4. Orchid breeding_Recent advances in biotechnology and

Transcription

3 Feb. 2015
Orchid breeding: Recent advances in
biotechnology and considerations
So-Young Park, Ph.D
Department of Horticultural Science, Chungbuk National University
Republic of Korea
1
Contents
• Introduction
• Micropropagation
: Regeneration and Development
• Breeding by in vitro biotechnology
• Consideration
: Somaclonal variation
• Conclusions
2
3
INTRODUCTION
Orchid Industry in Korea
 Total area utilized for floriculture was around 6,640 ha and the
economic value reached US$ 720 million. Among them 29% of the
total pot flower cultivation area is allocated for orchid production.
 The value of orchid production reached up 36.8% of the total pot flower
production value in Korea.
 The number of orchid growers is around 804 (7.5%) out of 10,690
flower growers.
 Orchid production is one of the most representative bio-agricultural
industries in Korea.
4
INTRODUCTION
(1,000$)
Import
Export
(1,000$)
5
INTRODUCTION
Orchid production
(RDA, 2009)
6
INTRODUCTION
Orchid Cultivation in Korea
Kyeongki
Gangwon-do
38.8 % (97.9 ha)
Incheon
Chungcheongbuk-do
3.6%(9.0ha)
Gyeongsangbuk-do
Daejeon
Chungnam
20.3 % (51.4 ha)
Jeollabuk-do
Daegu
Ulsan
Kyeongnam
8.6 % (21.6 ha)
Gwangju
Jeonnam
Jeonnam
6.7 % (16.8 ha)
Jeju-do
Jeju
11.9 % (30.1 ha)
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Micropropagation
- Regeneration and Development -
8
Conventional propagation
by cutting and division
Mother plants for
flowers and pseudo
bulbs for propagation
Dendrobium-Phalaenopsis
propagation (TOC group in
Thailand)
9
Dendrobium-Phalaenopsis propagation (TOC group in Thailand)
Conventional propagation
by cutting and division
New shoot production from old pseudobulb
10
11
12
13
Micropropagation
by seed germination
Orchid capsule and seeds
Protocorm
In vitro seed germination
14
15
Micropropagation
by clonal propagation
* Protocormlike body
PLB propagation
(Paek et al. 2011, Plant Embryo Culture)
Fig. Vegetative propagation of elite orchid via PLB multiplication.
16
Concept of mass production system via micropropagation
Breeding by in vitro biotechnology
1.
2.
3.
4.
Micropropagation
Polyploidy breeding
Mutation breeding
Genetic transformation
18
Micropropagation
• Mass propagation of elite genotype by
selection from seedling populations
- Flower color, disease resistance, flowering time..
• Regeneration ability of plant
• Essential of plant cell and tissue culture technique
Rapid establishment of new variety
19
Orchid MICROPROPAGATION
(Phalaenopsis)
Proliferation via PLB/multiple-shoots
PPF (µmol·m-2·s-1)
50
100
150
200
300
M
M
1H 1M
3H 3M 5H 5M 7H
C1
1H 1M 3H 3M 5H
C4
Acclimatization and
greenhouse cultivation
7M
5M 7H 7M
1H 1M 3H 3M 5H
C2
1H 1M 3H 3M 5H
5M 7H 7M
5M 7H
7M
C3
Variant detection
20
Mass production via solid/liquid culture
Ploidy breeding
• Polyploidy is plants or cells containing more than two paired sets
of chromosomes.
• Polyploidy is responsible for increases in cell size, a characteristic
that leads to larger vegetative and reproductive organs (Kim et al,
2004).
2n
4n
Phalaenopsis equestris var cyanochilus
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Solanum commersonii (Riccardo et al, 2012)
Characteristics of ploidy orchid
Polyploidy orchid
Chemicals for chromosome doubling
1. Colchicine(C22H25NO6)
• Narcotic alkaloid extraction from Colchicum autumnale
• Inhibition of microtubules during mitosis of plant cell
2. Oryzalin(C12H18N4O6S)
• A kind of herbiside
• The disruption of microtubules during mitosis
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Cymbidium Show Girl ‘Silky’
Breeding in National Institute of Horticultural & Herbal
Science

Species
: Cymbidium Elma ‘Orient Toyo’, C. Golden Elf ‘Sundust’, C. Show girl ‘silky’

Diploid and tetraploid plants by treatments of Colchicine 100-500 mg/L for
1-2 week
Cymbidium Elma ‘Orient Toyo’
Cymbidium Golden Elf
‘Sundust’
23
Analysis of DNA content for ploidy determination
Treatment of colchicine and oryzalin
Flow cytometry
Extraction
Chopping and extraction of nuclei
Filtering
Nuclei staining
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Cymbidium Elma ‘Orient Toyo’
Number of nuclei
80
2n
Diploid
Tetraploid
60
4n
40
4n
20
8n
50
100
150
200
50
100
150
200
Relative DNA content
Fig. Flow cytometric DNA histograms of diploid and tetraploid of Cymbidium
Elma ‘Orient Toyo’.
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Cymbidium Golden Elf ‘ Sundust’
Number of nuclei
80
Diploid
2n
Tetraploid
60
4n
40
4n
20
8n
50
100
150
200
50
100
150
200
Relative DNA content
Fig. Flow cytometric DNA histograms of diploid and tetraploid of Cymbidium
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Golden Elf ‘Sundust’.
A
B
C
Fig. Control plants (A), chromosome doubled plants (B), ploidy chimera
plants (C) of Cymbidium Show Girl ‘Silky’.
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Cymbidium Show Girl ‘Silky’
A(3x)
B(6x)
Fig. Guard cells of control and chromosome doubled plant of
Cymbidium Show Girl ‘Silky’. 400× magnification. Bars = 40㎛.
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Flower morphology by ploidy level
A
Diploid
B
Tetraploid
Fig. Diploid C and tetraploid D flowers of Cymbidium Elma ‘Orient Toyo’. (Hwang et
al. unpublished data)
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Lateral sepal (upper)
Petal
Stigma
Lip
Lateral sepal (under)
Fig. Diploid (Left) and tetraploid (Right) floral parts of Cymbidium Elma ‘Orient Toyo’.
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(Hwang et al. unpublished data)
Diploid
Tetraploid
Fig. Diploid A and tetraploid B flowers Cymbidium Golden Elf ‘Sundust’. (Hwang et al.
unpublished data)
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Lateral sepal (upper)
Petal
Stigma
Lip
Lateral sepal (under)
Fig. Diploid (Left) and tetraploid (Right) floral parts of Cymbidium Golden Elf
‘Sundust’. (Hwang et al. unpublished data)
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Fragrance pattern in ploidy plant
• Analysis of fragrance pattern using electronic nose
- MOS : High similarity with human olfactory sense
- Use : Fox2000 with Sensor Array System (Alpha M.O.S)
• Materials : Cymbidium Golden Elf ‘Sundust’ di-, tetraploid
• Alpha soft model : PCA (Principle component analysis),
DFA (Discriminant factor analysis)
33
Scent pattern of di- and tetraploid flower (PCA)
■ Control (air)
▲ Sundust 2n=2x
● Sundust 2n=4x
Fig. Principle component analysis (PCA) plot of the scent from the 2n and 4n flowers
in C. Golden Elf ‘Sundust’ using the electronic nose. (Hwang et al. unpublished
data)
34
Scent pattern of di- and tetraploid flower (DFA)
■ Control (air)
▲ Sundust 2n=2x
● Sundust 2n=4x
Fig. Discriminant factor analysis (DFA) plot of the scent from the 2n and 4n flowers in
C. Golden Elf ‘Sundust’ using the electronic nose. (Hwang et al. unpublished data)
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Mutation breeding
• X-ray, gamma ray irradiation, and chemical treatments
• Powerful tools for plant breeding as well as for physiological and
molecular studies
• Gamma (y) radiation
- a type of ionizing radiation
- produce free radicals in cells which damage or modify
DNA of plant cells and affect differently the morphology
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Mutation breeding
50 Gy
WT
Gamma ray effect on damaging DNA
(Wi et al., 2007)
37
Mother plants
Mutants
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Mutation breeding
(Taheri et al. 2014)
Curcuma alismatifolia varieties by gamma ray
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Mutation breeding of orchid
Genetic mutation
Some genetic variants
are popular and useful
Varients of Aerides
japonicum obtained from
asymbiotic seed cultures
Very popular in special
grower with high price
40
41
Genetic transformation of orchid
Delivering the gene to the plant
• Transformation cassettes are developed in the lab
• They are then introduced into a plant
• Two major delivery methods
- Agrobacterium
- Gene Gun (Particle bombardment)
Agrobacterium
Gene gun
42
by Agrobacterium
43
World Orchid Exhibition
2012. Okinawa in Japan
44
World Orchid Exhibition
2012. Okinawa in Japan
45
46
Control of flowering time by
transformation
FT2 Shortens the Juvenile Phase and Promotes Seasonal Flowering
HindIII
HindIII
XbaI
Sac I
RB
Pnos
NPT II
Tnos
(Hsu et al., 2008,
Plant Cell)
CaMV 35S
PagFT2
EcoRI
LB
Tnos
(Igasaki et al., 2008, Plant Cell Physiol)
47
Control of flowering time by
transformation
FT2 Shortens the Juvenile Phase and Promotes Seasonal Flowering
HindIII
HindIII
XbaI
Pnos
NPT II
Tnos
CaMV 35S
WT
- Phalaenopsis cv ‘Spring dancer’
- Destination vector pBI121
- 35S::FT2
EcoRI
Sac I
RB
PagFT2
LB
Tnos
Putative TP
Selection of transgenics on 50 μM
Kanamycin
(Park et al, unpublished data)
48
Somaclonal Variation
- Things to be consider -
49
SOMACLONAL VARIATION
50
Somaclonal variation of orchid
Somaclonal variation
 What’s somaclonal variation?


The variation seen in plants that have been
produced by long-term micropropagation
Somaclonal variation
- Genetic variation : caused by DNA changes
- Epigenetic variation : caused by temporary
phenotypic changes
51
Problems of somaclonal variation
 Morphological changes during floral development
in in vitro clonally propagated Orchid
 Decrease of plant quality in uniformity
Normal plant
Variations in flower morphology
52
What cause somaclonal
variation?
Micropropagation
Somaclonal
variants
- Changes in biochemical and molecular events
- Changes in DNA methylation pattern
- Activation of transposable elements or retroelements
Mother plant
- Chromosome remodeling
Clonally propagated
plant
53
Somaclonal variation – polyploid
Number of nuclei
Number of nuclei
2C
Diploid
4C
8C
A
Tetraploid
4C
8C
16C
B
DNA relative fluorescence intensity
Easily detected during
in vitro culture process
54
55
Various mutants in flowers (Phalaenopsis)
P. Baldans Kalaidescop
56
Detection of epigenetic variation
in tissue-culture-derived plants of Doritaenopsis
RAPD result
Methylation pattern analysis
(Park et al. 2009)
57
Morphological changes of flowers in P. Wedding Promenade
WP-N
WP-V1
WP-V2
WP-V3
58
Morphological changes of flowers in P. Spring Dancer
SD-N
SD-V1
SD-V2
59
Morphological characteristics of leaf and flower stalk
B
A
D
C
SD-N
SD-N
SD-V1
SD-V2
SD-V1
SD-V2
Fig. Leaves and flower stalks of P. Spring Dancer
(SD) : (A) Leaves, (B) Surface of leaves, (C)
Flower stalks, (D) Thickness of flower stalks. (Bar
of A,B,D = 1cm, Bar of C = 5cm)
60
Morphological characteristics of flowers
A
B
3.0
SD-N
SD-V1
SD-V2
Parameter (cm)
2.5
2.0
a
ab
a
a
SD-N
a
b
1.5
1.0
SD-V1
0.5
0.0
Width of petals
Length of petals
SD-V2
Fig. Width and length of petals(A) and flowers (B) in normal and variants in P. Spring
Dancer (SD). (Unpublished data)
61
Morphological characteristics of leaves
A
C
Parameter (cm)
18
15
SD-N
SD-V1
a
12
b
9
6
a
ab
3
a
b
0
Width of leaves Length of leaves
50
Leaf area (cm2)
B
40
a
a
b
30
SD-N
20
10
0
SD-N
SD-V1
SD-V2
SD-V1
SD-V2
Fig. Width and length of leaves (A) and
leaf area(B) and leaves (C) in normal and
variants in P. Spring Dancer (SD).
(Unpublished data)
62
Morphological characteristics of flowers and
leaves
A
Petal
12
SD-N
SD-V1
SD-V2
Thickness (mm)
10
a
8
Leaf
6
b
4
c
a
b
2
c
b
b
a
0
Petals
Flower stalks
Flower
stalk
Leaves
SD-N
SD-V1
SD-V2
Fig. Thickness of petal, flower stalk and leaf in P. Spring Dancer (SD). (Bar of petal and leaf
= 0.5 mm, bar of flower stalk = 1 mm). (Unpublished data)
63
Ploidy and endoreduplication analysis
A
N
V1
4C
8C
2C
16
C
4C
V2
8C
8C
4C
16
C
B
N
2C
V1
2C
4C
4C
V2
2C
8C
4C
Fig. The nuclear DNA content and distribution of endopolyploid nuclei in petals (A) and
leaves (B) of P. Spring Dancer (SD). (Unpublished data)
64
What determine floral structure?
 Genetic
mechanisms
that
establish floral organ identity
 MADS-box genes involved
most central genes in floral
organ identity
65
PMADS4 expression in flowers and leaves
Normalized expression
6
5
Leaf
Flower
4
Actin
3
PMADS4
2
1
0
N
V1
V2
N
V1
V2
Fig. Real-Time PCR analysis for PMADS4 expression level of normal and variant’s
flowers and leaves in P. Spring Dancer (SD). (Unpublished data)
66
PMADS4 expression in flowers
Normalized expression
16
14
12
10
8
6
4
2
0
P
S
S
P
S
L
Actin
Sepal
Petal
N
V1
Sepal
V2
N
V1
Petal
V2
N
V1
V2
Lip
Lip
Fig. Real-Time PCR and electrophoresis analysis for PMADS4 expression level of normal
and variant’s flower organs in P. Spring Dancer (SD). (Unpublished data)
67
Conclusions
Things to be improved in orchid breeding &
production
 Stable production of high-quality clones by micromropagation
 Development of new varieties for shorten juvenile phase
 Year-round culture system by applying hydroponic cultures
 Breeding new varieties by application of biotechnology
 Strict control of disease and insects
68
International Orchid Conference
World Orchid Conference (WOC)
2011 20th WOC in Singapore
69
Asia Pacific Orchid Conference (APOC)
2016 APOC in Bangkok, Thailand
70
2012 APOC in Okinawa, Japan
International Orchid Symposium (IOS)
2018 3rd IOS in Korea
2014 2nd IOS in Bangkok, Thailand
71
Thank you!
72

4. Orchid breeding_Recent advances in biotechnology and

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