3. Thailand

3. Thailand
Chitrapan Piluek1 and Arunee Wongpiyasatid2
1

Assistant Professor, Department of Horticulture, Faculty of Agriculture,
Kasetsart University

2

Professor, Department of Applied Radiation and Isotopes, Faculty of Science,
Kasetsart University

Introduction
Dendrobium hybrids are the major commercial orchid plants grown for cut flower and potted
plant in Thailand. The Dendrobium Sonia 'BOM' is a popular hybrid, fast growing, floriferous,
bright color and has long vase life. The original clone of Dendrobium Sonia 'BOM’ has
red-purple with white color at the central. After successive propagation by tissue culture, it
produced many mutants. The selected mutants for cutflower had developed clones such as
Dendrobium Sonia 'BOM 17K', Dendrobium Sonia 'BOM 17 Red', Dendrobium Sonia 'BOM
28' (large size flower), Dendrobium Sonia 'Kalya', Dendrobium Sonia 'Miss world'. And thesecond cross of the same parent produced the Dendrobium Sonia 'BOM Jo' which similar to
Dendrobium Sonia 'BOM'. and also produced mutant clones after successive propagation :Dendrobium Sonia 'BOM Jo Red', and Dendrobium Sonia 'Earsakul' which having superiordark color and obtained higher price than the original clone.
Thailand exported quantity of orchid flowers in 2003 was 439.86 million inflorescences or
17,411 metric tons, and of orchid plants was 27.12 million plants. The flowers were exported
26.59, 17.04, 16.53, 13.56, 6.57, 6.06 % to Japan, America, Italy, Hong Kong, China andTaiwan, respectively and only 10.29 % were shipped to other countries.

ThripsThrips are the important insect pest to the orchid
industry especially Dendrobium orchids. Thrips feed
inside newly expanding leaves and the developing
young inflorescence. Their feeding damage is notseen until leaves expanded and deformed flowers,
leaving plant unmarketable. Thrips have a wide hostrange and active all year in heat greenhouse.
- 33 -

Since the habitat of thrips is a flower petal, the plant quarantine agencies of many countries
will not allow to entry until all of the thrips are completely killed. The fumigation costs inimported countries are very expensive, time consuming and reduce the flower quality. It isalmost impossible to completely eradicate them in the growing area. To create resistanceclone will be the most valuable in the orchid industry.

Thrips are classified in Order Thysanoptera, Family Thripideae. Thrips being found on orchid
plants in Thailand were recorded as Dichromothrips corbetti (Priesner) and Thrips palmi
Karny by Kamjaipai (1984). Kajita et al (1992) recorded of Thrips sumatrensis Priesner,
Tusothrips teinostomus Okajima, Franklinella schultzei (Trybon) and Microcephalothrips
abdominalis (Crawford). Beside these species, Thrips hawaiiensis (Morgan), T. tabaci
Lindeman, Taeniothrips eucharii (Whetzel), Frankliniella intonsa (Trybom), and Selenothrips
dorsalis Hood were found by the Japanese plant quarantine from orchid imported fromThailand at the Japanese ports (Hayase, 1991; Itoh, 1990). Yano and Napompeth (1995)
reported that they collected and identified only 2 species of thrips; 1) Dichromothrips corbetti(Priesner) on genus Dendrobium, Mokara, Vanda and Oncidium and 2) Taeniothrips eucharii(Whetzel) on Dendrobium orchid. In 2001, Kienmesuk et al. reported that only Thrips palmi
Karny is the major insect pest in Thailand.

Normal flower

Infested flower by thrips

Characteristics of thrips
Thrip is a tiny yellow (young) or black (adult) insect with 0.8-1.0 mm in length. The adult
moves fast by flying while young thrips with short wing-pads can walks very quickly. They
hide from their enemies or sunlight in corners within the flowers. Thrips can be seen walking
on petals when they are over crowned. When they suck sap from the flower, the wounds will
- 34 -

be dry strips which make the flower look burnt. Thrips can spread very fast due to the highfecundity of female and short life cycle 2 - 3 weeks in 25 - 30 °C. The survival rate from egg
to adult is about 45 - 50 %. Thrips palmi Karny can not survive under 10 °C condition. Thrips
become active and reproduce in dry weather, in the hot season and in the rainy season. Whenit does not rain, they reproduce very quickly. High population of thrips was found during dry
season in January to April and in October to December. It is almost impossible to completely
eradicate them (Kienmesuk et al., 2001). Yano (1995) estimated that thrips probably occur at
almost all nurseries. The percentage of Dendrobium flowers attacked by thrips was 74 % (84from 113 flowers surveyed) in the nursery where no insecticide was applied. But most of theorchid nurseries were under heavy and regular application of insecticide resulting few insect
collection.

Damaged leaves of Dendrobium infested by thrips

Cost for control thrips
Approximately 2,240 hectares of growing area, the estimation of insecticide cost is about
1,500 US$/hectare/year. The most important insect pest is thrips.

The farmers control thrips by applying insecticide at 7 days interval. The recommended 5groups of insecticides for eliminate thrips are1) Imidacloprid (Confidore 100 SL 10% 20 ml/l, 1250 l/hectare)2) Acetamiprid (Molan 20% SP 5 gm/20 l, 1250 l/hectare)3) Abamectin (Jacket, Vertimec 1.8 % EC, 20 ml/20 l)4) Fipronil (Ascent 5 % SC 20 ml/20 l, 1250 l/hectare)5) Cypermethrin/phosalone (Parzon 28.75 % EC 40 ml/20 l, 1250 l/hectare)- 35 -

Problems of thrips on orchid export
Reports from the Plant Quarantine Section in Bangkok showed number of shipments thatfound thrips at the imported countries (Komson, 2003). The shipment that found thrips will
manage for fumigation or fire burn. The fumigated flower attains reduced vase life.

No. of shipments with thrips

No. of exported

No. of flower spikes

shipments(millions)

number

%1997

30,776

239.4

107

0.36

1998

35,708

302.9

90

0.25

1999

34,441

317.7

61

0.18

2000

38,573

283.1

70

0.18

2001

38,759

386.0

32

0.08

2002

39,907

421.6

26

0.06

Year

Thrips control and eradication
The Department of Agriculture, Ministry of Agriculture has many projects to eradicate thrips:
1) Research on irradicated thrips in the farms by insecticides.
2) Research on physical control using sticker pad. The results stated that the white or blue
sticker pad could trap more number of thrips than other colors. The suitable level for
hanging was 40-60 cm above ground and 4 m apart.
3) Research on integrated thrips control by insecticide, sticker pad and counting number ofthrips on flowers. It needs not to apply insecticide if the number of thrips on random
sampling flower is lower than 10/40 flower spike/1600 m2. This method can reduce halfcost of insecticide (Kienmesuk et al., 2001).
4) Produce booklet of GAP (Good Agricultural Practice) for recommending the farmer toirradicate thrips in the farm.
5) Postharvest research on fumigation of orchid cut-flowers with methyl bromide. All thrips
die when fumigate with methyl bromide at 20-22 g/m3 for 90 min.
The random sampling number of thrips by counting from 40 flower spikes/rai. If they
have more than 10 thrips/40 flower spikes, it needs to spray insecticide for irradicated(Kienmesuk et al., 2001).

- 36 -

Study on irradiation by gamma rays of orchids
Gamma irradiation was carried out to obtain the optimum doses on inducing mutation of
Dendrobium PLBs (Vajrabhaya, 1977; Angamnuasiiri, 2001), Brassolaeliocattleya PLBs
(Thammasiri, 1996); protocorm derived from seed of many orchids species (Piluek, 2002).
The results showed that most of the orchid protocorms or PLBs were tolerant to chronic
irradiation, they could survive and develop into normal plantlets.

The aims of this research work are:
1. To get the optimum doses of gamma rays for mutation induction of different growthstages of Dendrobium
2. To develop Dendrobium lines/varieties resistant to thrips using gamma radiation as amutagenMaterials and Methods
Eight experiments were carried out in this project.

Experiment 1

Evaluation and identification of breeding materials

Four clones of commercial cut flower orchids: Dendrobium Sonia ‘BOM 17 red‘,Dendrobium Sonia ‘Earsakul’, Dendrobium Pinky Sem ‘Rinnapa’ and Dendrobium hybrid‘White Sanan’ were evaluated.

Den. Sonia ’BOM 17 Red’

Den. Pinky Sem ‘Rinnapa’

Den. ’White Sanan’

Den. Sonia ’Earsakul’- 37 -

Experiment 2

Initiation of tissue culture of clones

Young shoots of Dendrobium Sonia ‘Earsakul’, Dendrobium Sonia ‘BOM 17 Red’,
Dendrobium Pinky Sem ‘Rinnapa’ and Dendrobium hybrid ‘White Sanan’ were collectedfrom commercial orchid nursery for tissue culturing. The lateral buds and terminal buds
excised from sterilized shoots were cultured in liquid Vacin-Went medium.

2 months
Excised lateraland terminal bud

Experiment 3

Solid Vacin-Went medium
for PLBs prolification

Exchange of material clones

The material clones are exchanged among Thailand, Malaysia and Indonesia.

Experiment 4
4.1

Determination of radiation sensitivity and optimum dose

Radiation effects on protocorm developed from seed of Dendrobium hybrid

Subculture
Protocorms

Seedling formation

To nursery

4.2

Radiation effects on PLBs of Dendrobium hybrids- 38 -

Experiment 5
5.1

Irradiation of PLBs for insect resistant clones

Effects of irradiation on plantlet growth of Dendrobium Sonia ‘ BOM 17 red’Protocorms like bodies (PLBs) of Dendrobium Sonia ‘BOM17 Red’ were irradiated withacute gamma rays at 0, 60, 70, 80, 90 and 100 Gy and subcultured. Large size seedlings weretaken out from aseptic culture and were evaluated for seedling weight and size before growing
in the nursery.

5.2

Effects of irradiation on plantlet growth of Dendrobium Sonia ‘Earsakul’

Protocorms like bodies (PLBs) of Dendrobium Sonia ‘Earsakul’ were irradiated with acuteand chronic gamma rays and then subcultured.
1.

High dose acute gamma irradiation

0, 60, 70, 80, 90 and 100 Gy

2.

Low dose acute gamma irradiation

0, 2, 4, 6, 8, 10 Gy

3.

Split dose acute gamma irradiation

0, 20, 20+20, 40 Gy

4.

Chronic gamma irradiation

0, 400, 800 Gy

Large size plantlets taken out from aseptic culture and were evaluated for seedling weight andsize before growing in the nursery.

Experiment 6. Study on the effects of single and split doses of acute and chronic
irradiation on in vitro plantlets and PLBs of Dendrobium Sonia ‘Earsakul’ andDendrobium Sonia ‘BOM 17 Red’

6.1

The in vitro plantlets- 39 -

A. Dendrobium Sonia ‘Earsakul’
8 treatments ofSingle doses (Gy)

0
50
100
200

Split doses(Gy)50+50
100+50100+100
200+100

B. Dendrobium Sonia ‘BOM 17 Red’6 treatments of chronic gamma irradiation of 0, 50, 50+50, 100+50, 100+100 and 200+100
Gy

6.2 The PLBs of Dendrobium Sonia ‘Earsakul’ and Dendrobium Sonia ‘BOM 17 Red’5 treatments of 0 Gy
acute 0 + chronic 50+50 Gy
acute 10 + 50+50 Gy
acute 20 + 50+50 Gy
acute 30 + 50+50 Gy
1. In vitro plantlet cultures of Dendrobium Sonia ‘Earsakul’ and Dendrobium Sonia ‘BOM
17 Red’ were chronic gamma irradiated in the Gamma Room at Gamma Irradiation
Service and Nuclear Technology Research Center, Kasetsart University.
2. For split doses, the interval between the first and the second irradiation was 1 month.

Experiment 7. Radiosensitivity study on acute gamma irradiation for determination ofLD50 of Dendrobium Sonia ‘Earsakul’ at 3 growth stages
Three different growth stages of Den. Sonia “Earsakul”, protocorm like bodies (PLBs),
plantlets and back bulbs, were acute gamma irradiated using Mark I Gamma Irradiator at
Gamma Irradiation Service and Nuclear Technology Research Center, Kasetsart University.
Radiation doses were 0, 40, 80, 160, 320, 640 and 1280 Gy. After irradiation, each material
was undertaken as follows:- 40 -

7.1.

Protocorm like bodies (PLBs)

Two replications of PLBs were irradiated. For the first replication, irradiated PLBs were
subcultured to the new medium, 20 pieces of PLBs were transferred to each bottle whereas
only 10 pieces/bottle was used in the second replication.

Survival of PLBs was recorded after two months of subculturing. LD50 was determined from
relationship between radiation dose and survival percentage.

Protocorm like bodies (PLBs) to be irradiated

7.2.

Plantlets

In vitro plantlets were irradiated and were transferred to the new medium. Four months after
irradiation, a number of survived seedlings was recorded, and LD50 was determined.

Plantlets to be irradiated

7.3.

Back bulbs

Twenty back bulbs of Dendrobium Sonia “Earsakul” were
irradiated for each treatment and were kept in nursery forinvestigation. Two months after irradiation, back bulbs
producing new shoots were checked as survival. Percent
survival of each treatment was calculated to get LD50.
Back bulbs to be irradiated- 41 -

Experiment 8. Evaluation of natural infestation of thrips in the nursery
1. The irradiated and the control plantlets from Experiments 5 and 6 were transplanted fromthe medium and grew in nursery to be naturally infested by thrips for 6 months.
2. A number of plantlets infested by thrips was recorded and percent infestation was calculated.
Infested and non-infested plantlets were tagged and were planted in pots for further investigation.
3. Irradiated plants as well as the controls were kept in the nursery until flowering for further
observation on morphological characters and would be selected for thrips resistance.

Results
Experiment 1. Evaluation and identification of breeding materials
Four clones of commercial cut flower orchids: Dendrobium Sonia ‘BOM 17 red‘,Dendrobium Sonia ‘Earsakul’, Dendrobium Pinky Sem ‘Rinnapa’ and Dendrobium hybrid‘White Sanan’ were evaluated. (Table 1-1 and Table 1-2)

Table 1-1. Plant height and number of flower spikes of cut flower orchids
Bulb characteristics
Clone

Bulb

Height(cm)

Number of spikes/bulb% of bulb

No. of% of bulb

No. ofwithleaves/produced

spikes/leavesbulb

flowersbulb

1. Den. Sonia

front bulb

57.5

100

7.2

100

3.0

‘BOM 17 Red’st

1 back bulb

47.5

100

7.2

100

3.3

Culture period:

2nd back bulb

2 years
2. Den. Sonia‘Earsakul’

39.8

100

5.4

100

2.4

rd

3 back bulb

31.3

100

4.2

90

1.5

front bulb

42.4

100

7.6

100

2.2

st

1 back bulb

41.2

100

6.6

100

4.4

nd

Culture period:

2 back bulb

36.0

100

5.8

100

3.2

2 years

3rd back bulb

32.0

100

4.6

100

3.0

3. Den. Hybrid

front bulb

62.3

100

10.1

100

2.7

‘White Sanan’st

1 back bulb

48.7

100

6.6

100

3.1

Culture period:

nd

27.6

70

2.0

60

0.9

rd

3 back bulb

20.2

10

0.1

30

0.4

front bulb

45.8

100

6.1

100

3.5

st

2 years
4. Den. Pinkysem

2 back bulb

‘Rinnapa’

1 back bulb

43.8

100

4.7

100

3.6

Culture period:

2nd back bulb

32.7

80

3.4

100

2.9

24.0

30

0.6

70

1.2

3 yearsrd

3 back bulb

- 42 -

Table 1-2. Flowering behavior, number of flower spikes/plants of cut flower orchids
Clone
Flowering behavior

BOM 17Red

Earsakul

White
Sanan

Rinnapa% of bulb which have 2 flowers
spikes from terminal buds
front bulb

70

100

70

60

1st back bulb

30

40

30

80

2nd back bulb

20

80

20

50

flower spikes/plant

10.2

12.8

7.1

9.9

-from terminal bud

5.2

7.4

4.1

5.8

-from lateral bud

5.0

5.4

3.0

4.1

-1st lateral bud

2.6

2.6

1.4

2.7

-2nd

1.3

1.8

0.7

1.5

-3rd

0.7

0.8

0.4

0.5

-4th

0.3

0.2

0.3

0.3

-5th

0.1

-

0.1

0.1

-6th-9th

-

-

0.1

0.2

Average number of

Average number of flower spikes
from lateral bud

Experiment 2. Initiation of tissue culture of clones
Young shoots of Dendrobium Sonia 'Earsakul', Dendrobium Sonia 'BOM 17 Red',
Dendrobium Pinky Sem 'Rinnapa' and Dendrobium ‘White Sanan’ were collected fromcommercial orchid nursery for tissue culture. The lateral and terminal buds were excised fromsterilized shoots and were cultured in liquid Vacin-Went medium. After two months underagitation condition, explants produced mass of protocorm like bodies (PLBs). The PLBs were
multiplied in liquid medium and were transflasked to solid Vacin-Went medium for PLBs
prolification.

- 43 -

Experiment 3. Exchange of material clones
Material clones, PLBs of Dendrobium Sonia ‘BOM 17 Red’, Dendrobium Pinky Sem‘Rinnapa’ and Dendrobium hybrid ‘White Sanan’ were exchanged among Thailand, Malaysia
and Indonesia. At the Mid term evaluation meeting, every country agree to research on theirvarieties. Thailand selected Dendrobium Sonia ‘Earsakul’ and Dendrobium Sonia ‘BOM 17
Red’.

Experiment 4. Determination of radiation sensitivity and optimum doses
4.1.

Radiation effect on protocorm developed from seeds of Dendrobium hybrids

Seeds of Dendrobium hybrid were in vitro germinated on modified Vacin-Went solid medium.
After germination for 20 days, seeds developed into tiny green protocorms. These protocorms
were irradiated with acute gamma rays of 0, 20, 40, 60, and 80 Gy. Two months after
irradiation, protocorms were subcultured to the new medium. All protocorms survived andshoot tips were developed. The second and third subcultures were done for seedling formation.
Six months after irradiation, the seedlings were taken out from sterile condition and theresults were recorded. The results show that the irradiated seedlings have shorter plant height
and leaf length but have more fresh weight, leaf number and root number than the control
plants (Table 4-1). The percentages of abnormal seedlings: 0, 21.33, 15.33, 11.33 and 9.33 arefound in the treatments of 0, 20, 40, 60 and 80 Gy, respectively (Table 4-2).
Table 4-1. Effects of gamma rays on seedling growth of Dendrobium hybrids derived
from in vitro acute irradiation
RadiationFresh weight

Plant height

dose (Gy)

(g)

(cm)

0

1.29 c20

Leaf number

Leaf length

Root number

7.90 a4.59 c5.76 a8.12 b

2.20 a6.46 b

5.27 a3.81 b

12.87 a40

1.63 b

5.29 c4.86 bc

3.21 c12.65 a60

1.73 b

4.97 cd

5.13 ab2.90 c12.05 a80
F-test1.43 bc**

4.69 d**5.28 a**2.89 c**11.53 a**CV (%)

26.79

14.81

10.69

16.30

26.84

- 44 -

Table 4-2. Abnormal seedlings (%) and survival (%) of Dendrobium hybrid seedlings
Survival rate (%)*Abnormal seedlings

Radiation dose (Gy)in vitroin vivo(%)

0

100.00100.00020

100.00

60.16

21.33

40

100.00

47.37

15.33

60

98.67

73.10

11.33

80

98.67

65.32

9.33

*Abnormal seedlings: rosette, thick leaf, malformation leaf, variegated leaf

4.2.

Radiation effects on PLBs of Dendrobium hybrids

The PLBs of 4 commercial cut flower clones: Dendrobium Sonia 'Earsakul', Dendrobium
Sonia 'BOM 17 Red', Dendrobium Pinky Sem 'Rinnapa' and Dendrobium hybrid ‘White
Sanan’ were irradiated with acute gamma rays at 0, 60, 70, 80, 90 and 100 Gy.

The irradiated PLBs were subcultured to the new medium. One month after irradiation, thenumber of survived and dead PLBs were inspected. The results show that all PLBs of
Dendrobium Sonia ‘Earsakul’ and Dendrobium hybrid ‘White Sanan’ were survived, while
PLBs of Dendrobium Pinky Sem ‘Rinnapa’ were tolerated to gamma rays than Dendrobium
Sonia ‘BOM 17 Red’. The non irradiated PLBs of Dendrobium Sonia ‘BOM 17 Red’ could
survived 97.74 % and after irradiating with 60, 70, 80, 90 and 100 Gy, the survived PLBs
were 77.45, 60.41, 62.39, 61.29 and 58.93 % respectively (Table 4-3). The PLBs were
subcultured for shoot multiplication and seedling formation, respectively.

Table 4-3. Survival (%) of Dendrobium hybrids after irradiated with gamma rays
Dendrobium

Dendrobium

Pinky Sem

Sonia

‘Rinnapa’‘BOM 17 red’

0

98.42

97.74

100

100

60

88.04

77.45

100

100

70

84.48

60.41

100

100

80

85.26

62.39

100

100

90

84.09

61.29

100

100

100

78.38

58.93

100

100

Radiationdose (Gy)- 45 -

Dendrobium Sonia‘Earsakul’

Dendrobium
hybrid‘White Sanan’Experiment 5. Irradiation of PLBs for insect resistant clones
5.1. Effects of radiation on plantlets growth of Dendrobium Sonia ‘BOM 17 Red’The survived PLBs were subcultured for 4 times to the new medium for multiplication andseedling development.

Subculture

PeriodGrowth(month)

1st

1

PLBs

2nd

3

multiplication of PLBs

3rd3

micro shoot formation with 3 - 4 leaves, 2 - 2.5 cm highculture 3 seedlings in one bottle until the seedling

4th

5

developed to large size with roots for transplanting to growin the nursery

The PLBs of Dendrobium Sonia ‘BOM 17 Red’ were 1st subcultured after irradiation

andwere continued to subculture for multiplication, seedling formation with 3-4 leaves, 2-2.5 cm.
high and for developing plantlets to large size with roots ready to be transplanted to grow in
the nursery.
After the 4th subculturing for 5 months, the plantlets were taken out from aseptic media forevaluation of weight, number of pseudobulb / plant, pseudobulb height and diameter, leaf
number and size. No significant difference in weight, pseudobulb diameter and leaf width.
The number of pseudobulb/plant, pseudobulb height, leaf number and leaf length of irradiatedplants were lower than the control. Irradiated treatments with 90 and 100 Gy reduced
pseudobulb height and leaf length (Table 5-1) and the abnormal rosette plantlets were found15.71 and 15.79 %, respectively (Table 5-2).

The irradiated plantlets were reduced in plant height. When the plantlets were ranked inheight for 4 levels, it was found that 49 % of non-irradiated plantlets had 6.1 - 11.0 cm highwith 51 % of 11.1 - 16.0 cm high. While the irradiated plantlets were in 6.1 - 11.0 cm. high.
These small plantlets had normal shape and large size plantlets but the growth rate was very
slow (Table 5-3).

- 46 -

The plantlets were grown in a rainproof nursery for one month. The 80 - 100 Gy irradiatedplantlets survived at 88.09, 85.71 and 86.84 %, respectively (Table 5-4). The normal plantlets
were alive while the rosette plantlets could not survive. The survived plantlets were cultured
for growth determination.
Table 5-1. Seedling weight and plant characteristics of Dendrobium Sonia ‘BOM 17 Red’Radiation

Weightdose (Gy)(g)

Pseudobulb

Plant

Leaf

size (cm)size (cm)Heightnumber(cm)/plantheightwidth/plantlengthwidth

number0

1.62

12.18a

2.85a

3.14a

0.29

10.9a

8.99a

0.55

60

1.19

8.669b

2.15b

2.40b

0.33

7.60b

6.50b

0.58

70

1.37

8.49b

1.85b

2.32b

0.33

6.40b

6.15b

0.55

80

1.11

7.92b

1.85b

2.23b

0.34

6.80b

5.51bc

0.62

90

1.42

6.77bc

1.98b

1.93b

0.37

7.05b

4.60cd

0.58

100

1.21

5.76c

2.20b

1.87b

0.37

7.20b

3.93d

0.62

F-test

ns

**

**

**

ns

**

**

ns

Table 5-2. Abnormal plantlets affected by radiation
Abnormal plantlets

Radiation dose
(Gy)%

Characteristics

0

0

non

60

7.51

rosette, thick leaf, short stem

70

8.57

rosette, thick leaf, short stem

80

11.91

rosette, thick leaf, short stem

90

15.71

rosette

100

17.79

rosette- 47 -

Table 5-3. Percentage of seedlings ranked in 4 levels of plant height of Dendrobium Sonia‘BOM 17 Red’
Plant height (cm)Radiation
Abnormaldose

Slow growth

Normal growth(small size)(Gy)
1.8-3.0

3.1-6.0

6.1-11.0

11.1-16.0

Total

0

0

0

49

51

100

60

2

14

61

22

83

70

2

16

69

13

82

80

2

19

66

13

79

90

4

25

64

7

73

100

4

33

60

3

63

Table 5-4. Total of survived plants after growing in the nursery for 1 month

5.2.

Radiation dose (Gy)Survival (%)

0

97.60

60

94.62

70

98.57

80

88.09

90

85.71

100

86.84

Effects of radiation on plantlet growth of Dendrobium Sonia ‘Earsakul’

PLBs of Dendrobium Sonia 'Earsakul' were irradiated witha) high doses of acute gamma rays : 0, 60, 70, 80, 90, 100 Gyb) low doses of acute gamma rays : 0, 2, 4, 6, 8, 10 Gyc) split doses of acute gamma rays : 0, 20, 20+20, 40 Gyd) chronic gamma rays : 0, 200, 400, 800 Gy
The PLBs were 1st subcultured after irradiation, then continued to subculture formultiplication, seedling formation with 3 - 4 leaves, 2 - 2.5 cm high and for developing theplantlets to large size with roots ready to be transplanted to grow in the nursery.
- 48 -

After the 4th subculturing for 3 months (8 months from irradiation), the plantlets were
removed from aseptic media for evaluation of weight, number of pseudobulb / plant,
pseudobulb height and diameter, leaf number and size. The irradiated plantlets at high doses
reduced in weight, height, pseudobulb height and number, number of leaf and leaf length. No
significant difference in number of pseudobulb/plant and leaf width (Table 5-5). The
percentage of plant height separate in 5 ranks were shown in Table 5-6. The low doses
irradiation had no effect on growth of plantlets (Table 5-7 and Table 5-8)
5.2.1

High doses of acute gamma irradiation

Table 5-5. Weight and plant characteristics of Dendrobium Sonia ‘Earsakul’
Radiationdose

Weight(Gy)(g)

Pseudobulb

Plant

Leaf

heightnumber(cm)/plantheightwidth/plantlengthwidth

size (cm)size(cm)

number0

2.0a

11.06a

2.8a

3.10a

0.42

8.9a

7.98a

0.60

60

1.2b

8.61b

1.7c

2.52b

0.41

6.3b

6.13b

0.61

70

1.4b

7.53bc

2.3b

2.33b

0.44

7.2b

5.53bc

0.67

80

1.4b

6.91cd

2.0bc

2.31b

0.44

7.2b

4.67cd

0.69

90

1.1b

6.86cd

1.9bc

2.16b

0.43

6.8b

4.59cd

0.63

100

1.0b

5.69d

1.6c

2.01b

0.44

7.1b

3.70d

0.74

F-test

**

**

ns

**

ns

**

**

ns

CV (%)

67.5

36.4

38.8

36.9

36.4

33.4

38.6

27.0

Table 5-6. Percentage of plant height separate in 5 ranks of Dendrobium Sonia‘Earsakul’
Plant height (cm.)

Radiation
dose

AbnormalSlow growth(Gy)

1.8-3.0

3.1-6.0

6.1-11.0

11.1-16.0

Total

0

0

0

52

48

100

60

1

4

74

21

95

70

4

30

57

9

66

80

3

29

65

3

68

90

1

31

64

4

68

100

4

33

62

1

63

- 49 -

Normal growth5.2.2Low doses acute gamma irradiation

Table 5-7. Weight and plant characteristics of Dendrobium Sonia ‘Earsakul’
Radiationdose

Weight(g)(Gy)

Pseudobulb

Plant

Leaf

heightnumber(cm)/plantheightwidth/plantlengthwidth

size (cm)size(cm)

number

0

1.9

10.84

2.0

2.75

0.42

7.5

8.00

0.71

2

2.0

11.04

2.2

2.77

0.40

7.3

8.39

0.70

4

2.0

10.83

2.3

2.68

0.40

8.3

8.24

0.74

6

1.8

10.44

2.3

2.67

0.39

8.3

8.03

0.69

8

1.8

10.15

2.0

2.66

0.41

7.2

7.64

0.72

10

1.8

10.11

2.1

2.56

0.39

8.1

7.51

0.70

F-test

ns

ns

ns

ns

ns

ns

ns

ns

CV (%)

40.9

23.0

31.6

23.7

66.2

26.5

23.5

18.1

Table 5-8. Percentage of seedlings ranked in 4 levels of plant height of Dendrobium
Sonia‘Earsakul’
Plant height (cm.)
Radiation dose
AbnormalSlow growth

1.8-3.0

3.1-6.0

6.1-11.0

11.1-16.0

Total

0

0

0

74

26

100

2

0

0

72

28

100

4

0

0

74

26

100

6

0

0

80

20

100

8

0

2

80

18

98

10

0

1

85

14

99

(Gy)

- 50 -

Normal growth5.2.3

Split doses acute gamma irradiation

Table 5-9. Seedling weight and plant characteristics of Dendrobium Sonia‘Earsakul’
Radiationdose

Weight(g)

Pseudobulb

Plant

Leaf

heightnumber(cm)/plantheightwidth/plantlength

width

(Gy)

size (cm)size(cm)

number0

1.9

10.84a

2.0

2.75a

0.42

7.5b

8.00a

0.71

20

1.9

9.37b

2.3

2.40b

0.39ab

9.1a

7.06ab

0.67

20+20

1.5

7.49c

2.1

2.27b

0.37b

9.8a

5.08c

0.64

40

2.0

8.88b

2.4

2.39b

0.42a

8.9a

6.56b

0.70

F-test

ns

**

ns

*

*

**

**

ns

CV (%)

41.3

27.8

37.1

26.1

74.7

30.4

32.2

19.4

Table 5-10. Percentage of seedlings ranked in 4 levels of plant height of Dendrobium
Sonia‘Earsakul’
Plant height (cm)Radiation
dose

AbnormalSlow growth(Gy)

1.8-3.0

3.1-6.0

6.1-11.0

11.1-16.0

Total

0

0

0

74

26

100

20

0

6

85

9

94

20+20

0

26

73

1

74

40

0

11

82

7

89

5.2.4

Normal growth

Chronic gamma irradiation

Table 5-11 Seedling weight and plant characteristics of Dendrobium Sonia Earsakul’
Radiation
dose
(Gy)

Weight(g)

Plant

Pseudobulb

Leafheightnumber(cm)/plantheightwidth/plantlengthwidth

size (cm)numbersize(cm)

01.9a10.84a

2.0

2.75a

0.42

7.5

8.00a

0.71

400

1.6ab

10.33a2.0

2.45ab

0.37

8.2

7.85a

0.65

800

1.3b

8.28b

1.8

2.35b

0.38

7.5

5.64b

0.68

F-test

*

**

ns

*

ns

ns

**

ns

CV (%)

49.3

24.0

25.9

25.3

75.5

23.0

27.4

22.4

- 51 -

Table 5-12. Percentage of seedlings separate in 5 ranks of plant height of Dendrobium
Sonia ‘Earsakul’
Radiation

Plant height (cm)Dose

AbnormalSlow growth(Gy)

1.8-3.0

3.1-6.0

6.1-11.0

11.1-16.0

total

0

0

0

74

26

100

400

0

3

78

19

97

800

0

20

74

6

80

Normal growthExperiment 6
Study on the effects of single and split doses of acute and chronic irradiation on in vitro
plantlets and PLBs of Dendrobium Sonia ‘Earsakul’ and Dendrobium Sonia ‘BOM 17 Red’

6.1

The in vitro plantlets

A. Dendrobium Sonia ‘Earsakul’

Table 6-1. Pseudobulb height of Dendrobium Sonia ‘Earsakul’ after grew in nursery for
6 and 12 months
Pseudobulb height (cm)Radiation dose (Gy)Single doses

Split doses

6 months

12 months

0

6.63

9.98

50

6.47

8.10

100

6.16

7.52

200

5.63

6.37

50+50

6.14

7.60

100+50

6.11

8.41

100+100

6.06

8.24

200+100

5.45

6.22

- 52 -

B. Dendrobium Sonia ‘BOM 17 Red’

Table 6-2. Pseudobulb height of Dendrobium Sonia ‘BOM 17 Red’ after grew in nurseryfor 6 and 12 months
Pseudobulb height (cm)Radiation dose (Gy)
6 months

12 months

0

6.95

11.07

50

6.44

9.55

50+50

6.21

8.89

100+50

6.36

8.91

100+100

6.60

9.06

200+100

6.12

8.62

Single doses

Split doses

6.2. The PLBs of Dendrobium Sonia ‘Earsakul’ and Dendrobium Sonia ‘BOM 17 Red’
A. Dendrobium Sonia ‘Earsakul’

Table 6-3. Plantlets characteristics of Dendrobium Sonia ‘Earsakul’ after transplantfrom aseptic condition

Radiation dose (Gy)

Pseudobulb

Fresh weight(g)

Leaf length

NumberHeightof plant

(cm)

(cm)

0

2.01

2

3.42a

6.75a

acute 0 + chronic 50+50

1.98

2.1

3.30ab

5.53bacute 10 + chronic 50+50

1.92

1.9

3.31ab

5.59bacute 20 + chronic 50+50

2.12

2.2

3.25ab

5.17bacute 30 + chronic 50+50

1.87

2.0

3.06b

5.11b

F-test

ns

ns

**

**

CV (%)

31.70

26.41

32.78

24.87

- 53 -

Table 6-4. Pseudobulb number/plant and height of Dendrobium Sonia ‘Earsakul’ andpercent natural infestation of thrips on seedlings after growing in nursery for 6 months
Pseudobulb
Radiation dose (Gy)
Number/plant

Height (cm)

0

4.67a

11.51aacute 0 + chronic 50+50

4.47ab

8.81bacute 10 + chronic 50+50

4.73a

9.29b

acute 20 + chronic 50+50

4.03b

9.04b

acute 30 + chronic 50+50

4.33ab

9.55b

F-test****CV (%)

13.93

22.86

B. Dendrobium Sonia ‘BOM 17 Red’

Table 6-5. Plantlets characteristics of Dendrobium Sonia ‘BOM 17 Red’ after transplantfrom aseptic condition
Fresh
Radiation dose (Gy)

Leaf

Pseudobulb

weightlength(g)

Number/plant

Height (cm)

(cm)

0

2.11a

2.2

3.61a

6.39a

acute 0 + chronic 50+50

1.91ab2.3

3.36ab

6.33aacute 10 + chronic 50+50

1.87ab2.0

3.33ab

6.01abacute 20 + chronic 50+50

1.85ab2.1

3.30ab

5.64abacute 30 + chronic 50+50

1.73b

2.2

3.03b

5.21b

F-test

*

ns

**

**

CV (%)

34.92

29.74

23.72

30.02

- 54 -

Table 6-6. Pseudobulb number/plant and height of Dendrobium Sonia ‘BOM 17 Red’ andpercent natural infestation of thrips on seedlings after growing in nursery for 6 months
Pseudobulb
Radiation dose (Gy)
Number/plant

Height (cm)

0

4.70b

13.32aacute 0 + chronic 50+50

4.80ab

11.01bacute 10 + chronic 50+50

4.97ab

11.11bacute 20 + chronic 50+50

5.23a

11.57bacute 30 + chronic 50+50

5.10ab

10.99bF-test**CV (%)

17.89

27.40

Experiment 7. Radiosensitivity study on acute gamma irradiation for determining LD50
of Dendrobium Sonia ‘Earsakul’ at 3 growth stages

7.1

Protocorm like bodies (PLBs)

The result showed that no survival of PLBs of Den. Sonia ‘Earsakul’ at the doses higher than
320 Gy (Table 7-1). Relationship between radiation dose and percent survival gave LD50 (at 2months after irradiation) of 70 Gy (Figure 1).

Table 7-1. Percent survival of Den. Sonia ‘Earsakul’ PLBs treated with different acute
gamma radiation doses (2 months after subculturing)
Survival (as % of control)
Radiation dose (Gy)Rep. 1

Rep. 2Ave.

0 (control)

100

100

100

40

100

100

100

80

47

40

43.5

160

17

10

13.5

320

0

0

0

640

0

0

0

1280

0

0

0

- 55 -

7.2

In vitro plantlets and back bulbs

In commercial orchid growing, the gardeners do vegetative propagation of sympodial orchid
by dividing. After culturing for 4 years the Dendrobium orchid plants produced 7-9
pseudobulbs per plant. The gardeners separate each mature psuedobulb or back bulb and leftthem produce new shoot from lateral bud before growing on coconut medium. After culturing
for 8 months these shoots produce the inflorescences.

Three months after irradiation, in vitro plantlets treated with 0 and 40 Gy produced newshoots while plantlets in the other treatments stopped to grow and die in the 4th month. For theback bulbs, all of non-irradiated pieces produced new shoots from lateral buds at rhizomes in
2 weeks whereas the buds treated with radiation still dormant. After 2 months, only 40 % ofbuds treated with 40 Gy produced small shoots. The shoots could not develop when treatedwith radiation doses at 80 - 1280. The results are shown in Table 7-2.
Table 7-2. Plantlets and back bulb survival of Den. Sonia “Earsakul” treated withdifferent doses of acute gamma irradiation
Survival (as % of control)

Radiation dosePlantlets

Back bulb(4 months after irradiation)(2 months after irradiation)

0

100

100

40

70.0

40

80

62.5

0

160

61.8

0

320

50.9

0

640

26.2

0

1280

0

0

(Gy)

Plantlet survival decreased as radiation dose increased. LD50 of plantlet at 4 months after
irradiation was 330 Gy (Figure 1).

For back bulb, radiosensitivity was higher than plantlets. Radiation doses over 40 Gy
prohibited shoot formation of irradiated materials. LD50 of back bulb at 2 months after
irradiation was 33 Gy (Figure 2).
- 56 -

PLBs survival (as % of control)

100
90
80
70
60
50
40
30
20
10
0

LD =70 Gy
50

0

200

400

600

800

1000

1200

1400

Radiation dose (Gy)Figure 1. Relationship between radiation dose and survival of protocorm like bodies (PLBs)
2 months after irradiation

100

Survival (as % control)

90
80

LD

= 33 Gy

Seedling
Back bulb

50

70

LD = 330 Gy

60

50

50
40
30
20
10
0
0

200

400

600

800

1000

1200

1400

Radiation dose (Gy)Figure 2. Relationship between radiation dose and survival of plantlet and back bulb
4 and 2 months after irradiation, respectively.

- 57 -

Experiment 8. Evaluation of natural infestation of thrips in the nursery
8.1

Plantlets from Experiment 5

After transplanting of plantlets from aseptic culture, the plantlets were grown in 1 inch pot
with coconut fiber as medium under the rainproof nursery. Water was applied in every
morning and fertilizer was sprayed once a week. No application of insecticide and fungicide.
The first evaluation was carried out for 4 months during November 2004-February 2005. The
plantlets of Dendrobium Sonia ‘BOM 17 Red’ and Dendrobium Sonia ‘Earsakul’ were grownin the nursery for natural infestation of thrips. The results showed that only 0 - 17.14 % ofplants were damaged by thrips. The non-irradiated plants showed 16.85 and 17.14 % damaged
which more than irradiated plants, since the control plants started to produce new shoots
earlier than irradiated plants. Thrips could damage more number of the control plants (Table
8-1).

Table 8-1. Percentage of seedlings which the first new shoots were damaged by thrips
Radiation dose

% of thrips damaged seedlings(Gy)

Den. Sonia ‘BOM 17 Red’

Den. Sonia ‘Earsakul’

0

16.85

17.14

60

4.55

5.00

70

5.26

5.56

80

7.14

5.81

90

0

3.57

100

2.04

3.27

After 10 months in the nursery during November 2004 - August 2005, a number of plants died,
whereas the living plants produced the 2nd and 3rd shoots. The data was recorded again fromthe living plants. The results showed that 67.27 - 92.31 % of plants were damaged by thrips(Table 8-2).

The number of damaged seedling were classified into 2 types:
1) damaged on the 2nd new bulbs with no damaged on the 3rd new bulbs2) damaged on the 2nd and the 3rd new bulbs (Table 8-3).

- 58 -

In the non-pesticide with hot temperature and high humidity condition, thrips gave noproblems to the plants. In rainy season, plants died by the infestation of worms, fungi andbacterial diseases. A lot of insect larvae were feeding on most of leaves during dry season,
thus the plants had no leaves for thrips.

The non-damaged plants by thrips were transplanted to grow in large size pots for evaluationof growth and flower size. The experiments on selection of thrip resistant clones in flowering
stage will be designed.
Table 8-2. Survival and percentage of the 3rd new shoots damaged by thrips
Radiation

Den. Sonia ‘BOM 17 Red’

Den. Sonia ‘Earsakul’

doseSurvival

Damaged seedlings

Survival

Damaged seedlings

(Gy)

(%)

(%)

(%)

(%)

0

42.0

92.31

45.0

86.40

60

30.5

83.78

33.0

87.88

70

10.5

80.77

43.5

88.51

80

3.0

83.33

40.5

87.78

90

0

--

27.5

67.27

100

3.0

83.33

45.0

85.66

Table 8-3. Percentage of seedlings which the 2 nd and the 3 rd new shoots were damaged bythripsRadiation

Damaged seedlings (%)

dose

Damaged on the 2 nd new shoots

Damage on the 2nd and(Gy)and no damaged on the 3 rd new shootsthe 3rd new shoots

0

37.60

48.80

86.40

2

37.06

56.65

93.71

4

36.89

45.08

81.97

6

40.63

53.12

93.75

8

40.79

40.79

81.58

10

39.89

38.95

76.84

- 59 -

Total

8.2. Natural infestation of thrips on chronic gamma irradiation of Dendrobium Sonia‘BOM 17 Red’ and Dendrobium Sonia ‘Earsakul’ plantlets (plantlets from Experiment 6).
1) The irradiated plantlets
The numbers of Dendrobium Sonia ‘BOM 17 Red’ and Dendrobium Sonia ‘Earsakul’plantlets infested by thrips of each treatment are shown in Table 8-4 and Table 8-5,
respectively.

Table 8-4. Percent natural infestation of thrips on plantlets of Dendrobium Sonia ‘BOM
17 Red’ treated with different doses of chronic gamma irradiation (6 months after
growing in the nursery)
Radiation dose

Number of plantlet

Infested

(Gy)

Total

Infested(%)

0 (control)

320

14

4.38

50

50

11

22.00

50+50

295

95

32.20

100

94

16

17.02

100+50

245

41

16.74

100+100

153

24

15.69

200

79

22

27.85

200+100

444

97

21.85

Table 8-5. Percent natural infestation of thrips on plantlets of Dendrobium Sonia‘Earsakul’ treated with different doses of chronic gamma radiation (6 months after
growing in the nursery)
Radiation dose

Number of plantlet

Infested

(Gy)

Total

Infested(%)

0 (control)

127

34

26.77

50

219

34

15.53

50+50

191

26

13.61

100+50

234

43

18.38

100+100

142

22

15.49

200+100

140

21

15.00

- 60 -

Natural infestation of thrips was more severe on Dendrobium Sonia ‘Earsakul’ than
Dendrobium Sonia ‘BOM 17 Red’. Percent infestation reduced on the irradiated treatments ofboth varieties.

For Dendrobium Sonia ‘Earsakul’, the lowest infestations were found in doses of 100+100,
100+50 and 100 Gy. Lower or higher doses seemed to be susceptible to thrips. However, theinfestation of irradiated plantlets was higher than the control.

The opposite result was noticed in Dendrobium Sonia ‘BOM 17 Red’. It appeared thatradiation doses gave no different effects on infestation of thrips but all radiation treatments
were shown to be resistant than the control.
2) Plantlets derived from irradiated PLBs
Table 8-6. Percent natural infestation of thrips on seedlings of Dendrobium Sonia ‘Earsakul’ andDendrobium Sonia ‘BOM 17 Red’ after growing in nursery for 6 months

Infested (%)

Radiation dose (Gy)‘Earsakul’‘BOM 17 Red’

0

6

6

acute 0 + chronic 50+50

810acute 10 + chronic 50+50

48acute 20 + chronic 50+50

86acute 30 + chronic 50+50

68

For growth and morphological characters, it was found that at the beginning, plantlets growthdecreased as radiation doses increased, but later, they returned to normal growth. At the same
total dose, split dose showed less effect than single dose. Some abnormal leaves and flowers,
as well as some other morphological changes e.g. flower color, flower size were observed.
Selection for thrips resistance needs to be followed at flowering stage.

Acknowledgement
The authors appreciate the research grant from Kasetsart University Research andDevelopment Institute (KURDI) and the expert service from Gamma Irradiation Service andNuclear Technology Research Center, Kasetsart University.
- 61 -

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- 63 -