Journal of Cell and Tissue Research Vol.

8(2) 1585-1588 (2008)

ISSN: 0974- 0910 (Available online at www.tcrjournals.com)

Original Article

MASS PROPAGATION OF ORCHIDS THROUGH IN VITRO SEED

CULTURE TECHNOLOGY
?

DAS, A. K., DAS, J., GOGOI, H. K. AND SRIVASTAVA, R. B.

Defence Research Laboratory, Post Bag No. 2, Tezpur- 784 001, India.
E-mail: ajit_drl@rediffmail.com
Received: August 8, 2008; Accepted: September 4, 2008
Abstract: Seeds of four orchid species from North-east India namely Aerides odoratum, Cymbidium
longifolium, Dendrobium aphyllum and Rhyncostylis retusa were germinated on four basal media,
Knudson, Burgeff, Vacin and Went and Phytomax. Best response was exhibited by Knudson medium
followed by Burgeff and Phytamax in terms of faster and higher frequency of germination. Rootshoot initiation was found in Knudson and Burgeff media only and in support with natural extracts
like 15 % coconut water and 6 % banana extract. Earliest shoot and root initiation was recorded
as 70 days in C. longifolium, D. aphyllum and R. retusa and 98 days in D. aphyllum and R. retusa
respectively. Plantlets with well defined shoot and root system were cultured on half strength
Knudson basal medium prior to transfer in to pots. About 170-180 days old in vitro plantlets were
transferred into pots. The survival rate of the plantlets was found to be 70-80 %. This technique
involves three sequential phases germination, protocorm formation and seedling development.
The nutrients and culture requirement are different in each phase.
Key words: Orchid seed, In vitro germination.

INTRODUCTION
North East Indian orchids occupy a place of pride in
floriculture for their aesthetic values with most
beautiful and long lasting quality. This region is one
of the richest regions in orchid diversity with 876
species under 150 genera, comprising of more than
50% of the total Indian orchids, out of total 85,000
species and 800 genera all over the world [2]. Several
species of Indian orchids, like Cymbidium, Dendrobium, Paphiopedillum and Vanda, available in
Northeast India, are of great demand in the country
and abroad [6]. Rhyncostylis retusa locally known
as Kopou phul in Assam is invariably adorned by
ladies during the famous Bihu festival for
ornamentation on their head. This glamorous orchid
has immense importance not only in the cultural front
of Assamese society but also has an impact on the
psyche of the population. Some other rare and showy
species like Coelogyne, Cymbidium, Dendrobium,
Paphiopedillum, Phaius, Rananthera, Vanda etc
of this region, have been listed as threatened and

point towards the endangered status. These precious

resources are, however disappearing alarmingly fast
due to rapid deforestation and industrialization. In
accordance with convention on International trade
in endangered species of wild flora & fauna, the
orchids are now treated as protected plants.
Another most important factor for rapid degeneration
of orchids is that natural propagation through seed is
rare, because the orchid seed has undifferentiated
embryo with little or no reserve food materials. For
germination, it requires symbiotic association with
specific fungus, which provides carbohydrates and
other nutrients. However this problem can be solved
by supplying suitable nutrients in vitro asymbiotically,
to enhance the seed germination potential. The
technique of raising orchid seedling through seed
culture has been applied to thousands of artificial and
natural hybrids and to certain rare, particularly
desirable species that can not easily obtainable in
adequate quantities. It has a great influence on the
orchid industries not only in India, but also all over

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the world. Many workers have reported in vitro
germination and regeneration of different orchid
species in culture media supplemented with natural
extracts as well as plant growth hormones [3, 4, 5, 7,
8, 10]. This paper reports successful asymbiotic
germination of seeds and regeneration of plants
through in vitro technique.
MATERIALS AND METHODS
Undehisced mature capsules of Aerides odoratum,
Cymbidium longifolium, Dendrobium aphyllum
and Rhyncostylis retusa were collected from Assam
and were washed properly with extran (1.0%) and
surface sterilized with 70% aqueous ethyl alcohol for
30 seconds followed by 0.1 % mercuric chloride
solution for 15 minutes and then rinsed thoroughly
with sterile double distilled water.Four different media
namely, Knudson C, Burgeff, Vaccin & Went and
Phytamax were selected for germination test. The
pH of the medium was adjusted at 5.6-5.8 before
autoclaving and the medium was gelled with agar.
Seeds were released in a known volume of sterilized
double distilled water and mixed thoroughly and 1ml
of this suspension was placed in each flask containing
the medium under aseptic condition. The cultures
were incubated at 25 2 0C under 16 hours photoperiod of 2000-3000 Lux fluorescence light intensity.
Observations on germination percentage after 40
days required for germination in each medium were
recorded.
The Protocorm developed from the germinated seeds
was sub cultured in knudson and Burgeff basal

media and also supplemented with 15.0% coconut

water and 6.0% banana extract for root-shoot
initiation and development of plantlets.
Well developed juvenile plantlets with roots and shoots
were transferred into Knudson C half strength
medium. At this stage the cultures were maintained
at 25 2 0C under 16 hours photoperiod of higher
intensity of 3000-4500 Lux fluorescence light for 170180 days.
The 170-180 days old plantlets were rinsed free of
agar under running tap water and dipped in 0.2%
Diathane-M-45 solution for 2 minutes and transferred
directly into pots, filled up with a mixture of sand,
vermiculate and shopped dry leaves (1: 1: 1), at room
temperature for acclimatization. It was reported that
for successful transplantation of in vitro orchid plants,
maintenance of 90-100% humidity for the first 10-15
days is most essential [1]. High humidity (90-95%)
was maintained for 8-10 days. N P K (17: 17: 17)
solution was sprayed for faster growth at an interval
of 7 days. After acclimatization the plantlets were
transferred into big pots (12"dia) and maintained in
an orchidarium of the laboratory.
RESULTS AND DISCUSSION
The first visible sign of germination is the swelling of
embryo followed by greening and emergence out of
the seed coats. At this stage they developed into
Protocorm. Among all the media, Knudson medium
was found best in which seeds of all four different
orchid species germinated. This medium was found

Table 1: Effect of media on in vitro germination of orchid seeds.

Orchid species

Knudson

G: Germination %, T: Time (days)

Vacin & Went

Burgeff

Phytamax

Dendrobium aphyllum

76-100

14

1-25

35

26-50

14

26-50

14

Rhyncostlis retusa

76-100

14

1-25

35

1-25

14

1-25

14

Aerides odoratum

1-25

35

Nil

Nil

1-25

42

1-25

42

76-100

35

Nil

Nil

26-50

35

1-25

35

Cymbidium Longifolium

Table 2: Effect of media on Root-Shoot initiation . * 15.0% , **6.0%

Knudson + coconut
water* + banana
extract**

Knudson

Burgeff

Burgeff + coconut
water* + banana
extract**

Duration (in days)

Orchid species
Shoot

Root Shoot

Root

Dendrobium aphyllum

77

105

70

Rhyncostylis retusa

77

105

70

Aerides odoratum

98

119

84

Cymbidium longifolium

77

126

70

119

1586

98

Shoot Root Shoot

Root

84

112

77

105

98

98

119

84

105

105

105

119

98

112

98

126

84

119

Das et al.

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Figs 1 to 5 are in vitro seed culture of orchids: Different stages:

Fig. 1: Protocorm
Fig. 2: Shoot formation
Fig. 3 In vitro hardening
Fig. 4: Hardening in pots
Fig. 5: In vitro plants in field

Fig. 5

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J. Cell Tissue Research

to be best best in terms of in vitro seed germination
followed by Phytamax and Burgeff. In Knudson
medium 76-100% germination was found in case of
C. longifolium in 35 days of inoculation whereas in
D. aphyllum and R. retusa after 14 days, 76-100%
germination was recorded (Table 1).
Protocorms were sub-cultured in Knudson basal
medium to get maximum number of Protocorms [9].
Protocorm itself shows exomorphic characters of
plantlets or seedlings.
In different media, shoots were initiated at varying
periods ranging from 70 days to 105 days after
inoculation of seeds. However, knudson and Burgeff
media, combined with coconut water (15%) and
banana extract (6%) was most effective in reducing
the shoot initiation period i.e., 70-98 days of seed
inoculation in different species. Similar root initiation
was also recorded in the same media. The time
required for initiation of root is 105-126 days in
knudson and Burgeff plain media, whereas the roots
initiated earlier i.e., in 98-119 days in both the media,
with the addition of coconut water (15%) and banana
extract (6%) (Table 2, Fig. 1). Among the four
different orchid species, germination and root-shoot
initiation in A. odoratum were slower than the other
three species. Shoot initiation in D. aphyllum, R.
retusa and C. longifolium took place first after 70
days of inoculation in coconut water and banana
extract incorporated knudson and Burgeff media
followed by root initiation after 119 days of inoculation
in C. longifolium and 98 days in D. aphyllum and
R. retusa on the same culture medium. It was
observed that within a period of 170-180 days from
inoculation of seeds, complete plantlets can be
obtained. The survival rate of in vitro plants under
natural conditions was observed to be70-80%.

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ACKNOWLEDGEMENTS
The authors are thankful to Director, Defence
Research Laboratory, Tezpur (Assam) and former
Director Dr. S. N. Dube for their constant guidance
and support to carry out the research work.

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