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Genetic transformation of the Indonesian black orchid (Coelogyne pandurata Lindley) through Agrobacterium tumefaciens for micropropagation

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Proceedings of NIOC 2010, Nagoya Dome, Japan
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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
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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
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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
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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.
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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
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 WentVW)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、
適培地(VWNew Phalaenopsis (NP)MurashigeSkoogMS)、Knudson CKC))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 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 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。
... Hitam' is an endangered wild orchid native to Kalimantan Island, Indonesia (Semiarti et al., 2010;Puspitaningtyas, 2020). The black orchid is also widespread on other Indonesian islands, including Sulawesi (Semiarti et al., 2011), which used in this research (Figure 1). ...
... However, it was reported that the media is good to cultivate the genus of the Dendrobium orchid (Akter et al., 2007). Semiarti et al. (2010) also found that after 12 weeks of seed sowing, NP medium was the best medium for shoot induction to obtain plantlets of C. pandurata with three leaves. This current research proved that the NP medium is suitable for the protocorm and plantlet growth of C. pandurata orchid as experimented with several alternative media. ...
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p>Due to the high cost of the most often used basic media in tissue culture, it is vital to identify more affordable alternatives. This research aimed to determine the best alternative culture media for the protocorm and plantlet growth of Coelogyne pandurata Lindl. It employed a completely randomized design, eight treatments and four replications. The treatments referred to the different types media and consisted of M1 = New Phalaenopsis (NP) medium, M2 = a medium made from foliar fertilizer (FFM), M3 = NP + 2 cc L-1 AB mix solution (a media for hydroponics), M4 = FFM + 2 cc L-1 AB mix solution, M5 = NP + 50 cc L-1 of tomato extract, M6 = NP + 50 cc L-1 of bean sprout extract, M7 = FFM + 50 cc L-1 of tomato extract and M8 = FFM + 50 cc L-1 of bean sprout extract. The M4 medium exhibited the best results in terms of average leaves count (4.80), average shoot length (2.68 cm), average root length (4.35 cm), the average fresh weight per plantlet (214.5 mg) and dry weight of plantlets (73.1 mg). The average number of roots per plantlet was 4.25, acquired using the less expensive M8 treatment, which also produces a negligible number of leaves (4.50). In conclusion, the M4 medium is the most appropriate medium for growing protocorm and plantlet of C. pandurata . The experiment also found that the FFM basic medium combined with 50 cc L-1 of bean sprout extract can be used as another cheaper alternative for growing protocorms of C. pandurata .</p
... Using plant tissue culture or in vitro culture techniques, large quantities of plants can be produced from a small size of explant in a short time and the plants are genetically identical with the parent [6,7]. In addition to plant tissue culture, the use of genetic engineering can be combined for special purposes [8]. Genetic engineering is a technique of modification for changing the genetic content of an individual by way of insertion of a foreign gene or altered by mutations that produce plants with novel properties as desired by the creator [9−11]. ...
... It was described that the growth and development of P. amabilis orchid seeds in vitro on New Phalaenopsis (NP) medium are divided in 6 phases. Phase 0, a ripe embryo contained in orchid seeds wrapped with a dead cell membrane called a testa; Phase 1, embryo grows larger still inside the testa, the Phase 2 embryo grows larger and opens the testa sheath; Phase 3, the embryo gets bigger and swollen, the color turns yellow showing the difference in color density on the apical and basal, the embryo development is called protocorm, in the basal part of an absorbing hair; Phase 4, protocorm enlarges color turns green, absorbing hair is increasing in number; and Phase 5, green protocorm forms buds in the apical part, and roots in the basal, seed germination occur [8]. It was also analyzed that the embryo development in Dendrobium phalaenopsis and found the similar pattern to that in P. amabilis [14] (Fig. 2). ...
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Modern biotechnology for orchid breeding, especially in vitro culture and transgenic technology for mass propagation has developed. Due to its attractive flower, Indonesian native orchids such as moth orchids Phalaenopsis amabilis (L.) Blume, Black orchid Coelogyne pandurata Lindley, Vanda tricolor Lindley are often taken from their natural habitat to be traded, or domesticated as a hobby. So the existence of orchids in Indonesia continues to decline. Modern technology integrated with classical breeding will support the orchid conservation strategy as well as the commercial orchid industry. Especially production of the somatic embryo and shoot formation as much as possible directly from explant or indirectly through callus that emerges from explant and regenerates into plants in the culture medium with an additional appropriate concentration of growth regulators cytokinin and auxin. In a model plant, Arabidopsis thaliana, AtRKD4 is a key gene for embryo initiation and KNAT1 gene for the initiation of shoot apical meristem for producing shoot. These two genes can be used to induce somatic embryogenesis and shoot development in orchid by inserted the genes into orchid genomes mediated by Agrobacterium tumefaciens. This strategy effectively increased the production of both native and hybrid orchid plants through somatic embryogenesis and shoot development, that hopefully the regenerated plants will meet commercial demand and support the conservation of the native orchids.
... The sepals and petals are lancet-shaped, pointed, and typically green. As an orchid, the seeds inside the fruit are microscopic in size due to the lack of endosperm [3]. Nowadays, the population of this orchid has greatly decreased in nature and is classified as a rare, endangered orchid [13]. ...
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Coelogyne pandurata Lindl. is an endemic orchid species of Indonesia from East Kalimantan and Papua. It is locally known as the “black orchid” due to the distinctive black coloration on its labellum with intricate green and hairy lines. Unfortunately, the population of this orchid has significantly declined in its natural habitat, rendering it a rare endangered species. Temporary immersion bioreactor systems have been proven to be efficient for endemic plant micropropagation with performance enhancements and innovations. This study aims to develop an optimized bioreactor design and innovate the automatic control performance of temporary immersion bioreactor systems based on previous research. The control system developed consists of three modules, namely Graphical User Interface (GUI) module, Sequence Control (SC) module, and Hardware Interface (HI) module. The GUI module receives information regarding time and duration of immersion and gas exchange, then the information is synchronized by the SC module, which plays the role of starting and stopping the processes, while HI module executes the order of the automatic control system in the immersion and gas exchange process. The developed bioreactor design and control system offer convenience, require less labor, and ensure precise control over the optimum conditions for black orchid micropropagation.
... 。文心兰(Oncidium Gower Ramsey)的花瓣和萼片(黄色带有红色斑点) 同时含有花青素苷和类胡萝卜素,类胡萝卜素主要 是堇菜黄质(violaxanthin)的全反和9-顺异构体,花 青素苷主要是矢车菊素(cyanidin)及其甲基衍生物 芍药花素(peonidin) (Hieber et al., 2006)。其它兰科 植物的花青素苷也主要是矢车菊素,其次是天竺葵 素(pelargonidin)或芍药花素 (Kuehnle et al., 1997;Fossen and Øvstedal, 2003;Tatsuzawa et al., 2010)。 植物花色素的生物合成途径与分子调节机理已比 较清楚 (Tanaka et al., 2008) (Mudalige-Jayawickrama et al., 2005;Liew et al., 1998a;Han et al., 2005;Pitakdantham et al., 2010;Han et al., 2006 (Zhang et al., 2010;Shrestha et al., 2007;Semiarti et al., 2010;Sjahail and Mii, 2006) Mori K., Goto-Yamamoto N., Kitayama M., and Hashizume K., ...
... There are 8 replicate to each treatment combination. Variable observed include start of growing time (measure when 25% of the seed produce green embryos), growing phase (1-5) (Semiarti et al., 2010) and number of shoot per week up to 12 week. 2. Acclimatization. ...
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Black orchid is an epiphytic orchid found mainly in Borneo Island, Indonesia. This orchid currently is facing a large conservation problem and threatened to extinction due to human exploitation. It is therefore an urgent requirement to develop efficient propagation technique to conserve this species. This research aims to determine growth response and the ability of black orchid seeds to grow in different medium and to establish the best media for acclimatization. The experiment was divided into 2 stages, seed growth in vitro, and acclimatization. Three different media were employed during seed growth in vitro, i.e. Western 3 media (W3), Knudson C media (KdC) and organic media (O). Each treatment consists of eight replicates. Acclimatization stage used 4 types of media; moss, fern stem, charcoal and combination of charcoal and coconut husk. Each consists of 8 replicates. Results shows that seeds started to grow at 3 weeks after sowing in W3 media, and 5 weeks after sowing in other medium. At 12 WAP, seed growth reached phase 5 (third leaf formed) on W3 medium, whilst on KdC and organic media, reached phase 4 (second leaf formed embryo). Within 3 months, the highest shoot produced was on W3 media (581 shoots), whilst on KdC and organic media produced 255 and 191 shoots respectively. Almost all plantlets (98%) survived during acclimatization period. Plantlets grow best on moss, compared to other media.
... Here too, transgenics could serve as a useful method to manipulate the growth rate and development of orchids for novel ornamental purposes. Semiarti et al. (2007Semiarti et al. ( , 2009Semiarti et al. ( , 2010a transformed Phalaenopsis amabilis and black orchid (Coelogyne pandurata Lindley) protocorms with a T-DNA vector containing the Arabidopsis class 1 KNOX gene, BP/KNAT1, which yielding multiple shoots (as many as 31 per protocorm) and thus the possibility of altering the architecture of orchids in vitro and use for micropropagation although the shoots showed malformation due to the effect of this gene. No molecular evidence for transgene integration was provided, however, casting the validity of these results into doubt. ...
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Breeding orchids through traditional means is a lengthy process. Therefore, it would be advantageous if transgenic technologies could be applied for orchids to improve important traits such as novel flower colour, fragrance and shape, cut-flower longevity and flowering control, abiotic stress tolerance and resistance to pests and diseases. Even though there are several genetic transformation techniques available to orchid breeders, only two (Agrobacterium-mediated transformation and particle bombardment) have been successfully and consistently used thus far. This review aims to capture the full range of studies conducted on orchid transformation with a view of providing new perspectives for future molecular breeding programmes.
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INTISARI Anggrek hitam (Coelogyne pandurata) dan anggrek mutiara (Coelogyne asperata) adalah anggrek dari habitat asli hutan Kalimantan yang memiliki bentuk dan warna yang menarik sebagai tanaman hias dan mulai banyak dicari dan dijual kepada masyarakat namun upaya budidaya masih sangat terbatas. Salah satu upaya yang dilakukan untuk mencegah jumlah populasi anggrek hitam dan anggrek mutiara semakin menurun adalah melalui metode persilangan interspesifik untuk mempertahankan atau menghasilkan jenis maupun varietas baru tanaman anggrek. Salah satu informasi yang diperlukan dalam pemuliaan tanaman adalah kompatibilitas. Kompatibilitas persilangan Coelogyne pandurata x Coelogyne asperata belum diketahui. Penelitian ini bertujuan untuk mendapatkan waktu yang tepat untuk melakukan persilangan. Pada penelitian ini penyerbukan dilakukan pada hari ke 2 dan hari ke 5 setelah bunga mekar. Penyerbukan dilakukan pada pagi hari. Dilakukan 3 kali ulangan penyerbukan pada bunga. Penyerbukan dengan metode anggrek hitam sebagai tetua jantan dan anggrek mutiara sebagai tetua betina. Hasil penelitian menunjukkan penyerbukan yang berhasil membentuk buah adalah hasil persilangan pada bunga mekar umur 2 hari. Buah dipanen umur 3 bulan akan tetapi buah belum matang sempurna. Perkecambahan biji menggunakan media Vacin & Went (VW) dan media VW modifikasi. Biji hasil persilangan C. pandurata x C. asperata berhasil berkecambah pada media VW modifikasi dengan penambahan auksin 0,1 mg/l sitokinin 0,1 mg/l dan berkecambah 9 minggu setelah di tanam. ABSTRACT Black orchid (Coelogyne pandurata) and Pearl orchid (Coelogyne asperata) are endemic orchid from Kalimantan forest, shape and color suitable as ornamental flower. Many people collected the orchids from the wild for commercial purpose but not much effort on their propagation. To prevent population decreased, propagation and production of new varities via interspesific hybridisation need to be done. An important step required in plant breeding is to obtain cross compatiblity. Cross compatibility of some orchids from Indonesia are not known, including compatibility of Coelogyne pandurata x Coelogyne asperata. Information on compatibility and fertility is very important to produce good quality seed as propagation material. In this research pollination was performed on orchid plants at
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Mass screening of plants for chemical compound has produced a vast library of chemical compounds from which scientists hope to find new remedies to slow the ageing process and health decline and to treat diseases. This chapter provides an outline of the areas of pharmaceutical interest and the processes by which compounds are discovered and evaluated. It also discusses methods for medicinal crop protection and genetic manipulation for better production of phytochemicals.
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Transgenic orchid (Dendrobium Madame Thong-In) plants were regenerated by inoculating thin-section explants from protocorm-like bodies (PLBs) with Agrobacterium tumefaciens strain LBA4404 harboring a binary vector that carried the orchid DOH1 antisense gene. The transformation was performed through two consecutive stages of cocultivation, with the first stage occurring on antibiotic-free medium for 3 days and the subsequent stage on medium containing 50 mg/l carbenicillin for 3-4 weeks. Proliferated PLBs were repeatedly selected for kanamycin resistance. The presence and expression of the transgene were assessed by molecular analyses. Expression of the DOH1 antisense gene caused abnormal multiple shoot development, indicating a role for DOH1 in the basic plant architecture in orchid.
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We compared the phenotypes of transgenic tobacco plants over-expressing various knotted1-type class1 homeobox genes. All transformants showed abnormal leaf morphology, with the degree of abnormality depending upon the Nicotiana tabacum homeobox (NTH) gene that was over-expressed. Tobacco plants over-expressing NTH1 or NTH9 showed a relatively weak phenotype, while NTH15 and NTH20 over-expressing plants exhibited severe alterations, with occasional ectopic shoot formation on the leaves. Plants over-expressing NTH22 had a relatively severe phenotype, but did not form any ectopic shoots. These results indicate that all of the NTH genes can influence leaf development from the shoot apical meristem, but that the effect varies with the gene. Based on phylogenetic analysis of the NTH genes and comparison of the phenotypes of plants over-expressing them, we suggest that the kn1-type class1 family can be divided into two subgroups, and that the differences in their ability to induce the abnormal phenotype corresponds to the structures of their conserved domains.
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This is a large and expensive book. Free copies are not available for distribution. Please do not ask. It can be purchased from Zip Publishing (info@zippublishing.com). This illustrated reference work provided a detailed scientific approach to orchid biology. There are 15 chapters: history (in Asia, Africa, Europe, New Guinea and Australia), including the history of the discovery of orchid reproduction; classification and naming of orchids; evolution of the Orchidaceae, and of plant parts individually; cytology; physiology; phytochemistry; morphology; anatomy; mycorrhiza (including orchid-fungus specificity, seed germination and root characteristics); pollination (with attention to attractants and pollinators); embryology; reproduction (including reproduction through seeds, germination, and sexual and asexual propagation); heredity and breeding; ecology (with an account of the habitats in which orchids exist, as well as notes on climate, carbon fixation, seed dispersal and conservation); and commercial and ethnobotanical uses. Each chapter has a bibliography. -J.W.Cooper This a book. The author cannot send copies. It is available for purchase at www.amazon.com. -Joseph Arditti
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Plant development depends on the activity of apical meristems, which are groups of indeterminate cells whose derivatives elaborate the organs of the mature plant. Studies of knotted1 (kn1) and related gene family members have determined potential roles for homeobox genes in the function of shoot meristems. The Arabidopsis kn1-like gene, KNAT1, is expressed in the shoot apical meristem and not in determinate organs. Here, we show that ectopic expression of KNAT1 in Arabidopsis transforms simple leaves into lobed leaves. The lobes initiate in the position of serrations yet have features of leaves, such as stipules, which form in the sinus, the region at the base of two lobes. Ectopic meristems also arise in the sinus region close to veins. Identity of the meristem, that is, vegetative or floral, depends on whether the meristem develops on a rosette or cauline leaf, respectively. Using in situ hybridization, we analyzed the expression of KNAT1 and another kn1-like homeobox gene, SHOOT MERISTEMLESS, in cauliflower mosaic virus 35S::KNAT1 transformants. KNAT1 expression is strong in vasculature, possibly explaining the proximity of the ectopic meristems to veins. After leaf cells have formed a layered meristem, SHOOT MERISTEMLESS expression begins in only a subset of these cells, demonstrating that KNAT1 is sufficient to induce meristems in the leaf. The shootlike features of the lobed leaves are consistent with the normal domain of KNAT1's expression and further suggest that kn1-related genes may have played a role in the evolution of leaf diversity.
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Protocorm like bodies (PLBs) derived from callus of Phalaenopsis utilized sucrose, maltose and sorbitol for their growth in vitro. These carbon sources affected differently and could control PLB growth. On sucrose supplemented medium a few PLBs produced plantlets and most others regenerated yellowish or greenish callus like body (CLB). Almost 80% of unrooted and 58% of rooted plantlets developed yellowish green CLB at the base of plantlets. On maltose supplemented medium, PLBs regenerated PLBs and a few plantlets. In subsequent culture, about 44% of unrooted and 24% of rooted plantlets initiated green PLBs at the base of cultured plantlets. On sorbitol supplemented medium, most of the PLBs developed plantlets and a few additional PLBs. Among the carbon sources tested, sorbitol supported plantlet development the best in vitro and proved to be the most suitable carbon source for plantlet initiation and development from PLB.
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The homeobox gene knotted1 (kn1) was first isolated by transposon tagging a dominant leaf mutant in maize. Related maize genes, isolated by virtue of sequence conservation within the homeobox, fall into two classes based on sequence similarity and expression patterns. Here, we report the characterization of two genes, KNAT1 and KNAT2 (for knotted-like from Arabidopsis thaliana) that were cloned from Arabidopsis using the kn1 homeobox as a heterologous probe. The homeodomains of KNAT1 and KNAT2 are very similar to the homeodomains of proteins encoded by class 1 maize genes, ranging from 78 to 95% amino acid identity. Overall, the deduced KNAT1 and KNAT2 proteins share amino acid identities of 53 and 40%, respectively, with the KN1 protein. Intron positions are also fairly well conserved among KNAT1, KNAT2, and kn1. Based on in situ hybridization analysis, the expression pattern of KNAT1 during vegetative development is similar to that of class 1 maize genes. In the shoot apex, KNAT1 transcript is localized primarily to the shoot apical meristem; down-regulation of expression occurs as leaf primordia are initiated. In contrast to the expression of class 1 maize genes in floral and inflorescence meristems, the expression of KNAT1 in the shoot meristem decreases during the floral transition and is restricted to the cortex of the inflorescence stem. Transgenic Arabidopsis plants carrying the KNAT1 cDNA and the kn1 cDNA fused to the cauliflower mosaic virus 35S promoter were generated. Misexpression of KNAT1 and kn1 resulted in highly abnormal leaf morphology that included severely lobed leaves. The expression pattern of KNAT1 in the shoot meristem combined with the results of transgenic overexpression experiments supports the hypothesis that class 1 kn1-like genes play a role in morphogenesis.
Article
Plant development depends on the activity of apical meristems, which are groups of indeterminate cells whose derivatives elaborate the organs of the mature plant. Studies of knotted1 (kn1) and related gene family members have determined potential roles for homeobox genes in the function of shoot meristems. The Arabidopsis kn1-like gene, KNAT1, is expressed in the shoot apical meristem and not in determinate organs. Here, we show that ectopic expression of KNAT1 in Arabidopsis transforms simple leaves into lobed leaves. The lobes initiate in the position of serrations yet have features of leaves, such as stipules, which form in the sinus, the region at the base of two lobes. Ectopic meristems also arise in the sinus region close to veins. Identity of the meristem, that is, vegetative or floral, depends on whether the meristem develops on a rosette or cauline leaf, respectively. Using in situ hybridization, we analyzed the expression of KNAT1 and another kn1-like homeobox gene, SHOOT MERISTEMLESS, in cauliflower mosaic virus 35S::KNAT1 transformants. KNAT1 expression is strong in vasculature, possibly explaining the proximity of the ectopic meristems to veins. After leaf cells have formed a layered meristem, SHOOT MERISTEMLESS expression begins in only a subset of these cells, demonstrating that KNAT1 is sufficient to induce meristems in the leaf. The shootlike features of the lobed leaves are consistent with the normal domain of KNAT1's expression and further suggest that kn1-related genes may have played a role in the evolution of leaf diversity.
Pengaruh Bahan Organik dan NAA terhadap Pertumbuhan Anggrek Hitam (Coelogyne pandurata Lindl.) dalam kultur in vitro
  • R Untari
  • D W Puspitaningtyas
Untari, R. and D. W. Puspitaningtyas. 2006. Pengaruh Bahan Organik dan NAA terhadap Pertumbuhan Anggrek Hitam (Coelogyne pandurata Lindl.) dalam kultur in vitro. Biodiversitas. Vol.7, No.3; 344-348.
Fundamentals of Orchid Biology Micropropagation of Orchid
  • J J Arditti
  • R Ernst
Arditti, J. 1992. Fundamentals of Orchid Biology. John Wiley & Sons, Inc. New York. Canada Arditti, J. and R. Ernst. 1993. Micropropagation of Orchid. John Wiley & Sons, Inc. New York.