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Proceedings of NIOC 2010, Nagoya Dome, Japan
- 16 -
Genetic Transformation of the Indonesian Black Orchid
(Coelogyne pandurata Lindley) through Agrobacterium tumefaciens for Micropropagation
Endang Semiarti1,2, Ari Indrianto1, Eko A. Suyono1, Rizqie Lingga Nurwulan2, Ratih Restiani1,
Yasunori Machida3, Chiyoko Machida4
1Faculty of Biology, Universitas Gadjah Mada, Jl. Teknika Selatan, Sekip Utara, Yogyakarta 55281,
Indonesia
2Graduate School, Center for Research of Biotechnology, Universitas Gadjah Mada, Barek, Yogyakarta
55281, Indonesia
3Division of Biological Sciences, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya,
464-8602, Japan
4College of Biotechnology and Bioscience, Chubu University, Kasugai, 487-8501, Japan
ABSTRACT
The black orchid (Coelogyne pandurata Lindley) is an endemic orchid from East Kalimantan, that is
characterized by the large size of flowers, with greenish sepals and petals, with a black labellum.
Recently, the population of this orchid was decreased and its extinction threatened to be extinct
because of overcollecting and/or habitat destruction. The mass propagation of this orchid is urgently
needed. Micropropagation of the black orchid was carried out by introducing the Arabidopsis gene
Knotted1-like Arabidopsis thaliana (KNAT1) using Agrobacterium tumefaciens. As the plant materials,
four-month-old protocorms that were maintained on Vacin and Went (VW) medium were used with 3
replications. The experiment was carried out in three steps: 1) Obtaining the most suitable basic
medium (Vacin and Went, VW; New Phalaenopsis, NP; Murashige dan Skoog, MS; and KNUDSON
C, KC); 2) Selection of the best phase of developing protocorms for gene transfer; 3) and Genetic
transformation of plasmid 35S-KNAT1 and pGreen (empty vector plasmid) using A. tumefaciens
strain LBA4404 into orchid protocorms. The results show that the best medium for in vitro culture of
the black orchid protocorm is a half strength of MS (1/2 MS) medium that could increase the growth
rate of 9.6% protocorms up to phase 6 after 12-week cultivation. But after 12 weeks, the best medium
for shoot induction to obtain plantlets is NP medium, so that 11.5% protocorms become plantlets with
three leaves. The best condition for gene transfer is five-month-old protocorm. The frequency of
genetic transformation using A. tumefaciens is 32.9% for pGreen vektor and 43.2% for p35S-KNAT1.
This work is in progress.
Key words: Black orchids, Coelogyne pandurata, basic medium, protocorm, genetic transformation,
KNAT1, Agrobacterium tumefaciens
INTRODUCTION
Black orchid (Coelogyne pandurata Lindley) is an
endemic orchid of the Province of East Kalimantan,
Indonesia, that is threatened by extinction. The
over-collection, habitat destruction, and difficulties
of cultivation through conventional methods are
the chief problem of this orchid (Arditti, 1992).
The uniqueness of this orchid is its very short (3-5
days) blooming period and difficult pollinazation
(Arditti, 1992). Moreover, in vitro seed
germination of this orchid needs special condition
such as 3-4 months incubation in a dark prior
germination (Wirakusumah, 2009, personal
communication). For successful cultivation, in
vitro seed germination is the key step (Arditti &
Ernst, 1993). In order to obtain the optimal
condition for in vitro seed germination of this
orchid, some experiments using various culture
media are needed to obtain the most suitable
medium.
In orchid tissue culture, the various
mediums for seed germination and shoot induction
are Knudson C (KC), Vacin and Went (VW), and
Murashige and Skoog (MS) with addition of some
organic complexes such as coconut water (Arditti,
1993, Widiastoety dan Syafril, 1993; Demasabu et
al., 1998; Untari et al., 2006). Islam et al. (1998)
used New phalaenopsis (NP) medium for callus
induction of Phalaenopsis. Semiarti et al. (2007)
also used the NP medium for growing
Phalaenopsis orchid before and after genetic
transformation of the orchid using A. tumefaciens.
The results of our previous experiment
(Semiarti et al., 2007) in which insertion of
Arabidopsis KNAT1 gene into Phalaenopsis orchid
Proceedings of NIOC 2010, Nagoya Dome, Japan
- 17 -
protocorm resulted in multishoots production
(about 31 shoots from one protocorm) will be
useful for the micropropagation of black orchids.
Multishoot occurrence in KNAT1 transgenic plants
has also been reported by Chuck et al. (1996) in
transgenic Arabidopsis plant and Nishimura et al.
(2000) in Nicotiana. In Dendrobium “Madame
Thong In” orchid, Yu et al. (2001) obtained the
multishoots from callus that derived from cut off
protocorms that transfered by DOH1 gene (KNAT1
homologous in Dendrobium). Each shoot could be
independently grown into a plantlet.
Here we report the genetic transformation of
KNAT1 gene under the control of Cauliflower
Mosaic Virus (CaMV) in pGreen vektor using
Agrobacterium tumefaciens strain LBA 4404 into
protocorms of black orchids for micropropagation.
MATERIALS AND METHODS
Plant materials and culture condition
Mature seeds and 4-month-old protocorms of black
orchids were used as plant materials. The
Protocorms were the generous gift of Mr.
Wirakusumah (the owner of Edward and Frans
Orchids Nursery, East Java). Seeds from fully
ripening fruit (five-months-old fruit) were sown in
various culture media: Knudson C (KC), Vacin &
Went (VW), New Phalaenopsis (NP) (Islam et al.,
1998), and Murashige & Skoog (MS) in half and
full strength concentration of macroelements with
and without 150 ml.l-1 coconut water. Protocorms
were transferred into four kinds of orchid medium:
VW, MS, NP and KC medium, each supplemented
with 150 mg.l-1 potato, 150 mg.l-1 banana, 150
ml.l-1 coconut water, and 1 ppm NAA. The in
vitro cultures were incubated at 25°C with 1000
lux continuous light. The growth of protocorms,
shoots and plantlets was examined every week.
Genetic Transformation
Genetic transformation of plasmid 35S::KNAT1
and pGreen vector into orchid was carried out
according to the method of Semiarti et al. (2007),
except that the liquid medium that was used to
rinse the protocorm was half strength VW medium
and 300 mg.l-1 Cefotaxim. SIM (Shoot Induction
medium; 0.15 µM NAA+ 5 µM 2iP) supplemented
with 100 mg.l-1 Kanamisin for selecting
independent transformant. Frequency of
transformation was decided by the ratio of the
number of surviving protocorms per total number
of transformed protocorms.
RESULTS AND DISCUSSION
Morphology of Black Orchid
The black orchid (C. pandurata) is an epiphytic
sympodial orchid. Some pseudobulbs grow parallel
with two leaves each. Five to seven flowers were
arranged in a raceme, fragrance, each flower is
7-12 cm in diameter. Sepals and petals are green
and the labellum (lip) is black. Seeds are
microscopic in size, inside the fruit (Fig. 1).
Fig. 1. Morphology of the Black Orchid. A. A Plant
with flower; B. Plant at the base of pseudobulbs; C.
Close up a flower; D. Black labellum; E. Mature
seeds; F. Fruit. Bars: 5 cm in A and B; 1 cm in C, D,
and F; 0,5 mm in E.
Developmental phases of black orchid
To analyze the growth rate of the black orchid
embryo during seed germination, we classified the
developmental period into six phases based on the
growth phases, namely phase 1-6: phase
1/yellowish embryo, phase 2/green embryo, phase
3/bipolar embryo phase 4/first leaf formed embryo,
phase 5/second leaf formed embryo and phase
6/third leaf formed embryo. The time course of
embryo development observation showed that the
color of the embryo started to change from
yellowish (phase 1) into green (phase 2) three
weeks after sowing. At four weeks, the green
embryo formed a bipolar structure (phase 3), with
one side darker than the other. The darker pole of
the embryo changed into leaf primordia (phase 4)
at the fifth week; protocorm with two leaves at
seven weeks (phase 5) and protocorm with three
leaves at eleven week (phase 6) (Fig. 2).
Twelve weeks after sowing, based on the
growth rate of embryos, the data revealed that 1/2
MS medium is the best medium to support and
accelerate the growth rate of black orchid embryos
(Table I). This was indicated by 84.62% of the
protocorms, which can grow up to phase 5 and
9.62% which grow up to phase 6 with the third leaf
Proceedings of NIOC 2010, Nagoya Dome, Japan
- 18 -
Table 1. Growth of Black Orchid’s Embryo in Various Medium at Twelve Weeks After Sowing
Percentage of growing embryo at each phase
Variation
of
Medium
Number of
Sowed
Protocorm
Phase 1 Phase 2 Phase 3 Phase 4 Phase 5 Phase 6 Death
protocorm
1/2 KC 215 0.00% 0.00% 0.00% 57.21% 27.91% 0.00% 14.88%
(123) (60) (32)
KC 174 0.00% 0.00% 0.00% 17.24% 70.69% 0.00% 12.07%
(30) (123) (21)
KC+CW 175 0.00% 0.00% 0.00% 41.14% 2.86% 0.00% 56.00%
(72) (5) (98)
1/2 VW 109 0.00% 0.00% 0.00% 59.63% 40.37% 0.00% 0.00%
(65) (44)
VW 134 0.00% 0.00% 0.00% 7.46% 40.30% 0.00% 52.24%
(10) (54) (70)
VW+CW 75 0.00% 0.00% 0.00% 9.33% 25.33% 0.00% 65.33%
(7) (19) (49)
1/2 NP 193 0.00% 0.00% 0.00% 13.47%
86.53% 0.00% 0.00%
(26) (167)
NP 112 0.00% 0.00% 0.00% 18.75% 57.14% 4.46% 19.64%
(21) (64) (5) (22)
NP+CW 105 0.00% 0.00% 0.00% 1.90% 72.38% 8.57% 17.14%
(2) (76) (9) (18)
1/2 MS 52 0.00% 0.00% 0.00% 5.77% 84.62%
9.62% 0.00%
(3) (44) (5)
MS 262 55.73% 0.00% 0.76% 20.99% 9.16% 0.00% 13.36%
(55) (24) (35)
MS+CW 73 0.00% 0.00% 0.00% 32.88% 50.68% 1.37% 15.07%
(24) (37) (1) (11)
v
Phase 1 Phase 2 Phase 3 Phase 4 Phase 5 Phase 6
Fig. 2. Developmental phases of The Black Orchid’s embryo
emerging from the shoot tip. These results indicate
that half-strength concentration of complete
elemens containing medium is needed for black
orchid seed germination.
When we started to use 4-month-old
protocorms as plant materials, based on the criteria
above, the best medium for the maintenance of the
developmental process of orchid protocorms and
shoots is NP medium. Three out of 26 protocorms
reached phase 5 within two months of subculture
on NP medium (Table II). This is consistent with
the results of Islam et al. (1998), who obtained the
best condition for callus induction on NP medium.
It because of the nitrogen and phosphate content in
the NP medium are higher than the others.
Proceedings of NIOC 2010, Nagoya Dome, Japan
- 19 -
Fig. 3. Shoots of transforman (s). (A), Non transforman; B. pGreen transformed shoots; C. pKNAT1
transformed shoots.
Table II. Growth of black orchid protocorms from 4 months in vitro culture, after 2 months transfered into
various culture media.
Medium Number of
transfered
protocorms Number of protocorms reach developmental phase
Phase 1 Phase 2 Phase 3 Phase 4 Phase 5
KC 15 0 7 8
0 0
VW 15 0 2 13 0 0
MS 23 0 3 18 2 0
NP* 26 0 8 13 2 3
*the best medium
Table III. Frequency of transformation of the black orchid (C. pandurata Lindley) using Agrobacterium
tumefaciens.
Replication Number of transformed
protocorms
Number of survival
protocorms
Non-transformant
3 - 45.97% (24/62)
pGreen (Vector) 3 54 32.96% (18/54)
P35S::KNAT1 3 35 43.25% (15/35)
Frequency of genetic transformation of black
orchid using A. tumefaciens
The transformation result of p35SKNAT1 and
pGreen vector show that the frequency of
transformation is relatively high, about 43.25% (15
of 35 protocorms are kanamycin-resistant), and
32.96% for pGreen vector (18 of 54 protocorms
are kanamycin-resistant) (Fig. 3 and Table 3). PCR
analysis to confirm the insertion of KNAT1 gene is
still in progess. The expression of KNAT1 gene in
the black orchid transformants might improve the
totipontency of the orchid to form shoots as has
occurred in another natural orchid, Phalaenopsis
amabilis (Semiarti et al., 2007). The results will
support both the conservation effort and orchid
farmers.
ACKNOWLEGDEMENT
The research was supported by Indonesian DGHE
Research Competition grant HB XVII 2009 No.
LPPM-UGM/604/2009. We thank the Bunga
Rintee Orchid Nursery for the generous gift of
Black Orchid fruit and Mr. Wirakusumah for the
gift of 4-month-old protocorms and valuable
discussion on black orchid culture techniques. This
work was supported, in part, by Grants-in-Aid for
Scientific Research on Priority Areas (No.
19060003) to Y.M. and C.M. from the Ministry of
Education, Culture, Sports, Science and
Technology (MEXT) of Japan, and by an
Academic Frontier Project for Private Universities
matching fund subsidy from MEXT 2005-2009.
REFERENCES
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Arditti, J. and R. Ernst. 1993. Micropropagation of
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Chuck, G., C. Lincoln, dan S. Hake. 1996. KNAT1
Induced Lobed Leaves with Ectopic Meristem
Proceedings of NIOC 2010, Nagoya Dome, Japan
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when Overexpressed in Arabidopsis. Plant Cell
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Semiarti, E., A. Indrianto, A. Purwantoko, S.
アグロバクテリウム遺伝子導入法を用いたインドネシア産 Coelogyne pandurataのマイクロプロパ
ゲーション法の開発
Endang Semiarti 1,2, Ari Indrianto1, Eko.A. Suyono1, Rizqie Lingga Nurwulan2, Ratih Restiani1,
町田泰則3, 町田千代子4
1.Faculty of Biology, Universitas Gadjah Mada, Jl. Teknika Selatan, Sekip Utara, Yogyakarta 55281,
Indonesia
2Graduate School, Center for Research of Biotechnology, Universitas Gadjah Mada, Barek, Yogyakarta
55281, Indonesia
3名古屋大学大学院・理学研究科・生命理学専攻 〒464-8602 名古屋市千種区不老町
4中部大学・植物バイオ研究センター, 応用生物学部 〒487-8501 愛知県春日井市松本町1200
摘要
ブラックオーキッド(Coelogyne pandurata Lindley)はインドネシア東カリマンタン産のランであ
り、花が大きくガク片と花弁は緑色で唇弁は黒色である。最近、このランは、過剰採取と生息地の
破壊によって絶滅の危機に瀕している。このランの人工増殖は緊急の課題である。シロイヌナズナ
のKnotted1-like Arabidopsis thaliana (KNAT1)遺伝子をアグロバクテリウム法により導入したブラッ
クオーキッドを用いてクローン増殖を試みた。植物材料として、Vacin and Went(VW)培地で 4
ヶ月培養したプロトコームを用いた。シュート形成の条件検討を3点について行った。第一に、最
適培地(VW、New Phalaenopsis (NP)、Murashige・Skoog(MS)、Knudson C(KC))の検討、第
二に、遺伝子導入に最適のプロトコームの生育段階の検討、第三に、A. tumefaciens LBA4404 を用
いてプロトコームへのプラスミド 35S-KNAT1 とpGreen ベクターの導入を行った。ブラックオーキ
ッドのプロトコーム培養の最適培地は 1/2 MS であった。12 週間の培養で 9.6 %のプロトコームが
フェイズ 6以上になった。 しかし、12 週間以降の幼葉殖物の生育には NP 培地が最適であり、11.5 %
のプロトコームが 3葉を形成した。遺伝子導入に最適なプロトコームは 5ヶ月培養したものであっ
た。遺伝子導入効率は pGreen ベクターについては 32.9%、p35S-KNAT1 については 43.2 %であっ
た。