Comparison of Different Culture Media For The in Vitro Culture of Dendrobium (Orchidaceae)

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INTERNATIONAL JOURNAL OF AGRICULTURE & BIOLOGY

15608530/2004/065884888
http://www.ijab.org

Comparison of Different Culture Media for the In Vitro Culture


of Dendrobium (Orchidaceae)
D. PUCHOOA
Faculty of Agriculture, University of Mauritius Reduit, Mauritius
Corresponding authors e-mail: sudeshp@uom.ac.mu

ABSTRACT
The potential of different media, supplements and conditions for reliable Dendrobium orchid plantlet regeneration from in
vitro grown leaf explants were studied. In all three media under investigation, regeneration was via the production of
protocorm-like bodies (PLBs). Maximum number of PLBs was obtained in Murashige and Skoog (MS) liquid medium
agitated at 80 rpm and supplemented with 0.1 mg L-1 of BA, 1.0 mg L-1 of NAA and 15% (v/v) coconut water. Shoot
regeneration was achieved when the PLBs were transferred onto solidified MS medium supplemented with 0.1 mg L-1 of BA,
1.0 mg L-1 of NAA and 15% (v/v) coconut water and 10 g L-1 potato homogenate. The shoots developed roots after four weeks
of culture on the same medium. Transferring them onto wood charcoal successfully acclimatized the in vitro grown plantlets.
The short duration of in vitro culture and its high efficiency makes this approach well suited for mass production of
Dendrobium orchids.
Key Words: Acclimatization; Dendrobium; In vitro culture; Plantlets; Protocorm-like bodies

INTRODUCTION
The genus Dendrobium was established by Olaf
Swartz (1799). Although most Dendrobiums have an
epiphytic growth habit, some are also found growing on
rocks and cliffs and terrestrial in grasslands. 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. The truly spectacular genus
Dendrobium contains the largest diversity of horticulturally
interesting specimens. More than 1,000 natural species
make Dendrobium the second-largest orchid genus (next to
Bulbophyllum) in the orchid family, which has over 700
genera. Although the color range of Dendrobiums is varied,
most hybrids are usually lavender, white, golden-yellow, or
combinations of these colors. Some of the more unusual
species and hybrids can be bluish, ivory colored, brilliant
orange or scarlet, or have exotic markings. Most of the
evergreen Dendrobiums are not fragrant, however the
deciduous species such as superbum, pierardii and parishii,
can have fresh citrus scent or smell of raspberries.
Dendrobiums can often bloom several times a year and the
flower sprays make excellent cut flowers for arrangements.
It is widely recognized that world production of potted
Dendrobium has increased in the last few years. Large scale
potted Dendrobium production is occurring in the
Netherlands, Germany, China, Taiwan, Thailand,
Philippines, Unites States, and Japan. However, orchid
growing has not fully achieved the transition from a hobby
to an industry in Mauritius. The main reason being that
conventional methods of orchid propagation are extremely

slow and the number of propagules produced by these


methods is low. Tissue culture techniques provide a solution
for producing large number of propagules within a limited
period of time. According to Morel (1974), more than four
million plantlets can be obtained in a year from a single
explant. Among the explants most commonly used are shoot
tips and axillary buds (Intuwong & Sagawa, 1974) although
stem, leaf, root and inflorescence can also be used (Homma
& Asahira, 1985). Also, a wide range of recipe has been
used in the in vitro culture of orchids (Lim-Ho, 1982). In a
study carried out by Wu et al. (1987), it was observed that
several factors influence shoot formation and in vitro
multiple shoot production in oriental orchids. When
growing the orchids in our laboratory, we found that the
plantlets of Dendrobiums do not grow fast enough in the
commonly used Vacin & Went (Vacin & Went, 1949) and
Knudson C (KdC) medium (Knudson, 1946) and do not
produce roots which are strong enough to withstand the
acclimatization conditions. As literature for the commercial
propagation of Dendrobiums specifically are scarce, due to
obvious reasons, a study was conducted to compare the
efficiency of different nutrient media, growth regulators and
medium supplements for the in vitro propagation of orchid
Dendrobium Sonia suitable for commercial exploitation.

MATERIALS AND METHODS


Plant materials. Leaves obtained from in vitro grown
protocorm-like bodies (PLBs) of Dendobrium Sonia were
used as starting material. The PLBs were cultured in liquid
MS medium (Murashige & Skoog, 1962) supplemented
with 30 g L-1 sucrose on an orbital shaker at 80 rpm and

IN VITRO CULTURE OF Dendrobium (ORCHIDACEAE) / Int. J. Agri. Biol., Vol. 6, No. 5, 2004
Fig. 2. Effects of BA and NAA on the initiation of
Dendrobium Sonia Protocorm-like bodies on three
different solid basal media
Mean number of PLBs

Fig. 1. Effects of BA and NAA on the initiation of


Dendrobium Sonia Protocorm-like bodies on three
different liquid basal media
Mean Number of PLBs

60
50
40

MS

30

VW
KdC

20
10
0
0.0
NAA /
0.0 BA

0.1
NAA /
0.5 BA

0.5
NAA /
0.1 BA

0.5
NAA /
1.0 BA

40
35
30
25
20
15
10
5
0

Mean Number of PLBs

Mean Number of P LBs

50
40
30
20
10
0
120

0.1
NAA /
0.5 BA

0.5
NAA /
0.1 BA

0.5
NAA /
1.0 BA

1.0
NAA /
0.5BA

Fig. 4 Effects of adding coconut water on the


propagation of Dendrobium Sonia PLBs. The
medium consisted of MS medium supplemented with
0.1 mgL-1 of BA and 1.0 mgL-1 of NAA. Values are
means of five replicates and five explants per treatment.

60

100

KdC

Plant Growth Regulators (mg/L)

Plant Growth Regulators (mg/L)

80

VW

0.0
NAA /
0.0 BA

1.0
NAA /
0.5BA

Fig. 3 Effects of the speed of agitation on the


propagation of Dendrobium Sonia. The medium
consisted of MS medium supplemented with 0.1 mgL-1
of BA and 1.0 mgL-1 of NAA. Values are means of five
replicates and five explants per treatment.

MS

140

80
60
40
20
0
0

10

15

20

Coconut water in MS medium (% - v/v)

Agita tion S pee d (rpm )

under conditions of 2620 C and 16 h of light daily provided


by cool-white fluorescent tubes (50 mol m-2 s-1). Subculturing was performed at 2-weeks intervals by the transfer
of terminal cuttings to the same medium and under the same
conditions.
Media and culture conditions. The basal media used were
Vacin and Went (VW) medium (Vacin & Went, 1949),
Knudson C (KdC) medium (Knudson, 1946) and Murashige
and Skoog (MS) medium (Murashige & Skoog, 1962). Five
leaf explants (~1 cm2) per flask were cultured in each of
these media supplemented with various amounts of BA (0
1.0 mg L-1) and NAA (01.0 mg L-1) both in liquid and
Phytagel solidified media (2 g L-1) for the determination of
the optimum plant growth regulator combination. Similar
experiments designed to assess the effects of the speed of
agitation (80140 rpm) and the effects of coconut water (0
20% v/v) on the propagation of the PLBs. The coconut
water was prepared from selected coconuts obtained from a
local market and processed to remove most of the proteins.

Any precipitate formed was removed by further filtration.


The pH of the media was adjusted to 5.4 with 1 N KOH or 1
N HCl and 25-mL aliquots of medium were dispensed into
125-mL bottles with subsequent autoclaving for 15 min at
1210C. For the solidified media, Phytagel (2 g L-1) was
added to the liquid media followed by warming. The
Phytagel dissolved media were then dispensed into 125 mL
bottles and autoclaved. The bottles were cultured in a
culture room at 2620C and 16 h of light daily provided by
cool-white fluorescent tubes (50 mol m-2 s-1). Bottles
containing the liquid media were also cultured in the same
culture room but on an orbital shaker (Edmund Bhler,
Swip SM 25 DIGI, Germany) at 80 rpm unless otherwise
stated. After six weeks in culture, explants were evaluated in
terms of the number of PLBs formation (>1mm in diameter)
per treatment.
Plantlet regeneration. In order to obtain plantlets, the
PLBs were transferred to the same medium as that for PLBs
initiation but solidified with 2 g L-1 Phytagel. The effect of

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PUCHOOA / Int. J. Agri. Biol., Vol. 6, No. 5, 2004


including potato homogenate (10 mg L-1) to the medium
was also investigated. The potato homogenate was prepared,
immediately before use, by grating 100 g of fresh potatoes
followed by blending with 500 mL of liquid medium for 2
min. The pH of the medium was adjusted to 5.4 before
autoclaving. The cultures were maintained in a culture room
at 2620C and 16 h of light daily provided by cool-white
fluorescent tubes (50 mol m-2 s-1).
Acclimatization. Plantlets were taken out from bottles and
gently washed with distilled water. They were then
separated, transplanted onto wet wood charcoal in a basket
at a temperature of 2620C and 16 h light: 8 h dark
photoperiod provided by white fluorescent tubes (500 mol
m-2s-1). The basket was covered with polyvinylidenechloride
film to avoid desiccation. Between 24 weeks after the start
of acclimatization, the film was gradually removed.
Experimental design. A completely randomized design
was used for all experiments with the only recognizable
differences between the explants being the treatments
applied to them. All treatments consisted of five replicates
and each replicate contained five explants. The number of
PLBs formed (>1mm in diameter) after six weeks of culture
as well as the number of plantlets regenerated per PLB after
a further six weeks of culture was recorded. The mean for
each treatment was calculated.

Fig. 5 Leaves protruding from PLBs

Fig. 6 Shoot and root formation on regeneration


medium

RESULTS
Effects of plant growth regulators and type of media.
The mean number of PLBs (>1mm) obtained with the three
combinations of BA with NAA in liquid and solidified
media is presented in Fig. 1 and 2, respectively. All explants
cultured in the liquid media started swelling after about one
week in culture. However, those cultured onto solidified
media took longer to swell. Similarly, PLBs formation were
visible in all the liquid media after a further two weeks in
culture while in solidified media they were visible only after
six weeks in culture. Explants cultured in media devoid of
plant growth regulators also responded, however, many of
them died after eight weeks in culture. The maximum
number of PLBs was obtained with liquid MS medium that
contained 0.1 mg L-1 of BA and 1.0 mg L-1 of NAA (54
PLBs). In all cases, it was found that cultures grown in
liquid medium gave better response than their counterpart
solidified medium. For example, the solidified MS medium
supplemented with 0.1 mg L-1 of BA and 1.0 mg L-1 of
NAA gave only 37 PLBs compared to 54 in the liquid
medium. MS medium was superior at all plant growth
regulator combinations followed by Vacin and Went
medium. Least PLBs development was obtained on unsupplemented solidified Knudson medium (only 2). Even
when supplemented with plant growth regulators and using
liquid medium, the response obtained with Knudson C
medium was lowest.
Speed of agitation. The influence of the speed of agitation
of the shaker was examined, from 80140 rpm, using MS

Fig. 7 Hardened Dendrobium plants

medium that contained 0.1 mg L-1 of BA and 1.0 mg L-1 of


NAA (Fig. 3). Although insignificant, a general decline in
the number of PLBs was noted with increase in agitation
speed.
Effects of adding coconut water to MS medium. Addition
of coconut water to the MS medium supplemented with 0.1
mg L-1 of BA and 1.0 mg L-1 of NAA proved to be an
important factor in the propagation of Dendrobium Sonia
PLBs (Fig. 4). In medium that contained 15% (v/v) of
coconut water, the highest number of PLBs was obtained.

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IN VITRO CULTURE OF Dendrobium (ORCHIDACEAE) / Int. J. Agri. Biol., Vol. 6, No. 5, 2004
immature fruits and seeds (Steward & Caplin, 1952;
Steward & Shantz, 1959), orange juice, malt extract, yeast
extract, casein hydrolysate, leaf extracts, sap from a number
of plants and tumour extracts. Park et al. (2003) have used
potato homogenate in the regeneration of Doritaenopsis.
For the hardening of the orchid plantlets, it was
essential to maintain a high humidity (about 90%) around
the plantlets for two weeks. Also, wood charcoal was used
as substrate as it provides good drainage and adequate
aeration to the roots, which is of primordial importance in
the culture of orchids. Substrates, which have the tendency
to retain too much moisture, should be avoided in all cases
for success in the hardening of orchid plantlets.
This study has shown that the orchid Dendrobium
Sonia can be regenerated on a large scale using a liquid
medium for the efficient initiation of PLBs and their
eventual regeneration into plantlets using a number of
supplements. The active ingredients of these supplements
and their role in promoting regeneration in Dendrobium
Sonia needs to be investigated so as the use of other
supplements such as bananas. Other substrates in the
hardening of the plantlets such as wood chips, sand,
rocksand, coconut coir, vermiculite or a mixture of these
could also be investigated.
Acknowledgements. The author wishes to thank Mr B
Rajkomar, Manager, Rose Belle Sugar Estate for providing
the orchid mother plants, Mr L Junye, Technical Assistant at
the Faculty of Agriculture, University of Mauritius, for his
invaluable help in looking after the in vitro cultures and the
hardening aspect, Mr A Maraye, Farm Manager at the
University of Mauritius for taking care of the hardened
plants and the Faculty of Agriculture, University of
Mauritius for supporting this work.

At higher (20% v/v) and lower (10% v/v) levels, the


propagation of PLBs was inferior.
Plantlet regeneration and acclimatisation. When the
PLBs were sub-cultured onto MS medium supplemented
with 1.0 mg L-1 NAA, 0.1 mg L-1 BA, 10 g L-1 potato
homogenate, 150 mL L-1 coconut water, 30 g L-1 sucrose
and 2 g L-1 Phytagel, they grew in size. After two weeks
leaves were seen protruding from the PLBs (Fig. 5). After
six weeks in culture, the medium supplemented with the
potato homogenate gave an average of ten shoots per PLBs
cultured compared to an average of only six in the medium
devoid of potato homogenate. Upon sub-culturing onto
fresh medium, plantlets with well-formed leaves and roots
were obtained (Fig. 6). These were potted into baskets
containing wood charcoal and acclimatized under the
conditions mentioned in the materials and methods section
to obtain normal Dendrobium plants (Fig. 7). Hardened
plantlets showed significantly high survival rate (84%) after
eight weeks.

DISCUSSION
This study deals with the mass propagation of
Dendrobium Sonia orchid. Among the three different
media and the different combinations of the two plant
growth regulators used in this study, liquid MS medium
(Murashige & Skoog, 1962) agitated at 80 rpm and
supplemented with 1.0 mg L-1 NAA, 0.1 mg L-1 BA, 150
mL L-1 coconut water and 30 gL-1sucrose was the most
efficient in inducing protocorm-like bodies (PLBs). This is
due to the fact that liquid medium provides better aeration
and optimum conditions for nutrient uptake. In orchids, PLB
regeneration has been suggested to be comparable to
somatic embryogenesis pathway (Morel, 1974). In other
orchid species, a callus stage has proved to be a necessary
condition for in vitro regeneration (Stewart & Button, 1978;
Kerbauy, 1984; Chen & Chang, 2001).
For the regeneration of plantlets, addition of potato
homogenate as well as coconut water proved to be
beneficial. Addition of naturally occurring supplements in
plant tissue culture has been investigated by a number of
workers. One of the earliest reports is that of Overbeek et al.
(1941), who succeeded in growing immature Datura
embryos in culture by including the liquid endosperm of
Cocos nucifera (coconut milk) in their culture medium.
Coconut milk was also shown to stimulate cell division in
other cultured tissues due to the presence of cytokinins and
its use as a supplement was adopted in many laboratories
(Duhamet & Gautheret, 1950; Morel, 1950; Nickell, 1950;
Duhamet, 1951; Henderson et al., 1952; De Ropp et al.,
1952; Archibald, 1954; Wiggans, 1954; Dix & van Staden,
1982). Other complex plant juices and liquid endosperms
have been shown to possess stimulatory properties more or
less similar to those of coconut milk. These include liquid
endosperm from immature corn (Ntien et al., 1951),
tomato juice (Nitsch, 1951; Straus & La Rue, 1954),

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(Received 18 May 2004; Accepted 10 August 2004)

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