6. Malaysia

Application of Mutation Techniques and Biotechnology for Minimal Water
Requirement and Improvement of Amylose Content in Rice
1

Rusli Ibrahim, 1Abdul Rahim Harun, 1Sobri Hussein, 2Abdullah Mat Zin, 3Sariam Othman, 4Marziah

Mahmud, 4Mohd Rafii Yusof, 1Siti Hajar Mohd Nahar, 4 Zarifth Shafika Kamaruddin, 5Ana Ling P.K
1

Agrotechnology & Biosciences Division, Malaysian Nuclear Agency, Ministry of Science,

Technology & Innovation (MOSTI), Bangi, Selangor; 2Universiti Malaysia Terengganu, 21030 Kuala
Terengganu; 3 Malaysian Agriculture Research and Development Institute (MARDI), Serdang,Selangor; 4Universiti Putra Malaysia (UPM), Serdang, Selangor; 5Department of Bioscience, Facultyof Engineering and Science, University Tunku Abdul Rahman, Kuala Lumpur
6.1

Introduction

Rice is the staple food crop in Malaysia. Malaysia managed to achieve 72% self-sufficiency level inrice with the current average rice yield of 3.7t/ha/season. In this situation, about 28% of the local
demand will have to depend on rice imports. In Peninsula Malaysia, rice production depends largelyon the irrigated lowland production system. Currently there are 241,741ha of irrigated rice inPeninsula Malaysia which contributes more than 85% of the national rice production. However,growing irrigated rice requires large amount of water. It was estimated that about 3,000 liters ofwater are required to produce 1kg of rice. Unfortunately, there are signs of declining water supply
that threatens the sustainability of irrigated rice production.
The largest water withdrawal (more than 75%) in Malaysia is for irrigation in the agriculture sector
and is mainly confined to irrigated rice production. However, the future agriculture’s share of water
will decline because of increasing competition for available water from urban and industrial sectors.
Water is becoming a scarce entity in the future and in some areas, it has become a limiting factor inrice production (Bhuiyan, 1992). Thus, rice will be the crop most affected by water scarcity as it
depends most heavily on irrigation. Therefore, there is an urgent need to develop rice varieties
adapted to water stress and still produce acceptable yield and posses all the other beneficial traits.
Water supply is affected by the loss of watersheds due to deforestration and soil erosion. There issevere depletion of valuable groundwater resources as water is taken up for agricultural andindustrial purposes. Some varieties of rice can be grown without irrigation, however their growth
totally depend. About 15% of the rice varieties in Sabah and Sarawak are rainfed. Rainfed rice
varieties are lower in yield due to uncertainty of water supply. In view of this water shortage, there isan urgent need to develop new varieties of rice with high yield potential and stability under water
stress conditions. Therefore, in an attempt to develop water stress tolerance lines, morphological andagronomic traits that are related to water stress condition, can be useful in establishing a successful
breeding program for rice improvement.
Induced mutation is an important supplementary approach to plant breeding, particularly when it isdesired to improve one or two easily identifiable traits in an otherwise good lines or varieties. The
successful use of plant breeding for improving crops requires the existence of genetic variation of
useful traits. Unfortunately, the desired variation is often lacking. However, radiation can be used toinduce mutations and thereby generate genetic variation from which desired mutants may be selected.
Mutation induction has become a proven way of creating variation within a crop variety. It offers the- 46 -

possibility of inducing desired attributes that either cannot be expressed in nature or have been lost
during evolution.
Heavy ion beams, such as carbon ions, are more effective in plant for inducing mutations comparedwith electron beam (Hidema et al., 2003). Novel mutant have been obtained by the carbon ion
irradiation in several plant species (Hase et al., 2000). To date, a number of rice mutants with various
significant improvements in morphological traits have been recommended for planting (Mohamed etal., 1988) but none was targeted for adaptive traits per se. Water shortage has become the bottleneckof Malaysia’s food security. The development of water-saving rice varieties to decrease water
consumption in rice production is inevitably a major goal in agriculture research. Thus, to achieve
long-termfoodsecurityandsustainable

development

in

Malaysia,‘Water-savingordrought-resistance’ (WDR) rice varieties are urgently needed.
Drought is one of the most important limiting factors in more than 65% of paddy fields in Malaysia
where super rice varieties cannot perform well under drought stress. Therefore, the development andproduction of drought-tolerant rice varieties, to stabilize and improve the production levels in the
low-middle-yielding fields, is needed. In recent years, the field drought-resistance screening facility
was established through mutation breeding program and the evaluation standard was developed.
Some advanced lines of drought tolerance rice varieties were identified and will be used in both
molecular mapping and breeding programmes. The objectives of this study, therefore, were to screen
for mutant lines derived from gamma rays and ion beam irradiation for high yield potential andstability under water stress conditions with improved quality trait such as amylose content.
6.2

Materials and Methods

6.2.1 Mutation Induction
Seeds of popular local variety MR219 were irradiated with 300Gy of gamma radiation from the60

Cobalt source at Malaysian Nuclear Agency in 2008. Irradiated seeds were sown at Mardi Research

Station in TanjungKarang, Selangor. The M1 seedlings were transplanted into the field with 25cm x
25cm planting distance. Ten thousand M1 seedlings were planted to produce M 2 seeds and a total of
5,250 plants were selected from which 2 panicles per hill were randomly harvested from each hill.
For ion beam irradiation, four rice cultivar, MR219, Q74, MR211 and PongsuSeribu 2 were irradiatedin Takasaki Japan. MR219 and Q74 were irradiated on 20 th Jan 2009 for sensitivity test. A total
number of 100 seeds per dose (0, 10, 20, 40, 60, 80, 100, 120, 160, 200 Gy) of MR219 and MRQ74
were irradiated with ion beam in Takasaki Japan. The seeds were evaluated at Malaysian Nuclear
Agency. The shoulder dose for MRQ74 was observed at 120Gy. The optimum doses of these cultivars
were identified and the second irradiation was done on 16 Jun 2009 whereby 1,500 seeds of each
cultivar were irradiated with doses of 60 and 80Gy. Meanwhile Seeds of MR211 and PongsuSeribu
were irradiated on 5th Feb. 2010 for sensitivity test.
A total of 1,500 M1 seeds of MR219 and MRQ74 irradiated with 60 and 80Gy were raised at
Malaysian Nuclear Agency green house. Only M2 seeds of MR219 with 80Gy were planted inAgriculture Department experimental Plot, TgKarang Selangor. About 10,000 of M2 individual plantsof MR219 were planted. About 5% of M2 populations were selected for further screening in M 3
including analysis of amylose content.

- 47 -

6.2.2 Green-house screening
Green-house screening was done using cement troughs filled with paddy soils. A water float control
was constructed to control the level of water from time to time by adjusting it in order to attain thesaturated water regime as require in the study. M 2 seeds of MR219 were hand seeded at a distance of
4cm between and within rows. Flooded water level was maintained at 4cm for 7 days after seeding toallow for good establishment of seedling. Then after, water was gradually drained to provide stress tothe plants. However, the water level was again raised to 4cm above soil surface at 21 and 45 days
after sowing. During this time, fertilizer was applied at the rate of 60:40:30 kg/ha. The troughremained flooded about 7 days after fertilization to allow optimum distribution of fertilizer and after
which the water level was again lowered to 5cm from soil level to provide additional stress to thewater. Second stress was done at 30 - 40 DAS where the water level was reduced to 10cm from soil
level and finally at 41 – 50 DAS until maturity, the water level was finally reduced to 15cm from soil
level. At maturiy, a total of 500 panicles with good filled grains were selected for field screening.

Figure 1. Greenhouse screening of mutant lines of MR219 irradiated with gamma rays
for minimal water requirement using water simulation technique.

Figure 2. Selection of mutant lines of MR219 irradiated with gamma rays tolerant
to minimal water requirement using water simulation in the greenhouse- 48 -

Figure 3. M1 plants of MR219 and Q74 irradiated with 60 and 80 Gy of ion beam planted
at Malaysian Nuclear Agency green house.

6.2.3 Field Screening
For field screening, the plot lay-out was constructed to simulate precise water stress regime. The
screening procedure (stress treatment, fertilization methods and etc) was similar to the green housescreening but 14 days old seedlings were transplanted instead. The seedlings were transplanted to thefield with planting distance of 10cm x 10cm to make up for 16 plants per entry. A total of 38 lines
were planted. Flooded field condition was maintained until 7 days after planting to allow for seedlingestablishment prior to water stress treatment at different stages of plant growth ie. at 41 - 50, 60 - 70
and 80 - 99 days after transplanting. During stress treatment the water level was maintained at 15 cm
below soil level. The 1st fertilizer application (100:40:30) applied was done at 21 days after
transplanting and the 2 nd fertilizer application was done at 53 days after transplanting. Sampling andharvesting was done at 85% ripening. Important agronomic traits were recorded at maturity from 5
plants, sampled at random from each plot.
After several series of selection and fixation, 12 potential lines with the required adaptive traits wererecovered at M4 generation. However, only two potential lines designated as MR219-4 and MR219-9
were selected for further testing under several stress environments. Selection was mainly based on
the percent filled grain because under water stress environment, it is the most sensitive indicator oftolerant lines.

- 49 -

6.2.4 Yield Trials
MR219-4 and MR219-9 were evaluated for yield and adaptation in MADA under saturated andflooded conditions. The aim was to study the interaction effects of rice variety and water
management on yield and yield components such as panicles/sq m, spikelets/panicle, percent filled
grains and the 1000-grain weight. The lines were also evaluated under aerobic soil condition in
MARDI Seberang. The same mutants were evaluated for adaptation and yield under flooded
conditions in KETARA, Besut, Terengganu.

Figure 4. Field screening and selection of advanced mutant lines of MR219 irradiated with gamma rays
for minimal water requirement using water simulation technique.

Figure 5. Field planting of M 3 mutant lines of MR219 irradiated with 80Gy ion beam at Tanjong Karang,
Selangor Experimental Station- 50 -

6.2.5 Analysis of Amylose Content
To determine the apparent amylose percentage a standard curve using amylose from potato wasapplied. The method described by Juliano (1971) was used to make a standard curve andamylose-iodine solution with rice flour was prepared manually by grinding rice grains in mortar andpestle. Then the samples were sieved with 45µm aperture. Using this method, 0.04g of standard
amylose was weighted and put into a volumetric flask. 1ml of 95% ethyl alcohol was added andgently mixed to spread the flour. 9ml of 2 N Naoh were added to the mixture. The standard solution
was then transfer to magnetic stirrer and stir for 10 minute after adding distilled water to 100ml. The
absorbance of the solution was measured at 620nm using a spectrophotometer, setting blank at zeroabsorbance. Data obtain were recorded and standard solutions against amylose content were plotted.
For the analysis of amylose content in a test sample, a portion (10g) from each of the sample wasreserved. Seeds from individual panicle were harvested and a portion was subjected to analysis ofamylose content. Apparent amylose content determination was performed using a near-infrared
reflectance spectrophotometer According to this procedure, 0.1g of rice flour was transferred into a
100ml dry volumetric flask and mixed with 1ml of 95% ethyl alcohol. 9ml of 2 N Naoh were added
to the mixture. The test solution was then transfer to magnetic stirrer and stir for 10 minute after
adding distilled water up to 100ml. In another 100ml volumetric flask was added in 70ml distilledwater, 2ml of 1N glacial acetic acid, 2ml iodine solution and 5ml aliquot of the test solution. The
solutions were added distilled water to the volume of 100ml and let stand for 10 minute.
In the presence of amylose, a blue-black color will be observed. The intensity of the color can betested using a spectrophotometer which reflects the concentration of starch present in the solution.
The test solutions were measured using a spectrophotometer at 620nm by setting the absorbance ofblank solution at zero. The blank solution was prepared by adding 2ml glacial acetic acid, 2ml iodine
solution and distilled water were adjusted to 100ml. The amylose percentage was evaluated with thestandard curve.
Methodology for determination of amylose content(By Juliano, 1971)
Method for determination of amylose content
1. Sample preparation (de-husking & grinding)
2. Weight 0.1g rice flour into 100ml dry Erlenmeyer flas
3. Dissolve in 1 ml 95% ethanol and 9 ml 2M Sodium Hydroxide (NaOH)
4. Boil the solution mixture for 10 min at 95ºC
5. Homogenize the solution and add distilled water till 100ml
6. Transfer 5ml of the sample aliquot into a new 100ml dry Erlenmeyer flask
7. Add 2ml 1M Acetic acid and 2ml Iodine solutions
8. Add distilled water till 100ml
9. Measure the absorbance of amylose-iodine complex at wavelength 620nm- 51 -

6.3

Results and Discussion

6.3.1 Radiosensitivity Test of MR219 irradiated with gamma irradiation

Figure 6. Radiosensitivity test of MR219

Figure 7. LD50 of MR219 irradiated with gamma rays wasusing sandwiched blotter techniquecalculated to be 310Gy

6.3.2 Dose-response curves of MR219 irradiated with different doses of with ion beam
100

(a)

100

(b)

90
80
Survival rate (%)

Germination rate (%)

120

80
60
40

70
60
50
40
30
20

20

10
0

0
0 Gy

10 Gy

20Gy

40Gy

60 Gy

0 Gy

80 Gy 100 Gy 120 Gy 160 Gy 200 GY

10 Gy

20Gy

40Gy

120

Percentage of ripening (%)

Growth rate (%)

80 Gy 100 Gy 120 Gy 160 Gy 200 GY

120

(c)

100

60 Gy

Dose rate

Dose rate

80
60
40
20

(d)

100
80
60
40
20
0

0
0 Gy

10 Gy

20Gy

40Gy

60 Gy

80 Gy 100 Gy 120 Gy 160 Gy 200 GY

0 Gy

10 Gy

20Gy

40Gy

60 Gy 80 Gy 100 Gy 120 Gy 160 Gy 200 GY
Dose rate

Dose rate

Figure 8. Dose-response curves of MR219 irradiated with different doses of with ion beam: (a)germination rate, (b) survival rate with different dose, (c) growth rate and d) percentage of ripening- 52 -

6.3.3. Germination rate of MRQ74 irradiated with ion beam

Figure 9. Germination rate of control and irradiated seeds of MRQ74 at day 1, 2 and 5
6.3.4 Plant typeThe general morpho-agronomic and genetic evaluation traits of mutants and parent are as tabulated inTable 1. Panicle length, flag leaf width, leaf width are longer or broader than the parent. Similarly,
the culm number for both mutants were comparatively or more than the parent.

- 53 -

Table 1. Morpho-agronomic characteristics of advanced mutant lines compared to its parents.
Plant Characters

MR219-4 MR219-9

MR219

Difference Over parent

Flag leaf length(mm)

34.2

36.4

40.1

lower

Flag leaf width (mm)

13.7

16

14.3

Higher(MR219-9)

Leaf length(cm)

41.3

40.6

41.4

Lower
Higher(MR219-4)

Leaf width (mm)

15.3

12.3

13.7

Culm length

(cm)

74.3

75

72

HigherCulm number (No)

15

13

14

Higher(MR219-4)

3.61

3.62

3.62

same

Culm diameter (mm)Blast Reaction

Resistant Resistant Resistantsame

BPH Reaction

Resistant Resistant Resistantsame

Culm Strength

StrongStrongStrongPanicle length

28.2

28.4

26.4

same
Higher

6.3.5 Yield and yield components
Yield trial in MADA, has indicated that the yields of MR 219-4 and MR219 -9 were comparable tothe yield of MR 220. However, lines MR 219-4 and MR219-9 had significantly higher percentage offilled grains and 1000-grain weight than MR 220 (Table 2). There was no significant interaction
effect of rice variety and water management on yield and yield components. Rice grown undersaturated condition yielded as high as rice grown under normal flooded condition. Similarly, the yieldcomponents (panicles/sq m, spikelets /panicle, percent filled grains and the 1000-grain weight) did
not differ significantly between flooded and saturated conditions. However, rice plants are
significantly taller (how many percent) when they are grown under saturated than flooded
condition.The growth and yield performance of MR 219-4 are similar to that of MR 219-9.The lines
were also evaluated under aerobic soil condition (sprinkler-irrigation) under dry land regime in
MARDI SeberangPerai and performed satisfactorily. Grain yield as high as 6.3t/ha for MR219-4 and3.4t/ha for MR219-9 were recorded under aerobic condition.
Table 2. Yield and yield components of MR219-4 and MR219-9 in Muda (direct seeding method)
Yield component
Treatment

Yield (kg/ha

Saturated
FloodedMR 219 – 4
MR 219 – 9
MR 220
Water
Management x
variety6480 a6799 a6542 a6460 a6815 ans

Spikelets/p
Panicle /sq.m
anicle
503 a
500 a
447 a
512 a
517 a
ns

107 a
104 a
107 a
99 a
109 a
ns

Percent
filledgrain

1000grain wt(g)

68.2 a70.2 a73.3 a77.7 a61.7 bns

28.4 a28.3 a29.0 a28.9 a28.1 bns

Plantheight(cm)
97.5 a95.1 b97.1 a98.6 a95.4 ans

Performance of yield components and yield for KETARA are as tabulated in Table 3. Both mutants
yielded lower than MR219 but they were better than MR219 for some traits such as grain width,
percent filled grain, early maturation.
- 54 -

Table 3. Growth and yield performance of MR219-4 and MR219-9 in KETARA (direct seeding method).
Parameters

MR219-4 MR219-9 MR219 Difference Over parent

Panicles /plant

6.5

7.4

9

lower

Plant ht

100.1

100.4

103.4

lower

9
73.4

higher98Percent Filled Grain

78.1

87.7

1000 grain wt(gm)

30.3

30.3

28

higherGrain Length(mm)

9.69

9.69

9.86

lower
higherNo. of tiller/hilllower

Grain width (mm)

2.38

2.41

2.35

Panicle length

n.a

n.a

n.a

n.a

Yield (kg)

5259

6000

6444

lower

Maturity

110

112

116

lower

Table 4. Yield performance of MR219-4 and MR219-9 at different field condition.

Field condition

MR219-4

MR219-9

Normal flooded conditions

4.8 t/ha

5.5 t/haSaturated soil

5.9 t/ha

6.8 t/haFlooded condition

7.2 t/ha

6.1 t/haAerobic soil condition

6.3 t/ha

3.4 t/haFigure 10. Evaluation of yield performance of advanced mutant lines MR219-4 and MR219-9 under low
water requirement- 55 -

Figure 11. Field Screening of mutants lines under

Figure 13. Advanced mutant of MR219-4

simulated water stress condition at MARDI Seberang.Peraiunder water stress (aerobic) condition

To produce a variety of rice mutants induced by carbon-ion beams (12C+6; 320 MeV), it is important
to investigate the effects of the carbon-ion on several biological end points such as germination,
survival, ripening and subsequent mutations in rice (Hidema et al., 2003). For MR219 which had
been irradiated with ion beam, a total of 31 mutant lines were selected in M 3 generation based on
their agronomic traits. However, physico-chemical characteristics were not significantly different
from the parental variety except for amylose content which showed high variation among all lines.
Based on overall data recorded, mutant line ML21 had the greatest performance in term of yield.
Further screening should be carried out in M 4 to select for potential mutant lines which are resistanceto blast disease and high yielding.
6.3.6 Analysis of amylose content
Traditional Malaysian rice varieties, such as Mahsuri and PongsuSeribu with 6 months of maturity
period have high amylose content of 25.4% and 22.2% respectively in comparison to low amylose
content of glutinous rice (13.3%), Carreon (13.3%), Columbia (16.6%). Original parental cultivar
MR219 has intermediate amylose content (20.0%), whereas MRQ74 has high amylose content(30.0%). Mutant lines from MR219 irradiated with gamma rays have higher amylose content (22.5 to
23.7%), whereas mutant lines irradiated with ion beam have low amylose content (MR219/I2 - 18.0%
and MR219/I12 - 18.6%). Parental variety MRQ74 with high amylose content (30.0%) after
irradiation with ion beam was able to produce mutant lines with low amylose content (MRQ74/8 21.4%, and MRQ74/26 - 22.8%). This shows that ion beam irradiation is much more effective than
gamma rays in producing lower amylose content.]
Table 5. Amylose content of Standard Rice samples

Standard rice samples% Amylose

Koshihikari (Japonica rice

17 (low)

MR219 (Local indica variety)20 (intermediate)

Mahsuri (Traditional indica variety)25 (high)- 56 -

Figure 14. Standard curve of amylose

Figure 15. Comparison of amylose content between otherusing UV-VIS Spectrophotometer

popular rice cultivars with Malaysian traditional rice

Figure 16. Comparison of amylose content between

Figure 17. Comparison of amylose content

ion beam and gamma rays

between MRQ74 control with mutant lines

Figure 18. Percentages of amylose content and comparison between all samples- 57 -

6.4

Conclusion

Main objectives of FNCA Plant Mutation Breeding Sub-Project on Composition or Quality in Riceare to select potential mutant lines of high yielding which are tolerance to minimal water requirement
and improvement of amylose content. Under flooded condition, mutant line MR219-4 recorded ayield of 7.2t/ha and MR219-9 recorded 6.1t/ha compared to 5.8t/ha from the parental variety MR219.
Under saturated condition, MR219-4 recorded 5.9t/ha whereas MR219-9 recorded 6.8t/ha of yield
performance. The most striking observation made was when both mutant lines were tested underaerobic condition and mutant line MR219-4 performed much better than MR219-9, recorded a yieldof 6.3t/ha in comparison to MR219-9 with 3.4t/ha. This shows that gamma irradiation was able toinduce potential mutant lines suitable for minimal water condition with improved yield. These twoadvanced mutant lines will be further evaluated for stability in yield performance and otheragronomic traits before they can be registered as new mutant varieties to be released to the farmers.
In terms of improvement of quality of rice with respect to amylose, ion beam is more effective than
gamma rays in producing lower amylose content. Mutant lines from MR219 irradiated with gamma
rays have high amylose content (22.5 to 23.7%), whereas mutant lines irradiated with ion beam have
low amylose content (18.0 - 18.6%). Similarly, when MRQ74 seeds with high amylose content of
30.0% were irradiated with ion beam, mutant lines recorded lower amylose content (21.4 - 22.8%).
Further screening and selection in M 4 to M 6 generation will be carried out to select for potential
mutant lines with high yield and resistance to blast disease.
6.5

Acknowledgement

The authors would like to thank Ministry of Science, Technology & Innovation (MOSTI) and
Malaysian Nuclear Agency for providing the fund to carry out this Sub-Project on Composition or
Quality of Rice coordinated by FNCA of Japan. The authors are grateful for the contributions of Dr.
Atsushi Tanaka from Japan Atomic Energy Agency (JAEA) for providing ion-beam irradiation
facilities. Special thanks go to Ms. Aki Koike from NSRA whose considerable patience, liaison andorganizational skills were very instrumental for the success of this project.

6.6

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