GAMMA RADIOSENSITIVITY STUDY ON RICE

(Oryza sativa)

NURUL NADIA BINTI JAAFAR

2017299046

DEPARTMENT OF AGRO-TECHNOLOGY AND BIOSCIENCE DENGKIL

COMPLEX

MALAYSIAN NUCLEAR AGENCY

MR. FAIZ BIN AHMAD

22th JANUARY 2019 – 15th MARCH 2019

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 ACKNOWLEDGEMENT

First of all, I would like to express my gratitude to Allah S.W.T for giving me the
blessings in completing this industrial training program. I would like to dedicate special thanks
to both of my parents, Encik Jaafar bin Ahmad and Puan Junaidah binti Omar for providing
support morally, financially and giving full encouragement to complete my industrial training
program. Thousands of thanks also I want to express towards my industrial training supervisor,
Encik Faiz bin Ahmad for sharing his valuable knowledge and guiding me throughout my
industrial training program. Not to forget, I would also like to thank other research officers such
as Dr Zaiton, Puan Norazlina, and Malaysian Nuclear Agency staffs such as Encik Ruzaini and
Puan Anis for the knowledge, experiences and help. Last but not least, I would like thank my
fellow friends Nurul Shafika, Muhammad Syukri, Sharmin Balqis, Ain Nur Najwa, Nurfarahin,
Nur Syafiqah and Nor Azah who really helped through the process of learning.

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Table of Contents
1.0 INTRODUCTION ................................................................................................................................. 5
1.1 Malaysia Nuclear Agency .............................................................................................................. 5
1.2 Vision and Mission ........................................................................................................................ 6
1.3 Slogan ............................................................................................................................................ 6
1.4 Objectives...................................................................................................................................... 6
1.5 Agrotechnology and Biosciences Division ................................................................................... 7
2.0 Literature Review.................................................................................................................................... 8
2.1 Rice ...................................................................................................................................................... 8
2.2 Mutation Breeding .............................................................................................................................. 8
2.3 Radiosenstivity test ............................................................................................................................. 8
3.0 Problem Statement ................................................................................................................................. 9
4.0 Objectives................................................................................................................................................ 9
5.0 Methodology......................................................................................................................................... 10
5.1 Acute Gamma Irradiation.................................................................................................................. 10
5.1.1 Plant Material............................................................................................................................. 10
5.1.2 Seed Irradiation.......................................................................................................................... 10
Materials and apparatus: ..................................................................................................................... 10
1. MR 284 seeds.............................................................................................................................. 10
5.1.3 Fungicide Application ................................................................................................................. 11
5.1.4 Sandwich Blotting Technique..................................................................................................... 12
5.1.5 Data collection ........................................................................................................................... 13
5.2 Chronic Gamma Irradiation............................................................................................................... 13
5.2.1 Soil preparation.......................................................................................................................... 13
5.2.2 Germination of rice seeds .......................................................................................................... 14
5.2.3 Seedlings planting ...................................................................................................................... 15
5.2.4 Data Collection .......................................................................................................................... 16
5.3 Experimental Design ......................................................................................................................... 16
6.0 Results and Discussion .................................................................................................................... 17
6.1 Acute Gamma Irradiation.................................................................................................................. 17

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 6.3 Chronic Gamma Irradiation......................................................................................................... 20
6.4 LD50 and LD20................................................................................................................................ 22
7.0 Conclusion ............................................................................................................................................. 23
8.0 References ............................................................................................................................................ 24
Other Activities ........................................................................................................................................... 25

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1.0 INTRODUCTION

1.1 Malaysia Nuclear Agency

Malaysia Nuclear Agency is a government institution that has a role to introduce and promote
application of nuclear science and technology for national development. Malaysia Nuclear
Agency was established in 19 September 1972 and it was known as Centre of Application
Nuclear Malaysia (CRANE) which was changed to Tun Ismail Atomic Research Centre
(PUSPATI). It was placed under patronage of Prime Minister Department and was called as
Nuclear Energy Unit (UTN) in June 1983. Then, in October 1990, it was placed under the
Ministry of Science, Technology and Environment. Later on the name was changed to Malaysia
Institute for Nuclear Technology Research (MINT) in August 1994. On 28 September, MINT
was given a new identity – Malaysia Nuclear Agency (Nuclear Malaysia). Its strategic location,
near higher learning institutions, besides its close proximity to the National Administration
Centre, Putrajaya and The Multimedia Super Corridor, Cyberjaya has stimulated Nuclear
Malaysia to meet its aspiration.

Malaysia Nuclear Agency (Nuclear Malaysia) is a reputable agency in research and

development (R&D) in science and technology. Its presence is synonymous with the
development, augmentation and advancement of nuclear science and technology in Malaysia.
From the time when it was established, Nuclear Malaysia has been given the responsibility to
introduce and promote nuclear science and technology for national development and until
present, Nuclear Malaysia maintains its significance as an outstanding organization in science,
technology and innovation.

Nuclear Malaysia also plays a pivotal role in providing quality and best-in-class research
towards comprehensively generating new technologies to meet the needs of nuclear technology’s
variety of application. This standing is acquired through professional workforce training and
discipline, well planned infrastructure and well-resources laboratories.

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1.2 Vision and Mission

Vision for Malaysia Nuclear Agency is Nuclear Science and Technology for Knowledge
Generation, Wealth Creation and Societal and National Well-being.

The mission is excellence in Research and Applications of Nuclear Technology for

sustainable development.

1.3 Slogan

Nuclear Technology propels the Nation Vision.

1.4 Objectives

1. To generate new products and technologies through research and innovation based on the
national development agenda.
2. To achieve an income, at minimum 30% of the annual operating budget, through transfer and
commercialization of technology.
3. To enhance organizational excellence through planning and quality management.

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1.5 Agrotechnology and Biosciences Division

Agrotechnology and Biosciences Division is one of the divisions under Research and
Technology Development Program which is located in Dengkil. It consists of three research
groups which cover the aspects of crop improvement, agroecosystem management and radiation
bioindustry. This division focused on research in agriculture and bioindustry. As a part of the
country premier nuclear center, this division is in full advantage of harnessing the nuclear
knowledge in agro and bio resources areas. Supported by qualifies researches and conducive
research environment, this division is capable to produce, evaluate and adopted the research
findings to the customers. Some of the products offered by this division include; banana tissue
culture seedlings, Kacip Fatimah, Tongkat Ali and Ginseng culture roots. It is also includes new
ornamental varieties seedlings, pesticide residue analysis and assessment of fertilizer efficiency
for smart agro management.

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2.0 Literature Review

2.1 Rice

Oryza sativa (O. sativa) and Oryza glabberima are the two species that have emerged as most
cultivated rice and O. sativa is the most widely produced. Rice is the principle staple crop of
Asia and any deterioration of rice production systems through climate change would seriously
impair food security in this continent (Wassmann R., 2009). Due to unstable grain production,
research on upland rice has been ignored though it is widely grown in the inner part of country (Aziliana,
2015).

2.2 Mutation Breeding

Nowadays, mutation breeding is a technique that is used to create new varieties by generating
and utilizing genetic variability through chemical and physical mutagenesis (Shu, 2012). In order
to obtain desirable traits in cultivated rice and determining the most effective mutation treatment,
bunch of attempts in mutation field research have been done by scientists (Sasikala, 2010).

2.3 Radiosenstivity test

The existence genetic varieties with useful traits are needed for plant breeding in improving
crops. However, the traits that always been desired always lacking. Nevertheless, radiation is
useful in prompt mutations and produce genetic variation by selecting from desired mutants.
Mutation induction is creating variation by selecting from crop varieties (Brunner, 1995)

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3.0 Problem Statement

In Malaysia, lack in rice variety that resists to bacterial leaf blight (BLB) disease is one
the major problem that constraint rice production. MR 284, the rice variety that is used in this
project is a high yielding variety but does not resists to BLB disease. Therefore, in order to
overcome this problem, mutation breeding acts as an alternative to improve the resistant to BLB
disease. Other than that, no data base on gamma radiosensitivity for MR 284 variety. That is why
this project was carried out in order to find the gamma radiosensitivity for MR 284 variety.

4.0 Objectives

1. To determine the optimum gamma irradiation dose (LD50 and LD20) for MR284 variety.
2. To determine the effect of gamma rays on morphological characteristic of paddy seedling
derived from irradiated seeds.

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5.0 Methodology

5.1 Acute Gamma Irradiation

5.1.1 Plant Material

Genotype Origin Characteristic

MR 284 Malaysia High yield

5.1.2 Seed Irradiation

Materials and apparatus:

1. MR 284 seeds
2. 8 petri dishes
3. Gamma-cell Biobeam Machine

The rice seeds were irradiated to the acute gamma irradiation using instrument called
Gamma-cell Biobeam Machine which used Caesium-137 as its source. 240 rice seeds
were placed in 8 petri dishes, 30 rice seeds each. The petri dishes were labeled with 0 Gy,
100 Gy, 200 Gy, 300 Gy, 400 Gy, 600 Gy, 800 Gy and 1000 Gy. The petri dishes were
positioned in between empty petri dishes and placed inside a BB13-5 irradiation beaker.
The Fricke dosimeter was placed in one of the empty petri dishes to determine the
accurate dose of irradiation that have been introduced to the rice seeds sample. The
higher the doses, the longer it will take to irradiate the rice seeds.

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 Petri dish contained seeds placed in container
Biobeam machine

5.1.3 Fungicide Application

Materials and apparatus:

1. Fungicide solution
2. Irradiated rice seeds
3. Tray

The fungicide solution was poured into petri dishes that contained irradiated rice
seeds to prevent the growth of fungi. The fungicide was poured until all rice seeds were
soaked in it. The rice seeds were left in a tray at a dark place for 24 hours.

Rice seeds soaked in fungicide Soaked rice seeds placed in dark place

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5.1.4 Sandwich Blotting Technique

Materials and apparatus:

1. Rice seeds (soaked in fungicide)

2. Blotter paper
3. Rack
4. Tray
5. Distilled water
6. Germination chamber

On wet blotter paper, 10 rice seeds (for each replication based on doses) were laid vertically
with embryo facing downwards. Another blotter paper covered the rice seeds. After the rice seeds
have been sealed with distilled water, the sandwich blotter was placed on a rack. The racks that
held the blotter paper were placed in a plastic tray. The third quarter of the plastic tray was filled
with water. The tray that contained the whole sample was placed in a germination chamber.

Sandwich blotter placed in germination chamber

Sandwich blotter technique

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 5.1.5 Data collection

Material and apparatus:

1. Ruler

On the 7th day, the shoots and roots for the seedlings were measured using a ruler. The data
collected were recorded. The data collection was done again on the 14th day.

Measuring seedlings on ruler

5.2 Chronic Gamma Irradiation

5.2.1 Soil preparation

Materials and apparatus:

1. 63 pots
2. Top soil

63 pots that came with no holes were obtained. The top soil were filled the pots until
it filled three quarters of the pots.

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 Soil preparation

5.2.2 Germination of rice seeds

Materials and apparatus:

1. 2 petri dishes
2. 80 rice seeds (MR 284)
3. Filter paper
4. Distilled water
5. Tray
6. Top soil

Filter papers were placed inside the petri dish and wet it with distilled water. The
seeds were placed on the wet filter papers. 40 seeds were placed in each petri dish. The
petri dishes were left in a dark place for a week. This germination test was carried out to
see the rate of germination or rice seeds whether it is acceptable or not (above 90%).

After a week, the seedlings were transferred into trays that have been filled with top
soil. The seedlings were planted with a distance between each other. The trays were filled
with water until the water level a bit higher than the soil surface. This method was done
to establish the seedlings roots.

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 Germination after 1 week
Germinate seeds in tray

5.2.3 Seedlings planting

Materials and apparatus:

1. 63 pots filled with top soil

Seedlings that have been germinated were transferred into pots. By making a hole
in soil, only a sprig of seedling was planted in each pot. 9 pots were placed in paddy
house as the seedlings were treated as control. Other 54 pots were transferred to Gamma
Green House (GGH) and placed at different rings, Ring 2, Ring 3, Ring 4, Ring 6, Ring 8
and Ring 10. The plants were always ensured soaked in water as the paddy plants need a
lot of water to grow.

Control treatment in paddy house

Plants in Gamma Green House 15
 5.2.4 Data Collection
Materials and apparatus:

1. Measuring tape

The plants height was measured on 7th, 14th and 21st day. The plant height were
measured by placed the measuring tape on the soil surface and measure the highest leaf
on the plant. The data collected was recorded.

Collecting data

5.3 Experimental Design

For acute gamma irradiation, 240 seeds of MR 284 variety were treated with eight
different doses which were 0 Gy, 100 Gy, 200 Gy, 300 Gy, 400 Gy, 600 Gy, 800 Gy and 1000
Gy. Each dose has three replications, 10 rice seeds per each replication.

For chronic gamma irradiation, 63 seedlings were placed at different ring in Gamma
Green House. 9 plants were placed at paddy house to be treated as control. Another 54 seedlings
were placed at Ring 2, Ring 3, Ring 4, Ring 6, Ring 8 and Ring 10 with 9 plants for each ring.

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6.0 Results and Discussion

6.1 Acute Gamma Irradiation

Dose Shoot length (cm) Root length (cm)

(Gy) 7th day 14th day 7th day 14th day
0 7.85 18.23 7.19 10.23
100 7.15 17.96 6.58 9.60
200 6.7 16.54 6.25 8.86
300 4.51 13.29 4.75 8.55
400 4.03 9.64 2.95 9.62
600 0.64 0.61 0.61 2.66
800 0.5 0.49 0.34 1.33
1000 0.51 0.57 0.17 0.70
Dose Shoot length (cm) Root length (cm)
(Gy) 7th day 14th day 7th day 14th day
0 7.85 18.23 7.19 10.23
100 7.15 17.96 6.58 9.60
200 6.7 16.54 6.25 8.86
300 4.51 13.29 4.75 8.55
400 4.03 9.64 2.95 9.62
600 0.64 0.61 0.61 2.66
800 0.5 0.49 0.34 1.33
1000 0.51 0.57 0.17 0.70

Table 1: Results of Sandwich Blotter Technique

Length (cm)
Shoot length 7th day
20
14th day

15 Linear (7th
day)
17
10

5
 Length
Root length
12

10

8
7th day

6 14th day
Linear (7th day)
4
Linear (14th day)
2

0 Dose (Gy)
0 200 400 600 800 1000 1200

Graph 2: Root length on 7th and 14th day

Based on the Graph 1, the shoot length keep on decreasing as the dose keep increasing.
This trend might be due to the high amount of irradiation that caused injuries to the plant cell and
DNA thus, shoots are stunted. The control treatment has the highest shoot which is 18.23 cm
while 1000 Gy treatment does not even have a shoot.

According to Graph 2, the root length also decreasing as the dose is increasing. However,
at the 400 Gy the root length increase a bit but drop again at the 600 Gy dose. The control
treatment has the longest root length which is 10.23 cm and the shortest roots found at 1000 Gy
dose which is 0.70 cm.

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Sandwich blotter results

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6.3 Chronic Gamma Irradiation

Treatment Height (cm) Dose (Gy) Height (cm) Dose (Gy) Height (cm)
0.00 26.96 0.00 37.87 0.00 62.78
2.70 25.83 5.68 32.53 8.42 39.67
3.60 26.13 7.58 36.00 11.22 51.44
6.30 25.67 13.26 32.83 19.64 35.78
15.30 24.53 32.21 36.17 47.69 52.20
27.00 24.84 56.84 37.28 84.15 53.61
60.29 24.64 126.93 32.72 187.94 47.39
th th st
Table 2: Plant height at 7 day Table 3: Plant height at 14 day Table 4: Plant height at 21 day

Height (cm)
Plant height (cm)
27.5

27

26.5
Plant height (cm)
26

25.5 Linear (Plant

25 height (cm))

24.5
Accumulated Dose (Gy)
24
0.00 20.00 40.00 60.00 80.00

Graph 3: Plant height at 7th day

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 Height (cm) Plant height (cm)
39

38

37 Plant
height (cm)
36

35

34

33

32 Accumulated Dose (Gy)

0 20 40 60 80 100 120 140

Graph 4: Plant height at 14th day

Height (cm) Plant height (cm)

70.00

60.00
Height
50.00 (cm)

40.00
Linear
30.00 (Height
(cm))
20.00

10.00

0.00 Accumulated Dose (Gy)

0.00 50.00 100.00 150.00 200.00

Graph 5: Plant height at 21st day

Based on the graphs, there are fluctuations in the plant heights. The accumulated doses
received at each ring are different and keep on increasing from 7th day to 21st day. From control
treatment to Ring 10, the plant height is decreasing but it increase at Ring 8. The plant height
decrease again at Ring 6 but increase once again at Ring 4 and Ring 3 on 14th and 21st day. Ring
2 shows decrease in plant height at all three weeks. Significant decreasing in plant height cannot
be shown as the data observed only 21 days. The data should be observed for more than 60 days.

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 Results for chronic gamma irradiation

6.4 LD50 and LD20

Graph 6: LD20 and LD50 for shoot length of acute gamma irradiation

Based on shoot length of acute gamma irradiation, both LD20 and LD50 can be
determined. LD20 is 257.46 Gy and LD50 390.74 Gy. However, LD20 and LD50 cannot be
determined as the data observed only for 21 days. Normally, optimum dose to create higher
percentage of mutant variety is in between range of LD20 and LD50.

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7.0 Conclusion

The LD20 for shoot length of acute gamma irradiation is 257.46 Gy and LD50 is 390.74 Gy. The
optimum dose range for MR 284 variety is between LD20 and LD50. As the gamma irradiation
does increase, the plants morphological traits such as shoots length, roots length and plant height
decrease. For acute irradiation, plants treated with more than 400 Gy of dose were stunted.

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8.0 References

Aziliana, M. S. (2015). The Effect of Chronic Gamma Irradiation on Malaysia Upland Rice (Oryza sativa)
Kuku Belang. Australian Journal of Basic and Applied Sciences, 9 (31): 1-6.

Brunner, H. (1995). Radiation induced mutations for plant selection. . Applied Radiation and Isotopes,
46(6-7), 589-594.

Sasikala, R. K. (2010). Sensitivity of rice varieties to gamma irradiation . Electronic Journal of Plant
Breeding, 1(4): 885-889.

Shu, Q. Y. (2012). Plant Mutation Breeding and Biotechnology. CAB International and FAO.

Wassmann R., J. S. (2009). Regional vulnerability of climate change impacts on Asian rice production and
scope for adaptation. Advances in Agronomy, 102, 91-133.

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Other Activities

Collecting data for paddy Field trip to Banghuris, Sepang

Collecting data for taro Surface sterilization

Radiosensitivity test for sorghum

25