2015
Contents
Introduction
Micropropagation
: Regeneration and Development
Conclusions
2
INTRODUCTION
The value of orchid production reached up 36.8% of the total pot flower
production value in Korea.
INTRODUCTION
Import
Export
(1,000$)
INTRODUCTION
Orchid production
(RDA, 2009)
INTRODUCTION
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)
7
Micropropagation
- Regeneration and Development -
Conventional propagation
by cutting and division
Dendrobium-Phalaenopsis
propagation (TOC group in
Thailand)
Conventional propagation
by cutting and division
Dendrobium-Phalaenopsis propagation (TOC group in Thailand)
10
11
12
13
Micropropagation
by seed germination
Protocorm
In vitro seed germination
14
15
Micropropagation
by clonal propagation
* Protocormlike body
PLB propagation
16
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
19
Orchid MICROPROPAGATION
(Phalaenopsis)
PPF (molm-2s-1)
50
100
150
200
300
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.
2n
4n
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
22
Cymbidium Show Girl Silky
Species
: Cymbidium Elma Orient Toyo, C. Golden Elf Sundust, C. Show girl silky
Flow cytometry
Extraction
Filtering
Nuclei staining
24
Number of nuclei
80
2n
Diploid
Tetraploid
60
4n
40
4n
20
8n
50
100
150
200
50
100
150
200
Number of nuclei
80
Diploid
2n
Tetraploid
60
4n
40
4n
20
8n
50
100
150
200
50
100
150
200
Fig. Control plants (A), chromosome doubled plants (B), ploidy chimera
plants (C) of Cymbidium Show Girl Silky.
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A(3x)
B(6x)
Diploid
Tetraploid
Fig. Diploid C and tetraploid D flowers of Cymbidium Elma Orient Toyo. (Hwang et
al. unpublished data)
29
Petal
Stigma
Lip
Fig. Diploid (Left) and tetraploid (Right) floral parts of Cymbidium Elma Orient Toyo.
30
(Hwang et al. unpublished data)
Diploid
Tetraploid
Fig. Diploid A and tetraploid B flowers Cymbidium Golden Elf Sundust. (Hwang et al.
unpublished data)
31
Petal
Stigma
Lip
Lateral sepal (under)
Fig. Diploid (Left) and tetraploid (Right) floral parts of Cymbidium Golden Elf
Sundust. (Hwang et al. unpublished data)
32
33
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
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)
35
Mutation breeding
X-ray, gamma ray irradiation, and chemical treatments
Powerful tools for plant breeding as well as for physiological and
molecular studies
36
Mutation breeding
50 Gy
WT
Mother plants
Mutants
38
Mutation breeding
39
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
Genetic transformation
41
Genetic transformation
Delivering the gene to the plant
Transformation cassettes are developed in the lab
They are then introduced into a plant
Agrobacterium
Gene gun
42
Genetic transformation
by Agrobacterium
43
44
45
46
HindIII
XbaI
Sac I
RB
Pnos
NPT II
Tnos
CaMV 35S
PagFT2
EcoRI
LB
Tnos
47
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 M
Kanamycin
Somaclonal Variation
- Things to be consider -
49
SOMACLONAL VARIATION
50
Somaclonal variation
Whats somaclonal variation?
51
Normal plant
52
Somaclonal
variants
Mother plant
- Chromosome remodeling
Clonally propagated
plant
53
Number of nuclei
Number of nuclei
2C
Diploid
4C
8C
Tetraploid
4C
8C
16C
B
DNA relative fluorescence intensity
55
P. Baldans Kalaidescop
56
WP-N
WP-V1
WP-V2
WP-V3
58
SD-N
SD-V1
SD-V2
59
D
C
SD-N
SD-N
SD-V1
SD-V2
SD-V1
SD-V2
3.0
SD-N
SD-V1
SD-V2
Parameter (cm)
2.5
2.0
ab
SD-N
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
Parameter (cm)
18
15
SD-N
SD-V1
a
12
9
6
ab
0
Width of leaves Length of leaves
50
Leaf area (cm2)
40
a
b
30
SD-N
20
10
0
SD-N
SD-V1
SD-V2
SD-V1
SD-V2
62
Petal
12
SD-N
SD-V1
SD-V2
Thickness (mm)
10
Leaf
a
b
2
c
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
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
65
Normalized expression
6
5
Leaf
Flower
Actin
PMADS4
2
1
0
V1
V2
V1
V2
Fig. Real-Time PCR analysis for PMADS4 expression level of normal and variants
flowers and leaves in P. Spring Dancer (SD). (Unpublished data)
66
Normalized expression
16
14
12
10
8
6
4
2
0
P
S
L
Actin
Sepal
Petal
V1
Sepal
V2
V1
Petal
V2
V1
V2
Lip
Lip
Fig. Real-Time PCR and electrophoresis analysis for PMADS4 expression level of normal
and variants flower organs in P. Spring Dancer (SD). (Unpublished data)
67
Conclusions
Things to be improved in orchid breeding &
production
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70
Thank you!
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