FNCA／MEXTTechnical Material

Achievement
Sub-Project on Insect Resistance in Orchid( 2003 – 2009 )Mutation Breeding Project
Forum for Nuclear Cooperation in Asia (FNCA)
March, 2010-1-

Foreword
Orchid is one of the largest families of flowering plants comprising an estimated 20,000 to
25,000 species. Orchids such as Dendrobium have been the major cut-flower export for
Thailand, Malaysia and Indonesia as well as for other Southeast Asian countries. Thailand is
the major orchid producer with an export value of US$ 80 million in 2006, or approximately
80 % of the country’s total ornamental export value. The export value of orchids from
Malaysia is estimated at RM 150 million per year, representing approximately 40 % of thetotal floriculture production. Problems regarding the infestation of insects in orchid flowers
have caused considerable losses to exporters due to strict quarantine regulations. Among them,mites and thrips are the most devastating pests of the orchid. Spraying with insecticides andmitecides are another option when dealing with orchid pests, especially when populations arehigh and spraying is the only effective means of controlling mite attacks. This practice leads
to heavy use of chemicals which requires government review and regulation. One of thestrategies that can be adopted to overcome the problem is to induce insect resistance in orchidthrough radiation breeding. The Mutation Breeding Project recently focused on the
improvement of insect resistance in orchid and represents one Sub-Project developed throughthe Forum for Nuclear Cooperation in Asia (FNCA), organized by Ministry of Education,
Culture, Sports, Science and Technology of Japan (MEXT), in 2003. This Sub-Project wasestablished with the collaboration of member countries such as Indonesia, Malaysia, andThailand. Mutation breeding through gamma-ray and ion beam irradiation is one of very
useful methods to induce favorable changes in germplasm when required breeding materials
are not otherwise available.
Activities of this project are focused on a wide array of important agronomic topics; of which
the following were deemed of highest importance: (1) exchange of breeding materials andpromising mutants; (2) exchange of information regarding efficiency of screening methods for
insect-resistance; (3) establishment of breeding techniques such as in vitro culture andidentification of optimum irradiation dose to plant parts. Each year, results and encounteredproblems within this project are discussed at the FNCA Workshop held in the member
countries as well as two meetings of specialists. In this project, promising lines have been
selected in Malaysia with the support of Indonesia and Thailand and evaluation of
performance and yield trials for the registration are being conducted. Since the cooperative
research activities fulfilled the project goals, the project was terminated in 2009. However,
several additional years of efforts are required for the plant breeders to release new cultivars
to the farmers. Following the identification of promising lines, yield trials, spanning at least 3igrowing seasons, are required for a new cultivar registration.
It is our hope that the promising lines developed in this project will benefit the orchid farmers
of Asia. We also hope that this book will be useful not only for the breeders interested in
gamma ray and ion beam induced mutation breeding, but also for the breeders of orchid.
I would like to state my appreciation to the contributing authors for their achievements in thisproject and the submission of their final reports.

Hitoshi Nakagawa
Project Leader of Japan, Mutation Breeding Project, FNCA
Director, Institute of Radiation Breeding
National Institute of Agrobiological Sciences
March, 2010ii

Content

1. Indonesia ··········································································· 1
2. Malaysia ·········································································· 13
3. Thailand ·········································································· 33

iii

1. Indonesia

1. Indonesia
Ismiyati Sutarto1, Suskandari Kartikaningrum2 and Yulidar1
1Center for Application of Isotope and Radiation Technology,
National Nuclear Energy Agency (BATAN)
2

Indonesian Ornamental Crop Research Institute,

Indonesian Agency for Agricultural Research and Development (IAARD)

Introduction
In Indonesia, demand for orchid cut flower is increasing due to the growth of population andtheir income. According to Soeroyo (1992), the world consumption value for orchid cut
flowers was US $ 1.935 billion, if Indonesia could contribute 2.5 %, it needs the availabilityof 129.43 million flower stalk for export quality. Whilst for those of 5 % for export quality, itneeds 32,357,500 plants (supposed each plant produced 8 flower stalk). Orchid industry needs
the support from science and technology based on the use of optimal natural resources.
Development of technology in culturing orchids is an opportunity to create desirable
characters of the flowers such as bright colour, beautiful shape and longer vase life.

Dendrobium is the most popular orchids in Indonesia (Figure 1) followed by Catleya, Vanda
and Phalaenopsis. Recently Dendrobium is mostly grown in Java and Bali islands. Bothsexual and asexual propagation are common for multiplication of Dendrobium. Orchid
consumers in Indonesia tend to prefer the new type. Micropropagation in orchid is very
common since the demand for potted plants and cut flowers increased.

Figure 1. Dendrobium Jayakarta for cut flowers is widely grown in Indonesia-1-Breeding of the genus Dendrobium has been gaining sufficient appreciation and development
in Southeast Asia as the most popular source of tropical cut flowers. A large number ofdifferent cultivars of distinct colours and shapes of this species have been created and grown
exclusively in farms in this area. Commercial plants produce cut flowers and cooled
inflorescences are exported throughout Europe and Japan.

The fact that orchids are, in terms of their development, a very young family of plants andtheir genetic instability. Therefore they tend to form mutation by means deviations. Usually
the frequency of mutation was very low and lack of desirable traits. In the wild, these
deviations are suppressed and gradually disappear. Orchid growers are able to stabilize themby further appropriate hybridisation and maintain the desirable traits such as colour and sizeof the flower (Jezek, 2003).

Sutater (1997) reported that exported orchid flower from Indonesia decreased from US $ 2,1
million (1993) to US $ 1.41 million (1995), whereas imported orchid flower increased from
US $ 28,831 (1994) to US $ 312,767 (1995). Based on the data mentioned above, it seems
that the demand was higher than the supply. Therefore, the opportunity for growing orchid inIndonesia is very promising.

Demand of orchid especially Dendrobium is increasing because of its unique, beauty, frequent
flowering, longevity and easier to be cultivated. These characters are leading compared withother orchids. Dendrobium could be used as indoor and outdoor potted plants, cut flowers or
corsages. However, Dendrobium is very susceptible to pets and diseases. Flower damage
caused by Thrips is one of the obstacles faced by orchid growers.

An effort to solve this problem had been initiated in FNCA Mutation Breeding ProjectWorkshop held in Yogyakarta, Indonesia from 30 August – 3 September 2004 (Hutabarat etal, 2004 and Piluek, 2004). The Orchid Group agreed to use Dendrobium Sonia ‘BOM 17
Red’, a leading variety from Thailand as the main material, Den. Sonia ‘BOM 17 Red’ is a
popular hybrid, fast growing, floriferous, bright colour and has long vase life.

In FNCA Mutation Breeding Project Workshop held in Bangkok from 5 - 9, September 2005,
the Orchid Group decided to irradiate different growing stages of young seedlings or shoots,
plantlets and PLBs of local variety from each country, such as Dendrobium Jayakarta from-2-

Indonesia should be irradiated in order to obtain the optimum dose for Dendrobium sp. fromdifferent member countries.

The most severe damage caused by irradiation was showed by PLBs compared with plantlets
and shoots. Three months after irradiation, survival rates from the dose 1,280 Gy up to thedose 40 Gy of irradiated PLBs, plantlets and shoots were 0 - 50 %, 0 - 78 % and 60 - 95 %respectively. Survival rate of untreated PLBs, plantlets and shoots remained 100 % after threemonths irradiation. Explants of Den. Sonia ‘BOM 17 Red’ cultured in VW enriched with
BAP was not able to form roots, whilst those enriched with coconut water and active charcoal
could form 2.2 - 4.7 roots (Sutarto et al 2006).

Materials and methods
Irradiated PLBs of Den. Jayakarta cultured in VW medium were regenerated to form
plantlets and each plantlet was planted in a single jar. While irradiated plantlets formerlygrown in community pots had been transferred to single pots. Irradiated potted plants started
blooming were grown under lath house. Potted plants were grown in the lath house andfertilized every week by applying 0.2 % liquid fertilizer.

Irradiated explants of Den. Sonia ‘BOM 17 Red’ grown at CAIRT were cultured by applying
modified VW medium enriched with coconut water (15 %), charcoal (0.1 %) and BAP (1ppm). Whereas those grown at IOCRI were cultured in VW medium. The last accession ofDen. Sonia ‘BOM 17 Red’ was Ac 10 + 50 +50 (Acute irradiation 10 Gy + chronic irradiation
50 + 50 Gy) and Ac 20 + 50 +50 (acute irradiation 20 Gy + chronic irradiation 50 + 50 Gy).

The use of different modified Vacin and Went (VW) media such as application of coconut
water, active charcoal and Benzyl Amino Purine (BAP) was also carried out to improve thegrowth and propagation of irradiated plantlet of Den. Sonia ‘BOM 17 Red’ and Den.
Jayakarta.

Application coconut water (Widyastoeti and Syafril, 1993) and active charcoal (Widyastoety
and Bahar, 1995) could stimulate the growth of Dendrobium plantlets. Kano and Fukuoka(1996) reported that coconut water contained complex organic materials such as plant growth
regulators that promoted plant growth.

-3-

Mass rearing Thrips was done at Entomology Laboratory of IOCRI by placing mature Thrips
into the flask with pumpkin inside (Figure 2), and let the Thrips lay eggs and multiply its
population, 25 - 30 days later mature Thrips were put on the flower of Spathoglottis sp(Figure 3) in order to see how the Thrips attack the flower since irradiated Dendrobium had
not bloomed yet. When the plants started blooming, insect tolerance trial will be conducted invivo in the green house.

Figure 2. Thrips were put in the flask with the pumpkin inside

(a)

(b)

Figure 3. a. Mature Thrips were placed on the flower of Spathoglottis sp.,b.Thrips attacked the flower of Spathoglottis sp.

The parameter observed were number of shoots per plant, number of leaves per shoots, plantheight, number of plantlet or plant per pot and number of potted plants. The data were taken
12 months after irradiation.

Results
After 12 months irradiation, PLBs of Dendrobium Jayakarta grew to form plantlets. The
highest number of plantlets and number of shoots per plant were found from untreated PLBs
followed by irradiated PLBs at the doses of 40 and 80 Gy. In contrast the highest number ofleaves per shoot was obtained from irradiated PLBs at dose 80 Gy followed by irradiated-4-

PLBs at the dose 40 Gy and untreated PLBs, while the highest plant was showed by untreated
PLBs followed by irradiated PLBs at the doses 80 and 40 Gy (Table 1 and Figure 4).

Irradiated plantlets of Dendrobium Jayakarta grown in the lath house showed better tolerance
to gamma rays than irradiated PLBs. Plantlets were able to grow well up to the dose 160 Gy(Table 2 and Figure 5), whereas irradiated young plants could only grow at the dose 40 Gy(Table 3 and Figure 6).

Untreated

80 Gy

40 Gy

Figure 4. Untreated (0 Gy) and irradiated PLBs of Den. Jayakarta
at the doses 40 and 80 Gy (12 months after culturing)

The most plantlets and clusters of PLBs formed were obtained from irradiated plantlets ofDen. Sonia ‘BOM 17 Red’ at the dose 70 Gy. PLBs was not found from those at the dose 30
Gy and untreated plantlets (Table 4 and Figure 7). Abnormalities were found in irradiated
PLBs of Den. Sonia ‘BOM 17 Red’ (Figure 8).

Acute irradiation 10 Gy followed by chronic radiation 50 + 50 Gy indicated better number ofplants and leaflets, whereas acute irradiation 20 Gy followed by chronic radiation 50 + 50 Gy
indicated higher number of shoots per plants and plant height (Table 5 and Figure 9).

Irradiated plantlets of Den. Sonia ‘BOM 17 Red’ at the dose of 90 Gy grown in the lath houseof IOCRI for acclimatization showed better performance compared to those at the dose 80 Gy(Table 6)-5-Untreated

160 Gy

80 Gy

40 Gy

Figure 5. Untreated (0 Gy) and irradiated plantlets of Den. Jayakarta

Compared to those after 3 and 6 months irradiation, all the parameter observed such as
survival rate, number of shoots per plant, number of leaves per shoot and plant height onirradiated PLBs of Den. Jayakarta were gradually decreased as the doses of gamma rays
increased. After 3 months irradiated plantlets were able to grow up to the dose 640 Gy
although the survival rate was only 39 %. Irradiated young plants grew well until 3 and 6
months after irradiation up to the dose of 1,240 Gy (Sutarto et al, 2006).

According to Broertjes and Van Harten (1988) the optimum dose for orchids varies fromapproximately 10 to 40 Gy. However, in view of the great number of very different generaand species, and of the material to be irradiated, the optimum dose should be determined inevery case before starting a mutation breeding program.

-6-

40 GyUntreated

Figure 6. Untreated (0 Gy) and irradiated shoot of Den. Jayakarta (12 months after planting)

Table 1. Growth of irradiated PLBs of Den. Jayakarta (12 months after irradiation)
cultured in jars at growth room of CAIRT
Dose of Gamma

Number of

Number of

Number ofrays (Gy)plantletsshoots/plantlet

leaves/shoot

0

63

2.97

4.67

5.95

40

38

2.69

6.05

4.62

80

3

2.67

6.97

4.75

Plantlet height (cm)

Table 2. Irradiated plantlets of Den. Jayakarta grown in the lath house of CAIRT (12months after irradiation)
Dose of

NumberGammaofrays (Gy)plants

0

26

3.54

2.46

5.39

1.50

14.30

40

16

2.69

2.26

4.23

1.23

8.89

80

20

3.00

2.50

6.38

1.83

13.92

160

6

3.17

2.50

5.20

1.40

13.90

Number of

Number ofshoots/plantleaves/shootLengthof leaf

Width ofleaf (cm)

(cm)

-7-

Plant
height(cm)

Table 3. Irradiated shoots (young plants) of Den. Jayakarta grown in the lath house of
CAIRT (12 months after irradiation)
Dose of

NumberGammaofrays (Gy)plants040

LengthWidthof leafof leaf

(cm)

(cm)

4.11

10.38

2.99

51.96

3.32

6.17

2.81

35.50

Number of

Number ofshoots/plantleaves/shoot

12

4.33

9

5.33

Plant
height (cm)

Table 4. Growth of irradiated plantlets of Den. Sonia ‘BOM 17 Red’ cultured inmodified VW medium with coconut water and active charcoal (CAIRT)
Dose of Gamma rays (Gy)Parameters
0

30

70

80

Number of plantlets

15

12

77

5

Number of shoots/plantlet

2.00

1.58

2.43

1.80

Plantlet height (cm)

2.05

5.00

2.64

1.96

Number of leaflets (avrg)

2.75

3.11

3.86

2.61

Number of PLBs (clusters)

0

0

13

2

Untreated

30 Gy

70 Gy

80 Gy

Figure 7. Untreated (0 Gy) and irradiated PLBs of Den. Sonia ‘BOM 17 Red’-8-

Table 5. Growth of irradiated plantlets of Den. Sonia ‘BOM 17 Red’ grown at the lathhouse for acclimatization (CAIRT)
Dose of Gamma rays (Gy)Parameters

Ac 10 + Chr (50 + 50)

Ac 20 + Chr (50 + 50)

Number of plants

27

24

Number of shoots/plants

4.59

5.33

Plantlet height (cm) (avrg)

1.38

2.28

Number of leaflets (avrg)

2.17

1.13

70 Gy

70 Gy

Figure 8. Abnormality found in irradiated PLBs of Den. Sonia ‘BOM 17 Red’Figure 9. Acute and chronic irradiation of Den. Sonia ‘BOM 17 Red’
Left (acute 10 Gy + chronic 50 + 50 Gy), Right (acute 20 Gy + chronic 50 + 50 Gy)

Table 6. Irradiated plantlets of Den. Sonia ‘BOM 17 Red’ grown in the lath house of
IOCRI for acclimatization.
Dose of

NumberGammaofrays (Gy)plants80
90

Length

Width

Plant

of leafof leafheight

(cm)

(cm)

(cm)

2.22

3.12

0.53

4.29

3.61

4.33

1.60

8.72

Number of

Number ofshoots/plantleaves/shoot

27

1.21

44

2.48

-9-

Figure 10. Irradiated plantlets of Den. Sonia ‘BOM 17 Red’ grown in the lath house of IOCRI(12 months after irradiation)

Conclusion
The results of this observation can be concluded that PLBs and young shoots of Den.
Jayakarta were very sensitive to gamma rays. The optimum dose for PLBs and plantlets ofDen. Jayakarta was 40 Gy since there was no PLBs and young shoots could grow at the doses
more than 40 Gy after 12 months of irradiation. Mass rearing Thrips can be done by using
pumpkin as artificial feeding. The most plantlets and clusters of PLBs formed were obtained
from irradiated plantlets of Den. Sonia ‘BOM 17 Red’ at the dose 70 Gy. PLBs was not foundfrom those at the dose 30 Gy and untreated plantlets. Irradiated plantlets of Den. Sonia ‘BOM
17 Red’ at the dose of 90 Gy grown in the lath house of IOCRI for acclimatization showed
better performance compared to those at the dose 80 Gy

References
Broertjes, C. and A. M. van Harten. 1988. Applied mutation breeding for vegetatively
propagated crops. Elsevier. Amsterdam. 345 p.

Hutabarat, D., L. Harsanti and Aryanti. 2004. Effects of gamma rays on protocorm like bodiesof three hybrids and one species of Dendrobium. FNCA Mutation Breeding ProjectWorkshop. Reproductive Pattern in Clonally Propagated Plants. Yogyakarta, 30
August - 3 September 2004.

Jezek, Z. 2003. The complete cyclopedia of orchids. Rebo Publishers. Prague.304 p.

- 10 -

Piluek, C. 2004. Study on radiation dose for insect resistant of cutflower orchids. FNCA
Mutation Breeding Workshop. Reproductive Pattern in Clonally Propagated Plants.
Yogyakarta, 30 August - 3 September 2004

Soeroyo, R. 1992. Chances for growing orchids and ornamental plants. Orchid Exhibition.
Indonesian Orchid Society. Surabaya. Indonesia

Sutarto, I., I. Dwimahyani, S. Kartikaningrum and Yulidar. 2006. Oservation of irradiation
doses on Den. Jayakarta and plantlet growth of Den. Sonia ‘BOM 17 Red’ in different
modified media for insect tolerance. FNCA 2006 MBWS. Takasaki Japan. 11 - 16
September 2006. 9p
Sutater, T. 1997. Developing cultural practices of orchids for industrial scale. Bull.
Indonesian Orchid Society . 5 (9) : 18 – 23

Widyastoety, D. and Syafril. 1993. The effect of coocnut water on the growth of PLBs of
Dendrobium orchids. Ind. Bull for Ornamental Plant Research. 1 (1) : 7 –12

Widyastoety, D. and Bahar. 1995. The effect of different carbihydrat source on the growth ofplantlets of Dendrobium orchids. Ind. J. Hort. 5 (3) : 76 – 80

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2.

Malaysia2. Malaysia
Zaiton Ahmad, Sakinah Ariffin, Ros Anita Ahmad Ramli,
Mohd Nazir Basiran, Affrida Abu Hassan
Malaysian Nuclear Agency (Nuclear Malaysia)

Introduction
Orchid is one of the largest families of flowering plants in the world comprising of an
estimated 20,000 to 25,000 species and untold number of hybrids of some 700 different
genera. Some estimates have put the registered number of hybrids at more than 50,000 entries,
with additional new ones at a rate of 1,000 per year. Orchids can be found growing at almost
every latitude and stratosphere, where plants can normally grow. The range of different
characteristics in shapes, sizes and colors have made orchids the plant for all types of interestsand sought for by collectors.

The successes of hybridization technology in producing vast number of orchid hybrids withattractive characteristics and the introduction of the in vitro technology to mass-propagate
clonal planting materials have been a tremendous boost for the orchid growing industry. Widerange of successful cultivars with attractive combinations of spray length, bud number, flower
color and form, vase life, fragrance, seasonality, and compactness have been produced
through hybridization. Meanwhile tissue culture has been the standard method of germinating
seeds and propagating seedlings for the industry. Through the meristem cloning technique,thousands of plants can be grown in a relatively short period of time and every regenerated
plant is exactly similar to the parent plant in every aspect. These two technologies have so far
been very reliable in supporting the orchid industry in the region. Commercially attractive
hybrids and varieties have been able to be mass propagated and supplied to the growers forthe market.

Orchids such as Dendrobium have been the major orchid cut-flower export for Malaysia as
well as for other Southeast Asian countries like Thailand and Philippines. Thailand is the
major orchid producer with an export value of US$ 80 million in 2006, or approximately
80 % of the country’s total ornamental export value (www.reedexpo.com/images/100266/documents/OrchidPavilion.doc). The export value of orchids from Malaysia is estimated at- 13 -

RM 150 million per year, representing approximately 40 % of the total floriculture
production.

Problems regarding the infestation of insects in the orchid flowers have caused a lot of losses
to exporters due to strict quarantine regulations. Presence of even one insect can cause thewhole export consignment to be rejected by importing countries. There were cases when thewhole orchid consignment had to be shipped back to the exporting countries due to thepresence of insect pests. Spraying with insecticides and miticides are the options when
dealing with orchid pests, especially when populations are high. This practice however leads
to heavy use of chemicals but the problems with insects still persist. Studies have been
conducted for post-harvest disinfestations of cut flower using irradiation. However, thepost-harvest irradiation treatment has shown detrimental effect on the quality of the cut
flowers. The strategy that can be adopted to overcome the problem is to breed for insect
resistance in orchid. Insect resistant orchid hybrid may minimize the use of chemicals andovercome the strict quarantine requirements of importing countries.

Mutagenesis is considered as an appropriate approach to induce resistance as hybridization islimited by the unavailability of a resistant genotype and problem of sexual compatibility.
Insect resistance has been induced by using mutagenesis approach in varieties of plants suchas mungbean and rice. Mutation induction by irradiation has effectively changed certain
characteristics of the plants to be ‘unattractive’ to insects.

Through biotechnological techniques, the insect resistance can be achieved via two
approaches. The first used genes that synthesize products, which are toxic to feeding insects(eg Bt insecticidal protein and the cowpea trypsin-inhibitor protein) (Perlac et al. 1991; Hilder
et al. 1987). The second approach used a cytokinin-synthesis gene 9ipt), which under thecontrol of a specific promoter will induce the synthesis of one or more insecticidal
metabolites (Smigocki et al. 1993). Several reports have shown that transformation of orchids
is possible using the particle gun (biolistic) and the Agrobacterium-mediated method (Knappet al. 2000; Wee et al 1999). Thus it is possible to introduce the genes for insect resistance into
orchids.

Following consultations with the local orchid growers, we have identified that mites andthrips are the main pests for orchids in Malaysia. Other pests include aphids, whiteflies,- 14 -

mealybugs, slugs and snails. At present, more than 30,000 mites have been identified all over
the world. Most of them are parasitic on animals and humans, with only a few feeding onplants. Among the most common mites for orchids are false spider mites or flat mites (Gough,1988). Flat spider mite or Tenulpalpus orchidarum is from the family of Tenuilpalpid. Flat
mites are native to tropical and subtropical habitats and hosts, and are moved globally by theplant trade. Flat mites are very difficult to see without magnification although they move very
slowly and they have a great resemblance with other mite species. Generally, adult flat spider
mite can only reach up to 0.03 mm in length. These mites are pale yellowish-green toorange-red color. Flat spider mites have a flat appearance somewhat resembling spider mites
and are often reddish colored with patterns of dark pigmentation.

Another common mite pest for orchids in Malaysia is Tetranychus urticae (twospotted spidermite). The pest is commonly found in the lower surface of leaves and sucks the sap of the leaf.
The leaves become pale yellow with numerous small spot which reduces the quality of theleaves. The pest can be controlled with 0.03 % sulphex spray.

Mites normally feed by sucking the sap from individual cells on the surface of the leaves.
Because of their feeding style, they may also be potential vectors for various diseases. Leaves
infested by mites appear silvery, especially on the underside, where the cells of the surface
layer were damaged. From the top, the leaf often has many tiny yellow spots. The false spider
mites do not make silk, and are extremely tiny and hard to see, but they produce the same
silvery structures on the leaves. A good way to know the presence of mites is to wipe the leaf
with a white tissue; if the tissue has red smears, then the plant has mites.

Thrips is another important insect pest for orchids in many countries including Malaysia, andcauses a lot of problems in flower production. Thrips normally infest young flower buds andnewly expanded leaves. Blooms of infested plants may become prematurely brown, whilst theinfested petals may either become spotted, streaked, silvery or discolored. Symptoms onleaves include chlorotic spots, wilting and eventually dropping. Plant growth can also be
stunted, and in a severe infestation case, the whole plant will die (Jones 2008). Examples ofmite and thrips infestation symptoms commonly found on infested orchid plants are shown in
Figure 1.

- 15 -

A

B

Figure 1 Common infestation symptoms by mites (A) and thrips (B) on orchid plants

Three orchid species used in this project were Dendrobium mirbellianum (Malaysia),
Dendrobium Sonia ‘BOM 17 Red’ (Thailand) and Dendrobium jayakarta (Indonesia).
Dendrobium mirbellianum is a species commonly found in the Pacific region. It is easy togrow, robust and produces long spray (up to 45 cm) with up to 30 flowers. It is flowering
throughout the year and the flowers last for about 4 weeks. Each flower measures between 3.5- 5.5cm across. This species is lacking in several qualities such as attractive color and long
lasting shelf life, thus making it as a good candidate for the mutagenesis project.

Dendrobium Sonia ‘BOM 17 Red’ is a very popular commercial hybrid in the region andwidely grown for the cut flower export market. This hybrid has purple flowers that can last up
to 14 days as cut flower. This commercial hybrid was chosen as it is one of the most popular
cut flower Dendrobium hybrids grown in the country. Several variations of this hybrid
differing in petal and sepal pigmentation are available such as Sonia and Sonia BOM Jo.
Meanwhile, Den. jayakarta is a popular commercial hybrid in Indonesia with white coloredflowers. Figure 2 shows the three orchid species used in this project.

A

B

C

Figure 2 : Orchid species used in the project;Den. mirbellianum (A), Den. Sonia ‘BOM 17 Red’ (B) and Den. jayakarta (C)- 16 -

Objective of the Project
The main objective of this project is to produce orchids which are resistant / tolerant to insect
pests, mainly mites and thrips.

Approach and Technology
In this project three main technologies were employed and developed. They were:
1. Mutagenesis using gamma irradiation and ion beam - Gamma and ion beam irradiation
technology were used to generate mutant lines with potential resistant to insects.
2. Selection of insect resistant orchids - The techniques of screening were developed inthis study to select plants resistant to insect infestation at both in vitro (tissue culture)
as well as in vivo (flowering) stages.
3. Agrobacterium-mediated genetic transformation was also used and optimized totransfer genes that may confer insect resistance in Dendrobium species/hybrids used inthis study.

Materials and Methods
Plant materials
Irradiated PLBs of Dendrobium Sonia ‘BOM 17 Red’ were obtained from Thailand (courtesyof Associate Professor Dr. Chitrapan Piluek, Kasetsart University, Bangkok), whilst
non-irradiated PLBs of Den. jayakarta was obtained from Indonesia (courtesy of Ms Ismiyati
Sutarto, BATAN, Indonesia). For Den. mirbellianum, PLBs were initiated from mature seedsof self-pollinated flowers. The seed capsules were surface sterilized by dipping them in 90 %ethanol and flaming. They were cut open with sterile tools and the seeds were germinated on
half-strength (½ MS) medium (Murashige and Skoog, 1962) at 26 °C with 16h photoperiod
until PLBs were formed. These PLBs were maintained in culture on ½ MS medium at thesame temperature and photoperiod, and subcultured every 4 weeks on fresh media.

Ion beam irradiation
PLBs of Den. mirbellianum and Den. jayakarta that were uniform in size were transferred into
sterile 6.0 cm Petri dish, covered with a sterile 8 µm-thick polyimide film (KaptonR Du
Pont-Toray, Japan) and sealed with Nescofilm. These PLBs were then irradiated with 220MeV
12

C6+ ion beam at Takasaki Ion Accelerators for Advanced Radiation (TIARA), JAEA, Takasaki

at doses 0, 0.4, 0.8, 1.0 and 2.0 Gy for Den. mirbellianum and 0, 1.0 Gy and 2.0 Gy for Den.
jayakarta. Two to three days after irradiation, PLBs were transferred onto fresh ½ MS medium.
- 17 -

The PLBs were allowed to proliferate and multiply by subculturing onto fresh medium every
four weeks. After four periodic subcultures, the irradiated PLBs were allowed to regenerate
into whole plantlets, transferred into pots and grown to maturity until flowering. For in vitro
insect resistance study, plantlets were placed individually in culture vials.

Gamma irradiation
Dendrobium mirbellianum
Den. mirbellianum plantlets with 3 to 4 expanded leaves
and almost similar in size were placed on petri dishes
containing ½ MS Agar. Each petri dish contains 5
plantlets. They were irradiated at doses of 0, 20, 40, 60,
80, 160, 320, 640 and 1,280 Gy using a gamma chamber(JL Shepherd). Irradiated plantlets were subsequently
removed from agar, cleaned under running tap water andtransferred onto wet tissue in the lab for one week toGamma chamber in Malaysia

allow the plantlets to be hardened before being planted in
the nursery. Assessment on the survival rate of the plantlets was done every week for 4
consecutive weeks.

Dendrobium Sonia ‘BOM 17 Red’
PLBs of Den. Sonia ‘BOM 17 Red’ from Thailand have been irradiated with gamma ray atdoses of 0, 60, 70, 80 and 90 Gy .The PLBs were multiplied, regenerated into complete
plantlets, and transplanted in the glasshouse.

Dendrobium ‘Jayakarta’
Irradiation of Den. jayakarta PLBs using gamma rays (JL Shepherd) was carried out using
two approaches;i.

A single dose of 30 Gy (acute radiation)ii.

Fragmented dose (10 Gy every week for 3 weeks)

The irradiated PLBs were cultured on ½ MS agar. Surviving PLBs were multiplied on thesame medium until the fifth generation. After five periodic subcultures, the irradiated PLBs
were allowed to regenerate into whole plantlets and transferred to glasshouse.

- 18 -

Mites culturing / rearing
Initially, mites were propagated in petri dishes containing clean Arachis pintoi leaves and wet
cotton, to study their morphology and reproductive behavior. They were segregated according
to their life stages; adult, deutonymph, protonymph, larvae and egg. Segregation according tosex was done to adult mites. Female and male adults were allowed to mate and the duration of
the development of different life stages produced were monitored and recorded.

The mites used for screening of irradiated orchid plantlets were later reared and multiplied invitro. Adult mites (Tenuipalpus pacificus) collected from nursery-grown orchid plants wereplaced in tissue culture jars containing Den. mirbellianum plants having at least two open
leaves. The jars were subsequently placed at room temperature (approximately under 10 hours
photoperiod), under rearing temperature at 27 °C and approximately 70 % relative humidity toallow the mites to multiply (Krantz 1978). Adult mites were transferred to new tissue cultured
Den. mirbellianum plants after 2 weeks
In vitro mites infestation and screening
Irradiated orchid plantlets used for in vitro screening were put in individual vials. Plantlets ofabout 4 cm with 4 open leaves were used in this study. Three female and two male adult mites
were then put in the vials containing the plantlets. Observations on the pattern of infestationand the multiplication of mites were made every week according to Zhang (2001). Plantlets
showing no or less symptom of infestation will be isolated and mass propagated.

For symptom analysis, each leaf was divided into five regions (Figure 1). Infestation was
detected using a dissecting microscope. Scoring was done for the first 3 consecutive weeks
and after 9 weeks, by estimating the area of infestation as shown in Figure 1. The infestationlevels were scored as follow; 1: no infestation, 2: less than 5 % infestation, 3: 5 - 10 %infestation, 4: 11 - 25 % infestation and 5: more than 25 % infestation. In cases where theinfestation occurred on different leaves, the areas of infestation were combined to get the total
infestation area.

After 3 months of infestation, surviving plants were transferred into small pots for hardening
and left to grow in the greenhouse until flowering. Fertilizers were applied to the plants for
growth, but not pesticides. Secondary screening at the flowering stage by infestation with thetarget insects were carried out on these potential mutants.
- 19 -

5% infestation

25% infestation

Figure 1 Illustrated scoring index for mite infestation on orchid leaf

In vitro thrips infestation and screening
In vitro screening on irradiated orchids for tolerance towards thrips was carried out using thesame procedure for mite screening (as discussed above). Thrips were collected directly fromheavily infested orchid plants.

In vivo thrips infestation
In vivo thrips infestation studies were carried out on flowering plants in a nursery dedicatedfor insect rearing and screening. Flowering plants of potential insect tolerant mutants
identified at tissue culture stage, as well as randomly selected irradiated plants were placedalongside heavily thrip-infested plants in the nursery. Observations on the flowers were madefor 30 days from the date of infestation. Non-irradiated plants were used as controls.

Orchid transformation and regeneration
Genetic transformation work has been initiated on Den. mirbellinium in 2005, using
Agrobacterium tumefaciens LBA4404 strains, carrying plasmid with a marker gene(hygromycin resistance), a reporter gene (GUS) and a chitinase gene. The Agrobacterium wasmaintained on LB agar plate containing 100 mg/L kanamycin. A single colony was inoculated
overnight in liquid LB medium at 28 °C with 100 mg/L kanamycin and 200 uM acetosyringone.
For transformation, the bacterial pellet was diluted in MS liquid in a ratio of 1:10.
Protocorm-like bodies (PLBs) of Dendrobium mirbellianum were generated as described above.
PLBs were pre-cultured for 5 days on ½ MS agar-solidified medium and incubated at 25 °C ±2 with 16 hours photoperiod. Prior to inoculation, 1 ml of overnight grown Agrobacterium
was centrifuged at 5,000 g and the pellet was dissolved in 10 ml ½ MS liquid medium (1:10
dilution). PLBs were inoculated by immersing in the diluted bacterial solution for 30 minutes- 20 -

with occasional shaking, blot dried and were placed back on ½ MS agar-solidified medium.
The inoculated PLBs were co-cultivated at 25 ± 2 °C in the dark for 3 days to allow thetransfer of genes from Agrobacterium into plant cells.

Following 3 days of co-cultivation, the PLBs were washed with sterile water and transferred
into conical flasks containing ½ MS liquid medium supplemented with 100 mg/L cefotaxime.
The flasks containing PLBs were placed on an orbital shaker for 4 hours to remove traces ofAgrobacterium. Subsequently, the PLBs were collected and blotted dry on sterile tissue paper
before being placed on ½ MS agar-solidified medium containing 100 mg/L cefotaxime for aweek. After one week, the PLBs were transferred again to ½ MS agar-solidified medium
containing 10 mg/L hygromycin for selection. Surviving PLBs were subcultured onto fresh
selective medium every 4 weeks until the formation of plantlets.

Results and Discussion
Orchid tissue culture
Plantlets of all varieties were successfully regenerated and planted in the glasshouse. Of these,
Den. mirbellianum and Den. Jayakarta plants have been flowering, whilst Den. Sonia ‘BOM
17 Red’ plants are still in growing stage and have not flowered.

Ion beam irradiation
Initial radiosensitivity data has shown that the optimum dose for ion beam irradiation oforchid PLBs was in the range of 0.8 to 1.0 Gy (Sakinah et al. 2005).

This data was obtained

through studies on orchid mutagenesis using ion beams under bilateral project between
Nuclear Malaysia and JAEA. Therefore, for Den. mirbellianum, PLBs irradiated at doses of
0.4, 0.8, 1.0 and 2.0 Gy were used for this project, whilst those from other doses will be used
for in vivo screening of other characteristics such as flower color and morphology. For ion
beam irradiated Den. jayakarta, a number of plantlets from all doses (0, 1 and 2 Gy) were also
infested with mites and thrips.

Gamma irradiation
Gamma irradiation of Den. mirbellianum plantlets showed that the plantlets were sensitive
gamma ray above 600 Gy. Most plantlets irradiated below 640 Gy were still viable after 1
week. However after four weeks, those irradiated at higher doses could not survive; i.e only
47.5 % plantlets at 640 Gy were viable and only 2.5% at 1,260 Gy. Table 1 shows detailed- 21 -

results on gamma irradiation of Den. mirbellianum plantlets. Gamma irradiated Den. Sonia‘BOM 17 Red’ and Den. jayakarta were also been planted in the glasshouse and used for invivo screening of insects.

Table 1. Effect of gamma irradiation on the viability of Den. mirbellianum plantlets
Viability after 1 week
Dose (Gy)No. of viable
plantlets%

Viability after 4 weeks
No. of viable
plantlets

%

0

40

100

38

95

20

37

92.5

37

92.5

40

37

92.5

37

92.5

60

38

95

34

85

80

37

92.5

27

67.5

160

38

95

32

80

320

33

82.5

27

67.5

640

30

75

19

47.5

1280

7

17.5

1

2.5

Note: The number of replicates per dose were 40

Mites culturing and rearing
Work on multiplying adult mites in large number has been carried out. It was observed thatthe complete life cycle of mites (from adults to adults) under Malaysian weather wasapproximately 9 weeks. They favour warm to hot dry conditions to multiply. Under normal
environment, a female mite would produce approximately 5 eggs per day. In this study, of thefive mites used to inoculate an in vitro orchid plant, two were male mites. Male mites weremorphologically smaller than female mites.

In vitro screening for mite tolerance
In vitro screening on ion beam irradiated Den. mirbellianum plantlets, has shown that after
one week of inoculation with mites, majority of the tested plants from all doses of irradiation,
showed a low level of infestation. Plants irradiated at doses above 1 Gy in particular, only
showed less than 5% infestation (Table 2). In week 2 and 3, more infestation on the plants’- 22 -

leaves was observed, especially those irradiated at doses below 0.8 Gy (Table 3 and Table 4).
However, there was no significant difference in the number of surviving adult mites, thenumber of eggs and the number of larvae produced after 3 weeks of inoculation on in vitro
plants. This suggested that the irradiated plants did not produce toxin that can kill mites, butrather develop internal resistance to mite infestation. Table 5, 6 and 7 show detailed results on
the number of surviving adult mites, eggs and larvae produced on the irradiated plants tested,
respectively.

Table 2. Severity of infestation after 1 week of inoculation with mites
No. of plants at different infestation scale (%)

Dose

# of

(Gy)

plants

1

2

3

4

5

0

52

43 (82.69)

4 (7.69)

5 (9.61)

0

0

0.4

50

44 (88.0)

5 (10.0)

1 (2.0)

0

0

0.8

57

51 (89.47)

4 (7.02)2 (3.51)

0

0

1.0

26

26 (100)

0

0

0

0

2.0

10

9 (90.0)

1 (10.0)000Note; 1 : no infestation, 2 : < 5% infested leaves, 3 : 5-10% infested leaves, 4 : 11-25% infested leaves
5 : > 25% infested leaves

Table 3. Severity of infestation after 2 weeks of inoculation with mites
No. of plants at different infestation scale (%)

Dose

# of

(Gy)

plants

1

2

3

4

5

0

52

33 (63.46)

6 (11.54)

7 (13.46)

5 (9.62)

1 (1.92)0.4

50

38 (76.0)

1 (2.0)7 (14.0)

4 (8.0)

0

0.8

57

47 (82.46)

3 (5.26)

2 (3.51)

5 (8.77)

0

1.0

26

21 (80.77)

4 (15.38)

1 (3.85)

0

0

2.0

10

7 (70.0)

2 (20.0)

1 (10.0)00Note; 1 : no infestation, 2 : < 5% infested leaves, 3 : 5-10% infested leaves, 4 : 11-25% infested leaves,
5 : > 25% infested leaves- 23 -

Table 4. Severity of infestation after 3 weeks of inoculation with mites
No. of plants at different infestation scale (%)

Dose

# of

(Gy)

plants

1

2

3

4

5

0

52

33 (63.46)

4 (7.69)

5 (9.62)

8 (15.38)

2 (3.85)0.4

50

37 (74.0)

2 (4.0)3 (6.0)3 (6.0)5 (10.0)

0.8

57

45 (78.95)

3 (5.26)

3 (5.26)

4 (7.02)2 (3.51)

1.0

26

21 (80.77)

04 (15.38)

1 (3.85)

0

2.0

10

7 (70.0)

1 (10.0)

2 (20.0)00Note; 1 : no infestation, 2 : < 5% infested leaves, 3 : 5-10% infested leaves 4 : 11-25% infested leaves, 5 : > 25%
infested leaves

Table 5. Number of surviving adult mites after inoculation on in vitro Den. mirbellianum
orchids
Dose (Gy)# of Adult Mites (%)# of inoculated
mites (initial)

Week 1

Week 2

Week 30260

166 (63.85)

162 (62.31)

165 (63.46)

0.4

250

147 (58.8)

146 (58.4)

141 (56.4)

0.8

285

184 (64.56)

182 (63.86)

176 (61.75)

1130

70 (53.85)

68 (52.31)

69 (53.08)

250

32 (64.0)

32 (64.0)

32 (64.0)

Table 6. Number of eggs after inoculation on in vitro Den. mirbellianum orchids
Dose (Gy)# of Eggs (%)# of inoculated
mites (initial)

Week 1

Week 2

Week 30260

93 (35.77)

116 (44.62)

122 (46.92)

0.4

250

87 (34.8)

92 (36.8)

102 (40.8)

0.8

285

54 (18.95)

124 (43.51)

114 (40.0)1

130

61 (46.92)

68 (52.30)

63 (48.46)

250

31 (62.0)

34 (68.0)

34 (68.0)- 24 -

Table 7. Number of larvae after inoculation on in vitro Den. mirbellianum orchids
Dose (Gy)# of Larvae (%)# of inoculated
mites (initial)

Week 1

Week 2

Week 30260

1 (0.38)

13 (5.0)

2 (0.77)

0.4

250

0

6 (2.4)3 (1.2)

0.8

285

0

2 (0.7)8 (2.81)

1

130

0

2 (1.54)

4 (3.08)

2

50

0

0

2 (4.0)The severity of infestation on the infested Den. mirbellianum plantlets was monitored for 9
weeks, or until a complete life cycle of mites (Table 8). At this stage, it was also observed that
all non-irradiated plants tested showed a minimum 5 % of the infestation symptom on theirleaves. Some of these control plantlets were also severely damaged and died. Irradiatedplantlets did also exhibit infestation symptoms at various scales, ranging from very mild toheavily infested, but there were also plantlets that showed no sign of infestation andcompletely healthy. The percentages of non-infested plantlets were gradually increased with
the increase in treated irradiation doses. The percentages of non-infested plantlets were 34 %,24.56 %, 50 % and 60 % for those irradiated at 0.4, 0.8, 1.0 and 2.0 Gy, respectively. These
results showed an increase in resistance towards mite infestation on irradiated seedlings.

Table 8. Severity of infestation after 9 weeks of infestation with mites
No. of plants at different infestation scale (%)

Dose

# of

(Gy)

plants

1

2

3

4

5

0

52

0 (0)

0 (0)

0 (0)

9 (17.31)

43 (82.69)

0.4

50

17 (34.0)

5 (10.0)

2 (4.0)1 (2.0))25 (50.0)

0.8

57

14 (24.56)

2 (3.51)

1 (1.75)

6 (10.53)

34 (59.65)

1.0

26

13 (50.0)

2 (7.69)

2 (7.69)

2 (7.69)

7 (26.92)

2.0

10

6 (60.0)

1 (10.0)

1 (10.0)

1 (10.0)

1 (10.0)

Note; 1 : no infestation, 2 : < 5% infested leaves, 3 : 5-10% infested leaves 4 : 11-25% infested leaves,
5 : > 25% infested leaves- 25 -

Overall, 50 potential Den. mirbellianum mutants were identified as tolerant to mite infestationand successfully planted in the glasshouse. Of these, 11 plants were from 0.4 Gy, 31 from 0.8
Gy and 8 from 1.0 Gy. These plants were further propagated in the glasshouse for subsequent
screening at flowering stage. Figure 3 shows the chronology for producing mite tolerant Den.
mirbellianum.

Figure 3. Development of potential mite resistant Den. mirbellianum mutants
A: Orchids PLBs used for irradiation

B: Ion beam irradiation facility, JAEA, Japan

C: Individual plantlets in vials for in vitro challenge-infestation
D: Infesting pest (mites)

E: Symptom of infestation on orchid leaf

F: Potential mite resistant orchid mutants in glasshouse

In vitro thrips infestation
Thrips have been known to cause injury to leaves and buds in infested orchid plants. Amongthe symptoms observed are browning or blotching of the leaves and bud destruction(http:www.ct.gov/caes/cwp/view.asp). Another symptom is severe flower break as a result ofvirus infestation transmitted by thrips (Pataky 1990).

An experiment was carried out to screen in vitro irradiated plantlets against thrips. Due to alimited number of thrips, only a small number of irradiated plants were used in thisexperiment. Table 9 and Table 10 show detailed results on the number of infested plants andthe severity of infestation with thrips, after week one and two, respectively.
- 26 -

Based on this work, thrips were seen to cause rapid and severe damages on the infested plants
as compared to mites. After only 2 weeks of infestation, all control or non-irradiated plants
showed symptoms of infestation. Of these, 100 % of Den. mirbellianum and 83.3 % of Den.
jayakarta, exhibited severe damage on more than 25 % of their leaf areas. A number ofirradiated Den. jayakarta and Den. mirbellianum plantlets displayed early tolerance to these
infestations. Overall from the in vitro screening, 2 Den. mirbellianum and 5 Den. jayakarta
plants were selected as potential tolerant to thrips. Figure 4 shows some infestation symptoms
on in vitro plantlets infested with thrips.

Table 9. Severity of infestation after one week of infestation with thrips
No. of Infested plants

Orchid species# ofDose (Gy)plants

1

2

3

4

5

0

5

3

2

0

0

0

0.4

5

5

0

0

0

0

0.8

5

3

2

0

0

0

1.0

5

5

0

0

0

0

0

6

1

1

4

0

0

10

5

5

0

0

0

0

30

5

4

1

0

0

0

1.0

5

3

1

1

0

0

2.0

5

2

3

0

0

0

Den. mirbellianum (gamma irradiated)

Den. jayakarta (gamma irradiated)

Den. jayakarta (ion beam)

Note; 1 : no infestation, 2 : < 5% infested leaves, 3 : 5-10% infested leaves 4 : 11-25% infested leaves,
5 : > 25% infested leaves- 27 -

Table 10. Severity of infestation after 2 weeks of infestation with thrips
No. of Infested plants

Orchid species# ofDose (Gy)plants

1

2

3

4

5

0

5

0

0

0

0

5

0.4

5

2

2

1

0

0

0.8

5

0

2

0

3

0

1.0

5

0

3

0

2

0

0

6

0

1

0

0

5

10

5

2

0

1

0

2

30

5

1

1

1

0

2

1.0

5

1

1

0

1

2

2.0

5

1

0

1

1

2

Den. mirbellianum (ion beams)

Den. jayakarta (gamma irradiated)

Den. jayakarta (ion beam)

Note; 1 : no infestation, 2 : < 5% infested leaves, 3 : 5-10% infested leaves 4 : 11-25% infested leaves,
5 : > 25% infested leaves

B

A

Figure 4.Thrips infestation symptoms on in vitro orchid leaves after 1 week (A) and 2 weeks (B)Potential mutant lines from in vitro screening
Den. mirbellianum
Based on in vitro mite screening tests, 50 Den. mirbelliannum lines, which were irradiatedwith ion beams, have been identified as potential mite tolerant mutants. These lines werealready planted in the glasshouse. Of these, 11 plants are from 0.4 Gy, 31 from 0.8 Gy and 8
from 1.0 Gy. From in vitro thrips screening, 2 plants were found tolerant.

- 28 -

Den. jayakarta
Based on in vitro thrips screening, 3 Den. jayakarta plants irradiated with gamma rays wereidentified as potential thrip tolerant, whereas 2 plants irradiated with ion beams were tolerant.

In vivo thrips screening
After 3 months of infestation, healthy and surviving plants were transferred into small pots for
hardening. Subsequently they were allowed to grow under shade in the greenhouse. The
plants were regularly sprayed with fertilizers for growth but were not sprayed with any
pesticides. In nature, expressions of disease or infestation symptoms in plants are influenced
by a combination of factors such as susceptibility of the host, infective pathogen andfavourable environmental conditions (Singh & Singh, 2005). Therefore, these potential
mutant plants were also screened at flowering stage by challenge-infestation with the target
insects, since insects like thrips especially feed on flowers. It was also a way to confirm thestability of the trait in natural growing conditions.

To date, the tests were carried out on irradiated Den. jayakarta plants and potential mite
resistant mutants of Den. mirbellianum only, since Den. Sonia ‘BOM 17 Red’ have notflowered yet. Infestation severity index was developed based on the level of damages causedby thrips to these flowers. The scale was between 1 and 5, where 1 was resistant and 5 wastotally susceptible (Figure 5).

1

3

2

4

5

- 29 -

Based on the in vivo thrips screening, all samples of Den. jayakarta plants from the control
and a fragmented dose (gamma, 3 x 10 Gy) populations, were found to be susceptible toinsects. Two lines from a single dose (gamma, 30 Gy) population exhibited minor damages(scale 2) on their flowers, and thus could be considered potential resistant lines. These plants
have been tagged and maintained in the glasshouse.

For mite tolerant Den. mirbellianum plants, the same index was applied and one plant was
found tolerant to thrips. All the thrips and mite tolerant mutants are now being propagated in
the nursery, to achieve large number of clones.
Genetic transformation
The objective of this genetic transformation work was to study the possibility of generatinginsect resistant plants through the insertion (transformation) of chitinase gene. From theexperiments on Den. mirbellianum using A. tumefaciens carrying a chitinase gene, 5.59% of
the putatively transformed PLBs were obtained after the first selection on hygromycin
selection media. These PLBs were transferred to fresh selection media to let them toproliferate. The PLB-to plant conversion was also carried on the same selection media. A totalof 13 hygromycin resistant plants have been successfully achieved. At present, the plants arebeing maintained, and further screening and evaluation will be carried out in the transgenic
facility, which is currently under construction.

Conclusion
Some observations and findings of this project were;
1. The optimum ion beam irradiation dose for PLBs was 0.8 - 1.0 Gy.
2. The method for in vitro rearing of mites was established and from this study, thecomplete life cycle of mites in Malaysian conditions was found to be 9 weeks.
3. An in vitro insect infestation procedure was developed for preliminary selection of
insect tolerant mutants at tissue culture stage.
4. In vitro mite infestation study on regenerated Den. mirbellianum orchids has showed
an increase in resistance towards mite infestation on ion beam irradiated plantlets. The
percentages of mite resistant mutant plantlets gradually increased with increasing
irradiation doses.
5. Through in vitro mite infestation study, a total of 50 potential mite tolerant plantlets- 30 -

were identified. Of these 50 plants, one plant was found tolerant to thrips whensecondary screening was carried out at flowering stage (in vivo).
6. Through in vitro infestation with thrips, 2 Den. mirbellianum plantlets and 5 Den.
jayakarta plantlets were tolerant. These plants are being propagated in the glasshouse,and secondary screening for thrip tolerance will be carried out once the plants areflowered.
7. In vivo thrip screening at flowering stage have identified 2 clones of Den. jayakarta
which were tolerant to this insect.
8. All identified mutants are currently being propagated to achieve large numbers ofclones, and will be transferred to a private collaborator for pre-commercialization
studies.
9. Genetic transformation study on Den. mirbellianum has generated a number ofputative transgenic plants which will be screened and evaluated in the transgenic
facility.

Acknowledgement
The authors would like to thank Mr. Shuhaimi Shamsudin, Mr. Suhaimi Musa and Ms. Zainab
Harun for technical assistance, JAEA counterparts; Dr. Atsushi Tanaka, Dr. N. Shikazono, Dr.
Yutaka Oono, and Dr. N. Hase, for ion beam irradiation and technical advice, Assoc. Prof. Dr.
Chitrapan Piluek of Kasetsart University for supplying irradiated Den. Sonia ‘BOM 17 Red’
PLBs and Ms. Ismiyati Sutarto of BATAN for supplying Den. jayakarta PLBs. We are also
grateful to Research Management Centre, Malaysian Nuclear Agency for providing financial
support.

References
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mechanism of insect resistance engineered into tobacco. Nature 330, 160-163
Krantz G.W (1978) A manual of Acarology. Oregon Sate University Book Store. Corvallis,
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Murashige,T. & Skoog, F.,A (1962) revised medium for rapid growth and bioassays with- 31 -

tobacco tissue cultures Physiol. Plant. 15,473-497
Gough, N. (1988) Mites: Their biology and control on ornamental plants. Queensland
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Knapp, J.E., Kausch, A.P. & Chandlee, J.M. (2000) Transformation of three genera of orchid
using the bar gene as a selectable marker. Plant Cell reports. 19, 893-898
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Sciences. University of Illinois at Urbana-Champaign
Perlac, F.J., Fuchs, R.L., Dean, D.A., McPherson, S.L. & Fischoff, D.A. (1991) Modificationof coding sequence enhances plant expression of insect control protein gene. Proc.
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Singh, D. P. & Singh, A. (2005) Disease and Insect Resistance in Plants. Science Publishers,
Enfield, USA. pp 396
Smigocki, A.C., Neal, J.W., McCanna, I. & Douglas, L. (1993) Cytokinin-mediated insect
resistance in Nicotiana plants transformed with ipt gene. Plant Mol. Biol. 23, 325-335
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Zaharah Hassan & Rozlaily Zainol (1991) Orchids Planting. Publish by Malaysian
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Auckland, New Zealand. 34pp

- 32 -

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 -

This report was prepared by the Nuclear Safety Research Association (NSRA) as a part ofactivities on Forum for Nuclear Cooperation in Asia (FNCA), organized by MEXT.