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.

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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.

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