AsPac J. Mol.

2010
Mol. Biol.
Biol.Biotechnol.
Biotechnol.
Vol. 18 (1), 2010
Vol. 18 (1) : 193-197

Germination of G. speciosum in vitro

193

Factors affecting seed germination of Grammatophylum speciosum cultured in vitro

Sudarat Khampa*, Preeya Wangsomnuk and Pinich Wangsomnuk
Department of Biology, Faculty of Science,Khon Kaen University, Khon Kaen, Thailand 40002.
Proceedings Asia Pacific Conference on Plant Tissue and Agribiotechnology (APaCPA) 17-21 June 2007

Abstract. Grammatophylum spreciosum is the largest epiphytic orchid distributed from Southeast Asia, Indonesia, New Guinea to
the Solomon islands. So far, there was no reports on seed germination or micropropagation of this species. Here, we investigated
seed germination on the five basic inorganic media which were Murashige and Skoog (MS) medium, a quarter of MS (1/4 MS)
medium, Gamborg B5 (B5) medium, Vacin and Went (VW) medium and Knodson C (KC) medium together with or without
0.5 g/l activated charcoal. MS medium with 0, 0.5, 1, 2, 3, 4 and 5 mg/l N6-benzyladenine (BA) and 0, 0.025, 0.05, 0.075,
0.10 mg/l thidiazuron (TDZ) and the light treatment were also tested their effect on the germination of this species in vitro for
12 weeks. The result showed that KC medium without activated charcoal gave the highest seed germination with no effect on
light treatment whereas MS medium showed the least seed germination efficiency. It was found that BA, TDZ, light and dark
treatment had no effect on seed germination unlike the activated charcoal which showed negative effect on seed germination of
G. spreciosum. This information should be useful for multiplication of G. spreciosum, its hybrid including genetically modified
G. spreciosum in the future.
Keywords: Grammatophylum speciosum; Germination; Micropropagation.

INTRODUCTION
Orchids are the largest family of flowering plants, of
which many are grown commercially world wide (Arditti,
1992). Grammatophylum spreciosum is the largest epiphytic
orchid distributed from Southeast Asia, Indonesia, New
Guinea to the Solomon islands. It is an important types of
commercial orchids used for potted plant. The increased
popularity of orchids has lead to a major increase in production and sale. Recently commercial production and cultivation of terrestrial orchids have slowly in seed germination.
So far, there were no reports on seed germination or micropropagation of G. spreciosum.
This work examined factors that influence in vitro germination of G. spreciosum. The effect of basic inorganic media,
activated charcoal, light, dark and growth regulator (BA and
TDZ) on the germination rate and protocorm formation
was investigated. This information should be useful for multiplication of G. spreciosum, its hybrid including genetically
modified G. spreciosum in the future.

MATERIALS AND METHODS

Seeds obtained from mature capsule were cultured on
the five basic inorganic media which were Murashige and
Skoog (MS) medium (Murashige and Skoog, 1962), a quar-

ter of MS (1/4 MS) medium, Gamborg B5 (B5) medium

(Gamborg et al., 1968), Vacin and Went (VW) medium
(Vacin and Went, 1949) and Knodson C (KC) medium
(Knodson, 1946), MS medium with 0, 0.5, 1, 2, 3, 4 and
5 mg/l N6-benzyladenine (BA) and 0, 0.025, 0.05, 0.075,
0.10 mg/l thidiazuron (TDZ) together with or without 0.5
g/l activated charcoal, 30 g/l sucrose and 2.5 g/l phyta gel
pH 5.7-5.8. One replicate consisted of on 4 culture jars containing 300-500 seeds per jar. After 12 weeks, the number of
protocorm was counted under a light microscope, and their
percentages were counted as well. Developmental post stage
were scored on 5 stages (Fig 1). Seed germination: 0: seed
(on germination); 1: embryo interrupt seedcoat (germination); 2: protocorm size < 0.25 mm.; 3: protocorm size
0.25-0.50 mm.; 4: protocorm size 0.51-1.0 mm. 5: protocorm size >1.0 mm.

RESULTS AND DISCUSSION

The effect of basic salt medium shown in Fig 2 presenting inducing seed germination and the number of protocorm; KC medium without activated charcoal gave the
* Author for correspondence:
Sudarat Khampa, Department of Biology, Faculty of Science,Khon Kaen
University, Khon Kaen, Thailand 40002, Tel:+6643 342 908, Fax:+6643
202317, E-mail: preeya11@yahoo.com.

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Germination of G. speciosum in vitro

Figure 1. Growth stages used to determine germination and development of G. spreciosum in vitro: A, seed (on germination);
B, embryo interrupt seedcoat (germination); C, protocorm size < 0.25 mm.; D, protocorm size 0.25-0.50 mm.; E, protocorm
size 0.51-1.0 mm. protomeristem denoted by the arrow; F, long-protomeristem protocorm. Scale bar = 0.25 mm. (A-C) and
0.50 mm. (D-F).

Figure 2. Percentage of seeds/protocorm in a given developmental stage 12 weeks after sowing for G. spreciosum: Effect of
basal salt medium on seed germination in 12 weeks 16-hr photoperiod (% Protocorm on X axis).

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Figure 3. Percentage of seeds/protocorm in a given developmental stage 12 weeks after sowing for G. spreciosum: Effect of
basal salt medium on seed germination in 12 weeks dark period (% Protocorm on X axis).

Figure 4. Percentage of seeds/protocorm in a given developmental stage 12 weeks after sowing for G. spreciosum: Effect of
TDZ on seed germination in 12 weeks 16-hr photoperiod (% Protocorm on X axis).

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Germination of G. speciosum in vitro

Figure 5. Percentage of seeds/protocorm in a given developmental stage 12 weeks after sowing for G. spreciosum: Effect of
basal salt medium on seed germination in 12 weeks dark (% Protocorm on X axis).

Figure 6. Percentage of seeds/protocorm in a given developmental stage 12 weeks after sowing for G. spreciosum: Effect of BA
on seed germination in 12 weeks 16-hr photoperiod (% Protocorm on X axis).

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Figure 7. Percentage of seeds/protocorm in a given developmental stage 12 weeks after sowing for G. spreciosum: Effect
of basal salt medium on seed germination in 12 weeks dark
(% Protocorm on X axis).
highest seed germination (100%) whereas MS medium
with activated charcoal gave the lowest seed germination
(92.51.3%). There is no effect of light and dark on the germination. The color of protocorms in which grown in light
was light green but the protocorms grown in the dark was
white. Stimulation of seed germination by auxin has been
reported in some orchid species (Withner, 1959; Arditti,
1979). In Calanthe, a high-concentration BA pre-treatment
inhibited seed germination but promoted protocorm formation (Miyoshi and Mii, 1995). However, we found no effect
of BA on both seed germination and protocorm formation.
The effect of cytokinin thus appears to be stage-specific, at
least for this species. In G. spreciosum, TDZ promoted protocorm formation but showed similar effect on seed germination. More detailed studies are therefore needed in this
species for elucidating the role of each phytohormone during orchid seed germination, particularly in respect to additional and/or synergistic effect. Though, activated charcoal
showed negative effect of seed germination, however, the
protocorms in this treatment had extended-longprotomeristem (Fig 1F) unlike ones treated in the medium without the
charcoal, they had slightly shorter extended-protomeristem.

REFERENCES

ACKNOWLEDGEMENTS

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We thank Raktin Uthai for helping to prepare the manuscript.

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