
60
80
60
30
20
20
40
50
20
20
50
70
20
40
50
10
30
50
20
30
-

INDIAN J BIOTECHNOL, APRIL 2007

260

Table 2 Response of protocorms grown on various culture media supplemented with different growth regulators Contd.
Culture
Media

Growth regulators (mg L-1)

BAP

NAA

2,4-D

IAA

% Response
CM

BP

Callus Plbs multiplication Shoot buds after 2 month

after 40 d
after 40 d
of Plbs inoculation
V&W
1.5
10
60
2.0
20
60
1.0
50
33
1.5
50
30
2.0
70
40
1.5
50
20
2.0
50
1.0
20
1.5
20
60
2.0
50
60
2.0
1.0
50
40
1.0
2.0
30
60
2.0
1.0
30
40
1.0
2.0
50
2.0
1.0
2.0
30
60
1.0
2.0
1.0
50
60
15%
10%
60
90
V&W
2.0
20
2.0
30
30
2.0
40
2.0
1.0
20
40
1.0
2.0
30
40
2.0
1.0
30
20
1.0
2.0
30
1.0
2.0
1.0
40
15%
10%
30
30
I&Y
1.5
20
50
2.0
40
50
1.5
30
2.0
30
1.5
20
2.0
50
1.5
20
20
2.0
40
50
2.0
1.0
30
1.0
2.0
20
30
2.0
1.0
30
40
1.0
2.0
50
2.0
1.0
2.0
40
30
1.0
2.0
1.0
40
60
15%
10%
50
40
I&Y
2.0
40
2.0
20
2.0
30
2.0
30
20
1.0
2.0
40
30
1.0
2.0
15%
10%
40
(-) Nil, BAP (6-Benzyl amino purine), NAA (Naphthalene acetic acid), 2,4-D (2,4-Dichlorophenoxy acetic acid), IAA (Indole-3-acetic
acid), CM (Coconut Milk), BP (Banana Pulp). Each concentration of hormone had 10 replicates, and the experiment was conducted
3 times.

SHADANG et al: PROTOCORM DEVELOPMENT OF H. PARISHII IN VITRO

promoted better response (Fig. 7). The survival rate of

these seedlings after in vivo transplantation in the
potting mixture (Brick:Charcoal:Tree fern::1:1:1) was
70%. The method thus suggested in this paper can be
used for large-scale propagation of this orchid
species.

6
7
8
9
10

Acknowledgement
Thanks are due to the Department of
Biotechnology, Govt. of India, New Delhi for the
financial assistance. We also thank the Director, State
Forest Research Institute, Itanagar for facilities.

12

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