Comparison of Different Culture Media For The in Vitro Culture of Dendrobium (Orchidaceae)
Comparison of Different Culture Media For The in Vitro Culture of Dendrobium (Orchidaceae)
Comparison of Different Culture Media For The in Vitro Culture of Dendrobium (Orchidaceae)
15608530/2004/065884888
http://www.ijab.org
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
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
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
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
60
100
KdC
80
VW
0.0
NAA /
0.0 BA
1.0
NAA /
0.5BA
MS
140
80
60
40
20
0
0
10
15
20
885
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
886
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.
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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