Biotechnology of Indonesian

Natural Orchids
Endang Semiarti

Faculty of Biology UGM

e-mail: esemiart@ugm.ac.id
 Indonesia is a hotspot of biodiversity

Rich of natural resources in the tropical rainforests, which

contain various flora, fauna and microbiota that support
people welfare

Nowadays, Indonesia is facing multiple problems in

biodiversity utilization and management due to increasing
human population

One of the critical problems is natural degradation that

threatening tropical biodiversity and also affecting the
community welfare

Need to be solved and maintained through

conservation toward sustainability
Benefits of Biodiversity
BIOLOGICAL RESOURCES

•Food for humans and for cultivated animals

•Medicinal and pharmaceutical resources
•Breeding stocks, population reservoirs
•Ornamental plants and animals
•Potential agents for crop improvement or biological
control
ECOSYSTEM SERVICES •Protection of water resources
•Nutrient storage and cycling
•Pollution breakdown and absorption
•Contribution to climate stability
•Maintenance of ecosystems

•Research, education and monitoring

SOCIAL BENEFITS
•Recreation & tourism
•Cultural values
Threats to Biodiversity
Hunting and overharvesting
Habitat loss
Pollution
Climate change
Natural disasters
How to save
them ?
Plant Breeding

Conventional
Breeding

Genetic
engineering

Clonal
micropropagation
Education for Sustainable
Development on Biodiversity

“Development that meets the present needs and

ability which compromise future generation
needs.”
 Orchids in the world
c. 800 genera (in SE Asia c. 240 genera)
c. 20000 species (in SE Asia c. 6500 spp.)
c. 5000 species in Indonesia
Indonesia kingdom of orchids

more than 5,000 species of orchids, these are native to almost every
part of the archipelago, growing at elevations from sea level
(Dendrobium striaenopsis) up to 3,000 meters (Dendrobium
cuthbertsonii) and temperatures between 8.7ºC and 32ºC.
Orchid habitat (Papua, 2700 m)
Vanda tricolor

V.Tricolor var suavis in the slope

of Mt Merapi are threaten to be
extinct because of pyrroclastic
flows and volcanic eruption at
2010
Black Orchid (Coelogyne
pandurata)

Over exploitation for illegal

market
Moth Orchid Phalaenopsis amabilis L. Blume
proposed to be used as a Model Organism for
Orchid’s Conservation
Phalaenopsis amabilis
Wild type

Phalaenopsis “yukimae”
(Hybrid)
Conventional Breeding
Conventional breeding
Clonal Propagation/Tissue Culture/
micropropagation

Plant Tissue
Culture

Protoplast
Culture

Microspore
Culture
Genetic engineering of Plant
Insersion of a foreign gene that responsible to
encode such desired character into a plant genome

GM- Depend on situation

Plant/BIOCROP and necessity

Drought tolerant

Rapid flowering
time

Different color,
shape, smell
The principal of creating GMO

DNA
Universal

Protein Protein
Bacteria
Bacteria Plant
Plant
Animal
Animal
SAME AMINO
ACID

Fluorescent
tobaco
Tobaco
Agrobacterium-mediated
transformation

E. Semiarti F. Biologi UGM 2010

Mechanism
Ornamental transgenic plants
Blue Rose Yoshikazu Tanaka et al 2007

Suntory Limited Research Center, Osaka, Japan

F3’5’H,
flavonoid 3,5-
Delphinidin
hydroxylase;
Accumulation
DFR,
dihydroflavono
l 4-reductase;
Blue color

2008

Stablee

comersial
E. Semiarti F. Biologi UGM 2012
Blue Orchids

flavonoid 3,5-hydroxylase; from

Commelina communis

Delphinidin
Accumulation
DFR,
dihydroflavono
l 4-reductase;
Blue color

Prof. Masahiro Mii & Team Chiba University-Japan

GM-Plant Research
in Gadjah Mada University (UGM)
Lab. of Biotechnology, Fac. of Biology
Micropropagation of Indonesian
endemic natural orchids  using a
key gene for shoot formation from a
Model Plant Arabidopsis thaliana
(KNAT1 gene)
Early Flowering Orchids 
acceleration of orchid flowering time,
using a Flowering Locus T-homolog
from Phalaenopsis amabilis orchids
(PaFT gene) in collaboration with Dr.
Step to create Transgenic
Plants
Explore target
gene
physical gen Detection
Transformation

(screening):
•GUS
chemical •GFP
Transformant
•PCR gen target
Selection :
biological • Antibiotic
Plant •Herbiside
•Enzym

Selection

Detection cultivation
 Structure of the T-DNA regions
that harbor 35S and 35S::KNAT1 DNA construct

35S
35S NPTII TNos

800 bp 260 bp

Fw 60 Rev 59

35S::KNAT1

NPTII
 Shoot Development of P. amabilis
Shoot Apical Meristem

Leaf
Primordia

Placental Ridge Procambial

Strand

Testa Absorbing hair Week-after sowing

Week-after sowing

(Semiarti et al., 2007)

 Transformation using
Intact protocorm Agrobacterium tumefaciens
Methods

Seed sowing Protocorms

3 weeks after sowing
on NP medium
Phenotypic & PCR
analysis
Co-cultivation
Regenerated 1-4 days
transformant
Rinse with liquid NP
medium+ Antibiotic

Selection of transformants
 Frequency of Shoot Formation on Kanamycin
containing selective medium

Non-transformant
NP0
8 weeks 24 weeks ( 6 months) Frequency of
shoot formation

Non-transformant 0.0%
NP + Kan
8 weeks

p35S
1.6%

NP + Kan
8 weeks 24 weeks ( 6 months)
KNAT1 gene of Arabdopsis thaliana induced multishoots
formation in P. amabilis orchid
pG35S

NP + Kanamycin

8 weeks 24 weeks

pG35SKNAT1

NP + Kanamycin

8 weeks 24 weeks
Multishoots
25.1 %
17.8 %

24 weeks ( 6 months)
23.7 %
Trumpet-like Rectangle Mediolaterally unopened
Detection of co-integration of
Transgene into Host genome
A. The presence of transgene B. Accumulation of transgene mRNA
in Orchid genom
 SOUTHERN BLOT ANALYSIS:
T-DNA that harbor GFP gene incorporated
into Orchid plant genomes
35S::KNAT1 transgenic orchid leaves produced
adventitious shoots from peripheral parts of leaves
in vitro on phytohormone-free medium

NT

35S::KNAT1

Vector

8 weeks after inoculated

 Phalaenopsis amabilis orchid transgenic
plants that harbor 35S::KNAT1

1.5 ys 1y

Advantages:
Multishoots: 1 embryo-> 91 plants
Shape: normal 1y

“Methods for Transforming Plant of Family Orchidaceae

World International Patent Organization (WIPO)-Japan
PCT/JP/2008/05622
 Transformation methods

Acetosyringone
Intact protocorm (AS)
2 -weeks after sowing

AS
Seed sowing on NP medium
Preculture in CIM For 4 days

Phenotypic & PCR analysis Transformation using

Agrobacterium tumefaciens
AS
Co-cultivation
1-4 days
Regenerated
transformant

Rinse with liquid NP medium + Selection of transformants

Antibiotic
Addition of Fruit extract and coconut water into basic medium,
accelerates the growth rate of early developmental stages of
Phalaenopsis amabilis orchid embryo

Fruit extract Coconut water

Addition of Fruit extract (FE) and Coconut Water
(CW) in pre-culture medium improved the efficiency
of Agrobacterium-mediated transformation in
P. amabilis orchid 10-fold
Number of
Number of
protocorms
protocorms
producing shoots
examined
(% of total)
Tomato
Plasmid Coconut water extractFruit
None ＋ ＋ 1557 0 (0%)
pBI121 (vector) ＋ − 1200 14 (1.2%)
pBI121 (vector) − ＋ 1200 159 (13.2%)
pBI121 (vector) ＋ ＋ 1557 260 (16.6%)
p35S::GFP ＋ ＋ 1557 210 (13.5%)

Patent Registration: by UGM to Patent Office

Registration No: P00201000142
22 February 2010.
Orchids
Black Orchid’s rapid
grow
Semiarti et al 2010

Fakultas Biologi UGM

KNAT1

High bud
rapid grow
multiplication
4. Frequency of transformation by pGreen and pKNAT1 on regeneration
medium, two-months after selection on kanamicyn containing medium

Percentage of Kan
Kan300 mg/l No of proto-
No. resistance plants
(+/-) corms
Survive Death
Non Transformant 98 % 2%
1. - 100
(NT) 98/100 2/100

37.6 % 62.4%
2. NT + 237
89/237 148/237

66.0 % 34.0%
3 pGreen + 707
467/707 240/707

p35S: 61.6 % 38.4%

4 + 701
:KNAT1 475/701 296/701
Table 3. Shoot Multipication of transformant on (1/2NP
+NAA 0.15 µM) medium

Average No of
No of transf
. Medium shoot emerged
observed
from explant

1 Wild type (NT) ½ NP 8 1

2 NT ½ NP SIM Kan 8 0

3 p35S::KNAT1 ½NP SIM Kan 8 7

The expression of KNAT1 gene in the
black orchid
transformantsmultishoots

Bar: 1 cm
Black orchid transformants. A. Two-months old candidate of transformants grow on
kanamycin antibiotic containing medium in selection plate; B. A shoot grows from a
non-transformant protocorm; C. Multishoots grow from one 35S::KNAT1 transformant
protocorm;
On going  DGHE Research
Grant STRANAS 2012 & 2013
Early Flowering Start 2011….
OOrchid

E. Semiarti F. Biologi UGM 2012

 DGHE Research Grant STRANAS 2012 & 2013

Generative
(Flower)

0 24 month
Vegetative 2 year

Biotechnology

Insertion
BA treatment accelerate
flowering gene
(PaFT)

Generative
Vegetative (Flower)

1 year
Conclusion

1. Some Natural Orchids in Indonesia are threatened to

be extinct because of habitat loss, overhunting and
excessive exploitation, pollution, climate change, and
natural disasters.

2. Orchid Plant breeding can be carried out through

biotechnology in three ways: Conventional Breeding,
Clonal propagation, and Genetic engineering.

3. Agrobacterium-mediated transformation in orchid using

KNAT1 gene of Arabidopsis increased the number of
shoots from embryo, that 1 embryo produced 50-91
shoots/multishoots give high benefit for conservation of
natural orchids.

4. Insertion of flowering time gene into orchid genome will

shorten vegetative stage and induce fowering.
Arigato
Gozaimasu

Thank You Very much

Collaboration:

UGM_Indonesia:
Dr. Endang Semiarti
Dr. rer.nat Ari Indrianto
Dr. Ir. Azis Purwantoro
Rindang Dwiyani, M.Agr.Sc.
Ixora S. Mercuriani, M.P
Rizqie L Nurwulan, S.Si.

Nagoya University-Japan:
Prof. Yasunori Machida, Ph.D

Chubu University:
Prof. Chiyoko Machida, Ph.D
Dr. Shoko Kojima