See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/234839449

IN Vitro multiplication and protocorm development of Dendrobium aphyllum

(Roxb.) CEC Fisher

Article · January 2011

CITATIONS READS

12 358

5 authors, including:

Abhishek Chowdhury Biman K. Dutta

Assam University Assam University
19 PUBLICATIONS   48 CITATIONS    48 PUBLICATIONS   518 CITATIONS   

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Studies on the dynamics of soil microbial biomass and nitrogen mineralization associated with tillage practices in rainfed agricultural systems View project

In silico drug designing View project

All content following this page was uploaded by Abhishek Chowdhury on 17 May 2014.

The user has requested enhancement of the downloaded file.

 SI
VER TY

NI Assam University Journal of Science & Technology : ISSN 0975-2773
ASSAM U

SI
LCHAR

Biological and Environmental Sciences

Vol. 7 Number I
57-62, 2011

In vitro multiplication and protocorm development of Dendrobium

aphyllum (Roxb.) CEC Fisher
S. Dutta1, A. Chowdhury1, B. Bhattacharjee1, P.K. Nath1 & B. K. Dutta2
1
Department of Botany & Biotechnology, Karimganj College, Karimganj-788710, Assam
2
Department of Ecology & Environmental Science, Assam University, Silchar-788011, Assam

Abstract
Fifteen weeks old seeds of D. aphyllum (Roxb.) CEC Fisher germinated on the MS media. The seed germination
(90.6%) was recorded. The seed showed development of protocorm like bodies (PLBs) within five weeks of
inoculation and the aforesaid viability was observed. The PLBs then started germinating in the basal MS
media and were subsequently transferred in the media supplemented with auxin & kinetin separately and
mixed through. The growths were recorded after two weeks of inoculation into supplemented media. The
PLBs showed development of distinct roots and shoots in media supplemented with IAA & KN. The PLBs
showed better germination and developed into multiple roots and shoots, when supplemented with IAA and
KN separately compared with the mixture of both IAA & KN application. The average length of roots and
shoots are measured and found to be IAA (0.38 cm & 1.68 cm), KN (0.43 cm & 0.45 cm) & IAA+KN (0.50 cm
& 0.78 cm) respectively.
Key words: Dendrobium aphyllum (Roxb.) CEC Fisher, In vitro, tissue culture technique, MS media

Introduction
Orchids are the most beautiful among the
flowering plants in the world and are favourites in
horticulture, and contain many genera. In recent
years there has been increasing interest among
botanists all over the world to collect and grow
this beautiful ornamental plant. The beauty of
flowers, variety of fragrance, brilliance in colour
and attractive habit has aroused highest admiration
among the people throughout the world. Many of
the orchids are expensive and difficult to cultivate
because the germination of the seeds is not possible
due to the shedding of the fruits before the
attainment of maturity, lack of mycorrhizal
association, inadequate nutrition etc.
Most of the orchids contain few-celled embryo at
the time of seed maturation and its proper
development takes place only during the
germination of seeds. However, as the seeds do
not have sufficient reserve food material (lacks
endosperm) to take care of the growth of embryo
during germination (Richardson et al., 1992), they
have to depend on some external source for
nutrients so as to make their undifferentiated
embryo to develop into a protocorm. The
association with mycorrhizal fungi helps to draw
the nutrient for orchids. Fungal endophytes in such
associations are believed to provide simple sugar
and other nutrients required for seed germination
by breaking down starch (Arditti, 1967).
Therefore, only 2-5 % of seeds germinate in the
environment, which is very less in comparison to
time.
The orchid seeds are the tiniest in the plant
kingdom. They are extremely light, more or less
fusiform and are produced in millions in each
capsule. The seeds at maturity are released by
the longitudinal slits in the fruits and are dispersed
to long distances by wind. A minute seed of orchid
travelling long distances through wind cannot
afford to carry enough food supplies for
independent germination and as soon as the seed
lands on a suitable substratum where conditions

- 57 -
In vitro multiplication and protocorm
are favourable for germination, it starts germinating
but is unable to grow further in the absence of a
suitable fungus and dies for the lack of food.
However, if a hypha from a highly specialized
mycorrhizal fungus enters the seed and penetrates
the germinating embryo and proper balance
between the embryo and fungus is established,
the germinating seedling gets enough food material
from the fungus to sustain itself and subsequently
develop leaves and roots to start photosynthesizing
its own food and in turn the fungus gets shelter.
Such a relationship between the fungi and orchid
is called “symbiosis” where the fungus provides
nutrition for orchid growth, while, the orchid
provides shelter to the fungi. In nature this
mycorrhizal association with an orchid seed is not
common and thus a high proportion of seeds fail
to survive. In order to overcome this association
and produce the seedlings of the desired orchid in
mass scale, mature seeds can be asymbiotically
germinated on a suitable culture medium under
controlled conditions in the laboratories (Sharma,
1998).
Micropropagation is particularly useful for
conservation of germplasm. Several valuable
species of Dendrobium have been reported to be
propagated through asymbiotic germination of
immature seeds or direct shoot regeneration of
PLBs from different explants (Vij et.al.,1981).
Dendrobium aphyllum is commonly known as
“leafless Orchid”. Plant blooms in the late spring.
Stems 30-70cm long, elongate, cylindrical, slender.
Leaves 9-11x3-4 cm, obtuse-lanceolate, apex
acute, flowers 3.3 cm wide. Flowers are fragrant,
bract ca, 5cm long, sepals 5-nerved, pale pinkish
white, dorsal sepals 2-3x0.7-1.5 cm, linear. Lateral
sepal 2-4x0.5-1cm, acute. Petals 2-4x1-2 cm
broadly acute, 5-nerved. Column ca. 5cm long,
white. Capsule (pod) ca. 4cm. Flowering time is
April-May. During these months the leaves shed
off and the whole branch gets covered by flowers
(Bose et al., 1999). It has been used in decorating
houses and as for its fragnance. Because of
continuous collection to meet the increasing
demand for their use in decoration, fragrant etc.,
the natural habitat of Dendrobium species
including D. aphyllum (Roxb.) CEC Fisher have
- 58 -
been largely destroyed. They are widely
distributed throughout the Asian and South Pacific
tropics and subtropics from lowland warm regions
in northern Australia, Papua New Guinea to
Thailand and Himalayan mountains.
Although the North-East India is reported to have
the richest reservoir for rare, ornamental and
threatened orchid resources, the resources of this
region are fast diminishing due to brutal
exploitation of orchid flora for export, trade and
increasing deforestation. On account of this, a few
species are extremely scant or perhaps already
extinct and many more are facing the danger of
being extinct. In this context, the Dendrobium
aphyllum (Roxb.) CEC Fisher is floriculturally
significant and comprises of important genetic
resource and is on the decline and has become
rare and threatened in Assam in general and
southern Assam in particular, due to habitat
destruction and over exploitation (Bhattacharjee
et al., 2008). Keeping in mind the conservation
and protection of the orchids from extinction,
present work was undertaken for large scale “in
vitro” propagation of Dendrobium aphoyllum.
Materials and methods
About fifteen-months old capsules of D. aphyllum
(Roxb.) CEC Fisher were collected from its wild
habitat near the Kalibari at Sarisha locality of
Karimganj District. All the experiments were
carried out aseptically in the transfer table of the
laminar flow. Before using the transfer table the
laminar cabinet was thoroughly scrubbed with
cotton soaked in 70% ethanol. The ultra violet
(UV) tube light of 30 watt was switched on for
15 - 20 min after which the transfer area was left
as such for about 5 min with the flow of ultra
filtered sterile air to strain out particles as small
as 0.3 mm providing a sterile atmosphere for
work. The velocity of the airflow which is 27±23m/
min helped in preventing air-borne contamination.
Forceps, needles, surgical blades etc. were
sterilized by dipping in 70 % alcohol and flamed
before use for proper sterilization. The collected
capsules were washed thoroughly with detergent
under tap water and surface-disinfected in 70 %
ethanol for 30 sec followed by surface flaming in
In vitro multiplication and protocorm
the transfer table. This process was repeated 3
times after which the capsules were rinsed five
times with sterile distilled water and dried in a
laminar airflow cabinet before dissection. The
capsules were then dissected longitudinally with
a surgical blade, aseptically. The seeds were
scooped out from sterilized capsules and
inoculated, spreading as thinly as possible over the
surface of the culture medium (Murashige and
Skoog, 1962) in 25 x 150 mm glass test tubes each
containing 20 ml of medium. The pH of the media
was adjusted to 5.8 prior to autoclaving. The
culture tubes were incubated at 22±2oC under 16h
photoperiod of 150µmol m-2 s-1 light intensity. The
inoculated seeds were examined regularly every
week. The percentage of seed germination of D.
aphyllum (Roxb.) CEC Fisher in MS media was
determined by examining the seeds microscopically
after five weeks of inoculation. The percentage
of germination was calculated using both green
and white protocorms. The MS media is
supplemented with auxin (IAA) and kinetin (KN)
separately and mixed in different cultures (Table
1).
Results and discussion
The seeds of D. aphyllum (Roxb.) CEC Fisher
germinated on the MS media. Seed germination
of 90.6% was recorded on MS basal medium.
After 3 weeks of inoculation protocorm like bodies
are found to have formed from the seeds (Plate-
1). The development of plantlets with shoots and
roots from the protocorms in medium was
observed in MS media after 8 weeks of inoculation
(Table-2; Graph-1; Plate-1).
In the in vitro regeneration study, the orchid seeds
responded successively in MS media. The
development of orchid seeds requires a balanced
supply of both organic and inorganic nutrients
(Arditti & Ernst, 1982). The seeds require a
nutrient rich medium which is ubiquitous in MS
medium containing optimal macro and
micronutrients, vitamins, inositol, glycine etc. This
has proved beneficial for seed germination as
already suggested by Devi et al., (1999). High
concentration of nitrogen (60.05mM) i.e.
ammonium nitrate & potassium nitrate present in
- 59 -
MS medium was required for the optimal
germination of seeds. A better efficacy of MS
medium in maintaining healthy growth of the
seedlings may also be attributed to their Fe-EDTA
contents, a growth promotory nature of Fe-EDTA
is already available on record (Lee et al., 1983).
The nutrient requirement of orchid seeds in terms
of quality as well as in form may vary at different
stages of development (Arditti and Ernst, 1984).
The importance of ammonium or nitrate ions
(individually or in combination) during the in vitro
germination of orchid seeds as a source of
nitrogen is well established. The growth regulators
(Auxin and Kinetin) in the medium play the role
of mycorrhiza which forms symbiotic association
with non-germinating seeds in nature and bring
about changes in the physiology which induces
germination in the seeds and protocorm
development (Kumaria and Tandon, 1991).
Kinetin helps in shoot regeneration and auxin
induces root development in shoots to make it a
complete plant. The effect of auxin and kinetin
vary from orchid to orchid (Arditti and Pridgeon,
1977). The promotory effect of growth regulators
such as IAA and KN on seed germination and
protocorm development in orchid species were
studied by Kano (1965), Mathews and Rao
(1980). Healthy growth of orchid protocorms in
medium containing balanced supply of organic and
inorganic nutrients has been reported by some
workers (Arditti and Ernst, 1982). Initiation of
seed germination, protocorm development and
subsequent growth and development of seedlings
seems to vary with the species and the medium
employed (Arditti and Pridgeon, 1977). The work
on tissue culture of D. aphyllum in MS medium
showed that the D. aphyllum seed responded
successively in different kinetin concentrations
(Mazumdar & Talukdar, 2007). In the present
work, the PLBs showed better germination and
developed into multiple roots and shoots, when
supplemented with IAA & mixture of IAA & KN
compared to the media supplemented with KN
only. The average length of shoots & roots are
measured and found to be IAA (0.38 cm & 1.68
cm), KN (0.43 cm & 0.45 cm) and IAA+KN
(0.50 cm & 0.78 cm) respectively.
In vitro multiplication and protocorm

Table 1: Different concentration of growth regulators supplemented with MS medium :

1. MS + 0.2 mL(50µg/100mL) IAA

2. MS + 0.3 mL(50µg/100mL) KN
3. MS + 0.05mL(50µg/100mL) IAA + 0.15mL ( 50µg/100mL) KN

Table-2: The effects of different supplemented media on shoot & root development of
D. aphyllum (Roxb.) CEC Fisher
Media Average Shoot Average Root
Length(cm) Length(cm)
MS + 0.2 mL(50µg/100mL) IAA 0.38 1.68
MS + 0.3 mL(50µg/100mL) KN 0.43 0.45
MS + 0.05mL(50µg/100mL)IAA+0.15mL( 50µg/100mL) KN 0.50 0.78
Length (cm)

Growth Medium
Graph-1: The effects of different supplemented media on shoot & root development of
D. aphyllum (Roxb.) CEC Fischer

- 60 -
In vitro multiplication and protocorm

- 61 -
 In vitro multiplication and protocorm
Acknowledgement
We thank the Principal of Karimganj College for
his permission for performing the work in the Plant
References
Arditti, J. (1967). Factors affecting the germination of
orchid seeds. Bot. Res. 7:421-55.
Arditti, J. and Earnst, R. (1982). Orchid seed
germination and seedling culture- A manual. In
Orchid Biology- Reviews and Perspective II (ed.
Arditti, J.) Cornel University Press, Ithaca pp. 243-
370.
Ariditti, J. and Earnest, R. (1984). Physiology of
germination orchid seeds. 177-222. In: Orchid Biology,
review and in perspectives III. (ed. J. Arditti). Cornell
University Press, Ithaca, New Delhi.
Arditti, J. and Pridgeon, A.M. (1977). Orchid Biology.
Reviews and Perspectives, VII KLuwer Academic
Publisher, Dordrecht, the Netherlands. pp 117-170.
Bhattacharjee, B., Dutta, B.K. & Tandon, P. (2008). The
genus Dendrobium Sw. in southern Assam (Barak
Valley), Cachar. J. Orc. Soc. India. 22(1-2):39-44.
Bose, T.K., Bhattacharjee, S.K., Das, P. and Basak, U.C.
(1999). Orchids of India, Naya Prakash, Calcutta. pp
1-77.
Devi, G., Damayanti, M. and Sharma, J. (1999). Aseptic
embryo culture of Vanda coerulea Griff. J. Orchid Soc.
India, 12(1 & 2): 83-87.
Kano, K. (1965). Studies on the media for orchid seed
germination. Mam. Fac. Argic. Kagwa Univ.20: 1-76.
Kumaria, S. & Tandon, P. (1991). Effects of growth
regulators on peroxides, polyphenol oxidase and IAA
oxidase activities and phenolic contents during
protocorm development of orchid Dendrobium
fimbriatum var. oculatum Hook. f. J. Orc. Soc. India
14(1-2):27-39.
- 62 -
View publication stats
tissue culture laboratory, Karimganj College. Our
thanks are also due to all the faculty members &
laboratory staffs of Department of Botany &
Biotechnology for their kind co-operation.
Lee, H.S., Peak, K.Y. and Lee, J.K. (1983). Studies on
the non symbiotic germination of seeds of Laelia
briegeri. II. Effect of anionic and cationic composition,
nitrogen and iron nutrition on the growth of seedling
In vitro. J. Korean. Soc. Hort. Sci., 24: 169-174.
Mathews, V.H. and Rao, P.S. (1980). In vitro
multiplication of Vanda hybrids through tissue culture
technique. Plant. 15:473-97.
Mazumdar, P.B., Talukdar, A.D. (2007). In-vitro
propagation of Dendrobium aphyllum in MS medium
showing different growth at different growth regulator
concentrations. Biodiversity Conservation-the post-
rio scenario of india (ed. B.K. Dutta, A.K. Das & P.
choudhury). pp. 270-274.
Murashige, T. and Skoog, F. (1962). A revised medium
for rapid growth and bioassay for tissue culture.
Physiol. Plant. 15:473-97.
Richardson, K.A., Peterson, R. L. and Currah, R. S.
(1992). Seed reserves and early symbiotic protocorm
development of Planthera hyperborean
(Orchidaceae). Can. J. Bot. 70: 291-300.
Sharma, J. (1998). Studies on Vanda: Effect of age of
capsules on in vitro seed germination J. Orchid. Soc.
India. 12(12):43-45.
Vij, S.P., Sood, A. and Plaha, K.K. (1981). In vitro
germination of some epiphytic orchids. In:
Contemporary Trends in Plant Sciences (ed. S.C.
Verma). Kalayani Publishers. New Delhi. pp.473-481.