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

Contents
Introduction
Micropropagation
: Regeneration and Development

Breeding by in vitro biotechnology

Consideration
: Somaclonal variation

Conclusions
2

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.

INTRODUCTION

Orchid Industry in Korea

(1,000$)

Import

Export

(1,000$)

INTRODUCTION

Orchid Industry in Korea

Orchid production

(RDA, 2009)

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)
7

Micropropagation
- Regeneration and Development -

Conventional propagation
by cutting and division

Mother plants for

flowers and pseudo
bulbs for propagation

Dendrobium-Phalaenopsis
propagation (TOC group in
Thailand)

Dendrobium-Phalaenopsis propagation (TOC group in Thailand)

Conventional propagation
by cutting and division
Dendrobium-Phalaenopsis propagation (TOC group in Thailand)

New shoot production from old pseudobulb

10

Dendrobium-Phalaenopsis propagation (TOC group in Thailand)

11

Dendrobium-Phalaenopsis propagation (TOC group in Thailand)

12

Dendrobium-Phalaenopsis propagation (TOC group in Thailand)

13

Micropropagation
by seed germination

Orchid capsule and seeds

Protocorm
In vitro seed germination
14

Dendrobium-Phalaenopsis propagation (TOC group in Thailand)

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 (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.

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

21
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

22
Cymbidium Show Girl Silky

Characteristics of ploidy orchid

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

Characteristics of ploidy orchid

Analysis of DNA content for ploidy determination

Treatment of colchicine and oryzalin

Flow cytometry

Extraction

Chopping and extraction of nuclei

Filtering

Nuclei staining
24

Characteristics of ploidy orchid

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.
25

Characteristics of ploidy orchid

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
26
Golden Elf Sundust.

Characteristics of ploidy orchid

Fig. Control plants (A), chromosome doubled plants (B), ploidy chimera
plants (C) of Cymbidium Show Girl Silky.
27

Characteristics of ploidy orchid

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.
28

Characteristics of ploidy orchid

Flower morphology by ploidy level

Diploid

Tetraploid

Fig. Diploid C and tetraploid D flowers of Cymbidium Elma Orient Toyo. (Hwang et
al. unpublished data)
29

Characteristics of ploidy orchid

Flower morphology by ploidy level

Lateral sepal (upper)

Petal

Stigma

Lip

Lateral sepal (under)

Fig. Diploid (Left) and tetraploid (Right) floral parts of Cymbidium Elma Orient Toyo.
30
(Hwang et al. unpublished data)

Characteristics of ploidy orchid

Flower morphology by ploidy level

Diploid

Tetraploid

Fig. Diploid A and tetraploid B flowers Cymbidium Golden Elf Sundust. (Hwang et al.
unpublished data)
31

Characteristics of ploidy orchid

Flower morphology by ploidy level

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)

32

Characteristics of ploidy orchid

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

Characteristics of ploidy orchid

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

Characteristics of ploidy orchid

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)
35

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

36

Mutation breeding
50 Gy

WT

Gamma ray effect on damaging DNA

(Wi et al., 2007)

37

Mother plants
Mutants

38

Mutation breeding

(Taheri et al. 2014)

Curcuma alismatifolia varieties by gamma ray

39

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

Genetic transformation

41

Genetic transformation of orchid

Genetic transformation
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

Genetic transformation of orchid

Genetic transformation
by Agrobacterium

43

World Orchid Exhibition

2012. Okinawa in Japan

Genetic transformation of orchid

44

World Orchid Exhibition

2012. Okinawa in Japan

Genetic transformation of orchid

45

Genetic transformation of orchid

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 M
Kanamycin

(Park et al, unpublished data)

48

Somaclonal Variation
- Things to be consider -

49

SOMACLONAL VARIATION

50

Somaclonal variation of orchid

Somaclonal variation
Whats 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

Somaclonal variation of orchid

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

Somaclonal variation of orchid

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 of orchid

Somaclonal variation polyploid

Number of nuclei

Number of nuclei

2C

Diploid
4C

8C

Tetraploid
4C
8C
16C
B
DNA relative fluorescence intensity

Easily detected during

in vitro culture process
54

Somaclonal variation of orchid

55

Somaclonal variation of orchid

Various mutants in flowers (Phalaenopsis)

P. Baldans Kalaidescop

56

Somaclonal variation of orchid

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

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

Somaclonal variation of orchid

Morphological characteristics of flowers

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

Somaclonal variation of orchid

Morphological characteristics of leaves

A

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

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

Somaclonal variation of orchid

Morphological characteristics of flowers and

leaves
A

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

Somaclonal variation of orchid

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

Somaclonal variation of orchid

What determine floral structure?

Genetic
mechanisms
that
establish floral organ identity
MADS-box genes involved
most central genes in floral
organ identity

65

Somaclonal variation of orchid

PMADS4 expression in flowers and leaves

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

Somaclonal variation of orchid

PMADS4 expression in flowers

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

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

International Orchid Conference

Asia Pacific Orchid Conference (APOC)

2016 APOC in Bangkok, Thailand

70

2012 APOC in Okinawa, Japan

International Orchid Conference

International Orchid Symposium (IOS)

2018 3rd IOS in Korea

2014 2nd IOS in Bangkok, Thailand

71

Thank you!

72