Mutation Breeding of Rice for Sustainable Agriculture in Malaysia
Sobri Bin Hussein1, Abdul Rahim Bin Harun1, James Mackester Anak Simoli1,
Muhammad Ruzaini Abdul Wahab1, Shakinah Binti Salleh1, Faiz Bin Ahmad1, Phua
Choo Kwai Hoe1, Shyful Azizi Bin Abdul Rahman1, Ahmad Nazrul A.W1, Latiffah Binti
Nordin1, Atsushi Tanaka2, Anna Ling Pick Kiong3, Koh Rhun Yian3, Mohd Rafii Bin
Yusop4, Asma Ilyani4, K, Kogeethavani R5, Yoshihiro Hase6, Aki Koike7, Noorman
Affendi M8, Rahiniza Kamaruzaman8, Nor’Aishah Hassan9, Noraziyah Abd Aziz
Shamsudin10 and Norainy Md Hashim11
1Agrotechnology

and Biosciences Division Malaysian Nuclear Agency (Nuclear

Malaysia), Bangi 43000 Kajang Malaysia, 2Kansai Photon Science Institute, National
Institutes for Quantum and Radiological Science and Technology (QST), Japan, 3Divisionof Applied Biomedical Sciences and Biotechnology, School of Health Sciences,
International Medical University, 126, Jalan Jalil Perkasa 19, Bukit Jalil, 57000 Kuala
Lumpur, Malaysia, 4Plant Breeding & Genetics, Institute of Tropical Agriculture andFood Security, University Putra Malaysia, 5Malaysian Agricultural Research andDevelopment Institute, 6Takasaki Advanced Radiation Research Institute, QST, Japan,
7International Affairs and Research Department Nuclear Safety Research Association(NSRA), 8MARDI Seberang Perai, Jalan Paya Keladi, Pinang Tunggal 13200 Kepala
Batas Pulau Pinang (Malaysian Agricultural Research and Development Institute), 9
Faculty of Applied Science, University Technology MARA, Negeri Sembilan, 10 School of
Biosciences and Biotechnology, Faculty of Science and Technology, UKM, 11Bahagian
Perkhidmatan Makmal, Jabatan Pertanian Negeri Kedah. State Department ofAgriculture Kedah.
Abstract
National Agriculture Policy (DPN 3) 1998-2010 and the National Agrofood Policy(NAP) 2011-2020 are the evidence of Malaysian government’s efforts in transforming aswell as improving the country's food quality. The National Agrofood Policy 2011-2020
was formulated with a special focus on improving the food production sector including
rice. Even though Malaysia is still depending on imported rice to fulfil consumer’s
demand, rice industry has always been a national priority based on its strategic
importance as a staple food commodity. Nevertheless, the rice industry in Malaysia ishampered by several challenges such as global climate change, lacking of new variety,insufficient certified seed, emerging of major diseases such as Bacterial Panicle Blight30

(BPB), Leaf Blast, drought season, flash flooding, decreasing of planting area, lostinterest of the farmers due to high cost of rice production and many others. Thus, the
NAP had highlighted that local rice production should be increased to ensure thecountry’s demand in the future. FNCA project is one of the initiatives that supported thenational policy. In 2019, Malaysian Nuclear Agency has moved one step forward in thearea of mutation breeding by signing the Memorandum of Understanding (MoU) with
Certified Seed Company HMN (M) Sdn. Bhd and Bayer Co. (MALAYSIA) Sdn. Bhd. With
this agreement, HMN (M) Sdn. Bhd. will collaborate with Malaysia Nuclear Agency to
produce certified seeds, multiply and commercialize the rice mutants. Meanwhile, Bayer
Co. (MALAYSIA) Sdn. Bhd. will be responsible in coating the mutant seeds with plant
growth promoters. This collaboration has added value to NMR 151 (PBR 0159) and NMR
152 (PBR 0156) mutant seeds and resulting in more competitive seeds as compared toother varieties in the market. In addition, from 2017 until 2020, several local verification
trails (LVT) were also conducted from the Northern part until the Southern part ofPeninsular Malaysia. The data obtained from Sekinchan, Selangor revealed that NMR
152 consistently produced between 7-10 t/ha in granary area as compared to 6 t/ha
produced by other varieties within the same planting areas. Field trials also showed thatthe production cost was reduced by 10%, mainly due to the reduction in fertilizer andpesticide usage. At the same time, the yield could be increased between 40 - 60%
depending on the planting areas. The preliminary studies on estimation of glycemic
index (GI) revealed that two mutant lines could be consumed by diabetic patients. The
two mutant lines (ML3 and ML30) were recorded to have normal glucose reading which
was identified to have a moderate GI of 65 and 66, respectively. As low and moderate GI
foods are recommended for diabetic patients, these two mutants (ML3 and ML30) havea high potential for their consumption. In addition, two mutant lines were successfully
granted with Certificate of Registration of New Plant Variety and Grant of Breeder’s
Right by Department of Agriculture Malaysia in Feb 2020 with registration number;
PBR0156 (for NMR152) and PBR 0159 (for NMR151). Apart from increasing the yieldand income of the framers, the mutant lines derived from FNCA project had also greatly
impacted the socio-economic status of the farmers as the mutant rice are adaptable tocurrent global climate change conditions. Furthermore, through the MoU with thecompanies, around 4000 metric tons of seeds will be produced in 2020 for the supply of
28,571 ha planting areas in Peninsular Malaysia. Basically, the project has addressed
the national agenda and policy in generating new rice varieties and thus, increase thewell-being and livelihood of the farmers.

31

Keywords: Gamma Irradiation, Ion Beam, Mutation Breeding, MINT, NMR152,
NMR151, Rice.
Introduction
In Malaysia, rice industry has always been a priority based on strategic importanceof rice as a staple food commodity. Although the production of rice is increasing towards
population increase, Malaysia still depends on imported rice to meet the consumer’s
demand. Malaysia managed to achieve 72% self-sufficiency level in rice 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 imported rice. In Peninsula Malaysia, rice production depends largelyon the irrigated lowland production system. Currently, there are 241,741ha of irrigatedrice in Peninsula Malaysia, which contributes more than 85% of the national riceproduction. However, growing irrigated rice requires large amount of water. It wasestimated that about 3,000 liters of water are required to produce 1kg of rice.
Unfortunately, there are signs of declining water supply that threatens thesustainability of irrigated rice production. The largest water withdrawal (more than75%) in Malaysia is for irrigation in the agriculture sector and is mainly confined toirrigated 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 alimiting factor in rice production (Bhuiyan, 1992). Water supply is affected by the loss ofwatersheds due to deforestation and soil erosion. There is severe depletion of valuable
groundwater resources as water is taken up for agricultural and industrial purposes.
Rice will be the crop most affected by water scarcity as it depends most heavily on
irrigation. Some varieties of rice can be grown without irrigation. However, their growth
totally depends on the rain fall. 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 is an urgent need to develop new varieties of ricewith high yield potential and stability under water stress conditions. Therefore, in an
attempt to develop water stress tolerance lines, morphological and agronomic traits thatare 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 is desired to improve one or two easily identifiable traits in anotherwise good lines or varieties. The successful use of plant breeding for improving
crops requires the existence of genetic variation of useful traits. Unfortunately, the32

desired variation is often lacking. However, radiation can be used to induce 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.
Heavy ion beams, such as carbon ions, are more effective in plant for inducing mutations
compared with electron beam (Hidema et al., 2003). Novel mutants have been obtainedby the carbon ion irradiation in several plant species (Hase et al., 2000). To date, anumber of rice mutants with various significant improvements in morphological traits
have been recommended for planting (Mohamad et al., 2006). In recent years, the fielddrought-resistance screening facility was established through mutation breeding
program and the evaluation standard was developed. Some advanced lines of droughttolerance rice varieties were identified and will be used in both molecular mapping andbreeding programme. Therefore, the objectives of FNCA rice mutation breeding project
were to screen for mutant lines derived from gamma rays and ion beam irradiation forhigh yield potential and stability under water stress conditions with improved quality
trait.
Materials and Methods

Part 1: Evaluation of mutant lines derived from gamma irradiation
The seeds of popular local variety, MR219 were irradiated with 300 Gy of gamma
radiation from the Cobalt-60 (60Co source) at Malaysian Nuclear Agency. Irradiatedseeds were sown at Mardi Research Station in Tanjung Karang, Selangor. The M1
seedlings were transplanted into the field with 25cm x 25cm planting distance. A totalof 10,000 M1 seedlings were planted to produce M2 seeds and a total of 5,250 plants wereselected from which 2 panicles per hill were randomly harvested from each hill. After M6generation, the best selected mutant lines were further evaluated for MLT (Multi
Location Trial), LVT (Local Verification Trial), disease screening, morphology andagronomy characteristics. The best selected lines were planted for 4 seasons and the dataon culm height, panicle length, number of panicle per plant, flowering time, day ofmaturity, weight for 1000 grain seed, seed length, width length and yield in t/ha werecollected. This study was conducted in collaboration with MARDI (Malaysian
Agriculture Research and Development Institute), MADA (Muda Agricultural
Development Authority), KADA (Kemubu Agricultural Development Authority), IADA
(Integrated Agricultural Development Area), Department of Agriculture Malaysia(DOA), HMN (M) Sdn Bhd), BAYER Co (M) Sdn. Bhd. and farmers. The locations forfield trial were inclusive of Northern part until Southern part of Peninsular Malaysia
33with the planting area of approximately one hectare per variety.

Part 2: Evaluation of mutant lines derived from ion beam irradiation
As for ion beam irradiation, several cultivars such as MR219, Pongsu Seribu 2,
Tongkat Ali, MARDI(B001), MARDI(C002), UKM-1, UKM-2, UKM-3, UiTM-1, UiTM-2
and UiTM-3 were irradiated in the ion beam facility in Takasaki, Japan. The Irradiation
Apparatus for seed, connected to a vertical beam line of the AVF-cyclotron (TARRI,
Takasaki), were used for the 320 MeV carbon-ion irradiation. The carbon ion irradiation
with the doses of 0, 10, 20, 40, 60, 80, 100 and 120 Gy were performed under atmosphericpressure within 3 min (Hidema et al., 2003). The seeds were evaluated at Malaysian
Nuclear Agency, UKM, UiTM and MARDI. The optimum doses of these cultivars wereidentified. The 31 potential mutant lines derived from MR219 rice that produced through
Carbon ion radiation (60 Gy) were screened and selected at Malaysian Nuclear Agency
up to six generation (M6) based on the targeted traits. These mutant lines (ML1 to ML31)
were evaluated on morphological characters, yield and yield components, and comparedto the parental variety, MR219. Meanwhile, another 10 potential mutant lines derived
from Pongsu Seribu2 were further evaluated at farmer’s field in Sekinchan, Selangor.
Data on culm height, panicle length, number of panicles per plant, flowering time, dayof maturity, weight for 1000 grain seed, seed length, width length and yield in t/ha werecollected. This study was conducted in collaboration with MARDI, IADA, UKM (National
University of Malaysia), UPM (Universiti Putra Malaysia), DOA, HMN (M) Sdn Bhd,
BAYER Co (M) Sdn. Bhd. and the farmers.

Part 3: Evaluation and characterization of advanced mutant lines of MR219-4 (NMR151/ PBR 0159) and MR219-9 (NMR 152/ PBR 0156) under drought conditions
The seeds of popular local variety, MR219 were irradiated with 300 Gy of gamma
radiation from the Cobalt-60 (60Co source) at Malaysian Nuclear Agency. Two advanced
rice mutant lines MR219-4/NMR151 and MR219-9/NMR152 and cultivars MR211,
MR219 and ARN1 were grown in a greenhouse at the Malaysian Nuclear Agency(Nuclear Malaysia), Bangi. The MR219-4 and MR219-9 and check varieties MR211,
MR219 and ARN1 were assigned in a randomized complete block design (RCBD) withthree replications. The pre-germinated seeds were sown in trays containing wet soil.
Healthy seedlings were transplanting 26 days after germination. The seedlings wereplanted in a row consisting of nine plants each genotype were replication three
represented 15 rows, all together. The planting distance was 23 cm within and 23 cm
between rows. The space between two adjacent troughs was 1 m. The water was drained34

at 30 days after transplanting (DAT) and was re-irrigated periodically when soil watertension fell below -50kPa. The control was continued with standing water until maturity.
Morphological traits evaluated on single plant basis were plant height, days toflowering, number of tillers, flag leaf area, panicle length and days to maturity. The
plants were scored for leaf rolling and leaf drying by observing visually using 0-9 scale(Table 1 & Table 2) based on Standard Evaluation System adopted for rice (IRRI 1996).
The process of harvesting the grains was done manually when the plants reachedmaturity. Grains from each plant were packed in an envelope. The culms and leaves were
cut at ground level and wrapped with newspaper. The grains and plant parts were driedin an oven at 37°C for 48 hours.
The measured agronomic traits for each plant were grain weight, grain yield, 100grain weight, dried plant weight, biomass and harvest index. Data were analysed using
the statistical analysis system (SAS 9.1.3) for windows software. All the data obtained
were subjected to a two-way analysis of variance (ANOVA) and the mean differenceswere compared by least significant differences (LSD).

Part 4: Evaluation of Nitrogen uptake in advanced mutant lines of MR219-4 (NMR151 /PBR 0159) and MR219-9 (NMR 152/ PBR 0156)
The pot trial was conducted at Nuclear Malaysia in April 2013. Mutant rice varieties
MR219-9 and MR219 (check variety) with two water regimes, non-flooded at soil water
potential of 0-30 kPa and flooded at 5cm water level were tested in a completely
randomized design. Nitrogen was applied at 120 kg N ha in three splits at 5, 30 and 60
days after transplanting using 15N-labelled urea with 5.14 % atom excess. Total N
content and 15N abundance in the plant samples was determined by Kjeldahl method
and emission spectrometry, NOI7. A field trial was conducted at MARDI and crop
establishment was conducted in August 2013. Four rice varieties, MRIA 1, mutant
MR219-4 (NMR151 / PBR 0159), MR219-9 (NMR 152/ PBR 0156) and MR 219 weregrown under aerobic condition. Each treatment is tested in a 5 x 5 m plot arranged in arandomized complete block design with five replications. 15N-labelled urea at 10% atom
excess was applied in microplots of 0.5 x 0.5 m and 1 x 1 m.

Part 5: Screening of Foliar blast disease in selected potential mutant lines
The evaluation of 5 test lines from MINT (NM 1= ML 30, NM 2 = ML3, NM 3 = ML
10, NM 4 = NMR 152 and NM 5= NMR 152) against foliar blast disease resistance wereconducted in blast nursery during off season 2016. The screening was done under naturalinfection from the inoculum bed according to method established by IRRI. Inoculum bed
35

for maintenance of natural airborne spores was maintained continuously by planting
mixture of susceptible varieties at 3 stages with interval of one to two weeks. Dry test
lines seeds were sown in rows in between two inoculum beds. Bombardment rows which
consist of mixture of susceptible varieties planted along both side of the test lines. This
is to ensure sufficient inoculum for spreading disease spore to the test lines. Seedlings
were watered frequently by sprinkler at every three hours throughout the day. Fertilizer
was applied at 2 weeks after sowing using N: P: K at the rate of 140:70:80 kg/ha. Disease
reaction was assessed at 21 and 28 days after sowing (DAS) using a 0-9 scale as describedby IRRI 3rd edition of the ‘Standard Evaluation System for rice (SES)’ (1988) (Table 1)
and the ability of disease resistance was interpretation based on the General Disease
Scale as in Table 2.

Table 1: Standard Evaluation System for rice

Table 2: General disease scale interpretation
Disease scale

Disease Reaction0Highly resistant (HR)

1-2

Resistant (R)3-4

Moderately resistant (MR)

5-6

Moderately susceptible (MS)7-8

Susceptible (S)9Highly susceptible (HS)36

Part 6: Evaluation on effects of MR219 mutant rice on glycaemic responses in BALB/c
mice
Plant Materials
The MR219 seeds were irradiated with a carbon-ion beam (60 Gy) using AVFCyclotron in a collaborative effort by the National Institute of Quantum and Radiological
Science and Technology, Japan and Malaysian Nuclear Agency, Bangi, Selangor. Afterseveral series of selection and fixation, 31 potential lines with the required adaptive
traits were recovered at M4 generation during the 2009 - 2012 seasons (M0 - M4). The
M4 seeds of these 31 mutant lines were evaluated including the parental variety MR219.
Samples of rough rice were dehulled using a dehusking machine (Motion Smith Co.,
Singapore). The dehulled rice was passed through a 500 μm sieve screen on a Sample
Mill (Cyclotec 1093, Foss analytical, Sweden) to obtain rice powder.
Evaluation of mutant lines for glycaemic responses through in vivo study
Female BALB/c mice of 8 w old were used in the present work and carried out in aCompletely Randomised Design with the guidelines of Research and Ethics Committee
of International Medical University (IMU). A total of 48 mice were divided into eightgroups having six mice each. The study started after one week of mice arrival to allow
them to adapt to the new environment. Normal diet was given to them throughout theperiod of adaptation.
Oral feeding and glucose monitoring of blood in mice
After the adaptation period, the mice were fasted (overnight fasting) for 12 h.
However, distilled water was offered ad libitum. The blood samples were taken at time
zero before given the test food to measure the glucose level. Blood collection was done
following the standard procedure described by IACUC (2011). Blood sample was obtainedby snipping not more than 1 mm of mice tail and gently milking the blood from thesnipped tail. One droplet of blood was placed on a glucose test strip and was read using
a glucometer (Accu-Check glucometer). Then the mice were given the test food samples
by force feeding using the feeding tube. The size of the feeding tube used for feeding was
18 gauge. The mice were restrained by grabbing them by the scruff of their neck andgrasping the skin over the shoulders with the thumb and middle fingers during force
feeding. This extended the fore-legs to the side, thus keeping the front feet from pushing
the gavage tube away. The blood glucose levels were determined again at 30, 60, 90 and
120 min after feeding. Data were subjected to statistical analysis such as analysis ofvariance (ANOVA) and mean comparisons using SAS 9.4 Software.
37

Estimation of Glycaemic Index
The percentage of GI was calculated using the formula prescribed by FAO-WHO(1998):
GI = [area under curve (test food) / area under curve (glucose)] × 100
where the area under curve was calculated based on Trapezoid technique in MS Excel toreflect the total rise of blood glucose levels after eating test foods.

Results and Discussion

Part 1: Evaluation of mutant lines derived from gamma irradiation
Malaysian Nuclear Agency has moved one step forward in the area of mutation
breeding by signing the Memorandum of Understanding (MoU) with Certified Seed
Company HMN (M) Sdn. Bhd and Bayer Co. (MALAYSIA) Sdn. Bhd. on 29 April 2019.
With this agreement, HMN (M) Sdn. Bhd. will collaborate with Malaysia Nuclear Agency
to produce certified seeds, multiply and commercialize the rice mutants. Meanwhile,
Bayer Co. (MALAYSIA) Sdn. Bhd. will be responsible in coating the mutant seeds withplant growth promoters. This collaboration will add value to NMR 151 and NMR 152
mutant seeds and result in more competitive seeds as compared to other varieties in themarket. In addition, from 2016 until 2020, several local verification trails (LVT) andmulti-location trial (MLT) were also conducted from the Northern part until theSouthern part of Peninsular Malaysia (Figure 1). The data obtained from the field trial
at Sekinchan, Selangor revealed that NMR 152 consistently produced between 9-10 t/ha
in granary area as compared to 6 t/ha produced by other varieties within the sameplanting areas. Field trials also showed that the production cost was reduced by 10%,
mainly due to the reduction in fertilizer and pesticide usage. At the same time, the yield
could be increased between 40 - 60%, depending on the planting areas (Table 3). Table 4shows the yield of NMR 152 using different evaluation methods in the farmers’ field. The
data revealed that Wheel method that introduced by the Agriculture Department is themost accurate methods in estimating the yield of mutant rice in an open field.
In 2015, Malaysian Nuclear Agency introduced new innovation in the area of rice
breeding technology. The innovation was called as “Nuclear Malaysia Rice Agronomic
Package”, in which the package was aimed to further stimulate the early maturationand yield of the advanced mutant line (Figure 2). The data revealed that the cost ofadditional fertilizer, pesticide and field management could be reduced up to 5-10% of
total expenditure. Results obtained from northern part of Malaysia indicate that mutant
38

variety (MR 219-9/NMR152) treated with the combination of Biofertilizer, Oligochitosan
and liquid smoke produce higher yield (13 t/ ha-using CCT Method) as compared to thecontrol (Figure 3a). The data obtained also revealed that similar pattern was observedwith mutant line MR 219-4/NMR151 where by 15 t/hectare (using CCT method) was
produced. In another farmer’s plot treatment with liquid biofertilizer was significantly
increased the grain yield of mutant MR219-4 up to 25% as compared with othertreatments over the control. A biofertilizer is a substance composed of living
microorganisms that colonise the rhizosphere or the interior parts of plants and promote
growth by increasing the supply or availability of primary nutrients to the host plant(Vessey, 2003). According to Phua et al. (2019), those microorganisms can improve theuptake of N 2 fixation and P solubilisation. Therefore, the yield for both NMR151 and 152
increase up to 15 t/ha and 13 t/ha respectively (Figure 3b). The yield obtained was higher
than the national average yield (4.207t/ha-actual yield from factory). Meanwhile, MR
219-9 (NMR152) showed more tolerant to drought as compared to MR 219-4 (NMR151)
and MR 219-control.

Figure 1: NMR 152 yield increment at different location in Peninsular Malaysia.

39

Table 3: Yield increment in different state of Malaysia as compared with data from
MOA
Location

Average Yield from

Average Yield

Yield Increment(State)

MOA (Agrofood

NMR152(t/ha) / (%)

Statistics 2018)(PBR 0156)

Perlis

5 t/ha7 t/ha2 (40%)Kedah

5 t/ha8 t/ha3 (60%)

Penang

5 t/ha7 t/ha3 (60%)Perak

3 t/ha5 t/ha2 (67%)

Selangor

6 t/ha10 t/ha4 (67%)

Johor

3 t/ha5 t/ha2 (67%)(Note: PBR = Certificate of registration of new plant variety and grant of breeder’s right,
MOA =Ministry of Agriculture and Food Industries, Malaysia)
Table 4: NMR 152 Local Verification Trial (LVT)
Location

Planting
SeasonYield (t/ha)
Actual Yield

CCT-Method(From factory)(Yieldestimation)

Wheel -Method(Yieldestimation)

Sekinchan

Main Season

10

15

8.2

(IADA)Off Season

9

13

7.5

Jenun (MADA)

Main Season

10

-

-

Off Season

9

10

7.1

Pendang

Main Season

8

13

-

(MADA

Off Season

7

9

-

Changlun

Main Season

8

-

-

Non-granary

Off Season

7

7.3

6.1

Main Season

7

10.0

-

(MADA)Off Season

6

9.0

-

Seberang

Main Season

5

7.0

6.5

Perak

Off Season

4

-

-

area
Utan

Aji

(IADA)(Note: IADA- Integrated Agricultural Development Area, MADA - Muda Agricultural

Development Authority & MADA – Muda Agricultural Development Authority)40

Figure 2: NMR152/ PBR 0156 field trial in 2015/16 under Malaysia Social Innovation
Fund project at Northern Part of Malaysia (Code Project MSI 16010).

Figure 3a: Effect in yield production on MR219-9/NMR152 using different fertilizer
treatment.

Figure 3b: Effect in yield production using different fertilizer treatment.

41

Upon completing the evaluation in the field including yield trial, disease screening
and DUS trial (Distinctness, Uniformity & Stability), two mutant lines were successfully
granted with Certificate of Registration of New Plant Variety and Grant of Breeder’s
Right by Department of Agriculture Malaysia in Feb 2020 with registration number;
PBR0156 (referring to NMR152) and PBR 0159 (referring to NMR151). As formorphology and agronomy characteristics, there was no significant difference between
NMR 151 and NMR 152 as both varieties performed similarly in terms of flowering time
(82 days), day of maturity between 100 – 110 days and weight for 1000 grain seed ofaround 31 g (Table 5). The data obtained also showed that NMR 151 produced a highnumber of tillers per plant and high number of panicles per plant as compared to NMR
152.
Table 5: Morphology and Agronomy Characteristic NMR 152 and NMR 151
NMR 152

NMR 151

Farmer Variety(PBR0156)(PBR0159)

MR219

Characteristics

Remarks

Remarks

Remarks

Flowering time

82 days

82 days

94 daysCulm height

82 cm

73 cm

76-78 cm

Panicle length

30 cm

29 cm

23 cm

No of panicle per plant

17

21

13

No of tiller per plant

20

22

15

Day of Maturity

100-110 days

105 – 110 days

105 - 111

Weight for 1000 grain

31.1 g31.6 g27.1 gSeed length11.1 mm9.85 mm

6.58 mmWidth length

2.75 mm2.67 mm

2.35 mmYield

7- 11 t/ha

7 – 9 t/ha

6.5-10.7 t/ha

(50% flowering)seed

A total of 4 seasons yield trial at Sekinchan, Selangor recorded that NMR152 showeda high yield (between 9 to 10 t/ha) as compared to other mutant lines (Figure 4). Farmers
observation also showed that NMR 152 is resistance to bacteria leaf blight disease(Xanthomonas oryzae pv oryzae) as indicated in Table 6. Meanwhile, the inspection
conducted by Agriculture Department of Malaysia indicated that NMR 152 could survive
under several rice disease conditions such as Leaf Blast (Pyricularia oryzae Cavara),
42

Panicle Blast, Brown Leaf Spot (Helminthosporium oryzae Breda de Haan), Sheath
Blight (Rhizoctonia solani Kuhn), Brown Planthopper (Nilaparvata lugens Stal), False
smut (Ustiloginoidea virens Cooke), Stem Borers (SB), Rice Ear Bug (Leptocorisa

oratorius Fabricius)), Green leafhoppers (Nephotettix virescens Distant) and Rice
leaffolder (Cnaphalocrocis medinalis sp). In addition, farmers in Sekinchan also
indicated that NMR 152 is resistant to the lodging problem. Nevertheless, the area ofinfected is very much depending on the farmers’ practice.

Figure 4: NMR 151 and NMR 152 rice mutant in Sekinchan, Selangor.

Table 6: Screening of NMR 151 and NMR 152 Varieties for Bacteria Leaf Blight(Xanthomonas oryzae pv oryzae)(Source :Dr Nor’ Aishah-UiTM)

43

Part 2: Evaluation of mutant lines derived from ion beam irradiation
In 2015, a total of 31 potential mutant lines were produced through mutagenesis of
MR219 seeds with Carbon ion radiation (60 Gy). They were then planted at the
Malaysian Nuclear Agency up to five generation (M5). These mutant lines (ML1 toML31) were evaluated on morphological, yield and yield components, and compared tothe parental variety, MR219. About 5% of M2 populations were selected for further
screening in M3. After several series of selection and fixation, 31 potential lines with therequired adaptive traits were recovered at M4 generation. The data obtained revealed
that the best dose (Shoulder dose) was observed at 60 Gy for Pongsu Seribu2. In termsof growth rate, the data showed that the pattern started to decline from 60 to 120 Gy(Figure 5) and germination rates were more than 90% in the range of 0 – 120 Gy (Figure6). This observation was similar to those reported by Hidema et al. (2003). However,variety PS2 could still be able to survive at 120 Gy as shown in Figure 7. As for MARDI(B001) the optimum dose was observed between 40 and 80 Gy while the shoulder dose
response curve for MARDI C002 was observed at 40 Gy (Figure 8). In terms of spikelet
fertility, declining pattern was significantly observed from 60 to 120 Gy for all samples
tested (Figure 8), similar pattern was also observed in variety Pongsu Seribu2 (Figure6). Studies for other varieties are currently in progress.

Figure 5: Dose response curves for percentage growth rate, plant height andripening (Pongsu Seribu2).

44

Figure 6: Dose response curves for germination rate, survival rate and spikelet fertility(Pongsu Seribu2).

Figure 7: Biological effect of carbon ion on rice (Pongsu Seribu2).

45

Figure 8: Biological effect of carbon ion on rice (B001 & C002).
The data obtained after M6 generation showed that the highest yield wassignificantly observed in MINT 4 (10.04 ± 3.38 t/ha) and followed by MINT 3 (9.52 ± 2.80t/ha), MINT 10 (9.42 ± 3.24 t/ha), MINT 5 (8.68 ± 2.99), MINT 9 (8.46 ± 0.43). In themeantime, similar pattern was observed in MINT 2, MINT 7 and MINT 8 which only
produced 7 t/ha (Table 7). In terms of weight for 1000 seeds, most of the potential mutant
lines recorded a value between 26g to 32g. The data also showed some correlation
between weight for 1000 seeds and actual yield as indicated in MINT 4 with 32.20 ± 0.07
g of seeds were produced around 10.04 ± 3.38 t/ha. This result showed that the highest
weight of 1000 seeds will contribute to the highest yield. Contrarily, the number of tillers
was not correlated to a high yield in mutant line. Figure 9 shows the potential mutant
line derived from ion beam at Sekinchan Selangor.

Figure 9: Mutant derived from ion beam at M6 generation (Sekinchan, Selangor).
46

Table 7: Morphology and Agronomy Characteristic MINT 1 -MINT 10 (Ion Beam)
Mutant Line

Actual Yield

Weight 1000

No of Tiller

Average Height(t/ha)

Seeds(From factory)(g)

MINT 1

8.13 ± 0.03

30.02 ± 0.09

27 ± 3100.40 ± 3.40

MINT 2

7.86 ± 1.66

29.40 ± 0.11

28 ± 2115.00 ± 0.90

MINT 3

9.52 ± 2.80

29.30 ± 0.16

23 ± 2124.40 ± 3.30

MINT 410.04 ± 3.38

32.20 ± 0.07

21 ± 2115.80 ± 4.50

MINT 58.68 ± 2.99

29.40 ± 0.07

25 ± 3116.40 ± 1.30

MINT 66.87 ± 1.32

29.40 ± 0.08

16 ± 3123.00 ± 2.00

MINT 77.00 ± 0.42

30.70 ± 0.11

19 ± 3126.40 ± 6.40

MINT 87.22 ± 0.30

31.40 ± 0.17

14 ± 3118.00 ± 2.80

MINT 98.46 ± 0.43

29.10 ± 0.12

20 ± 3102.00 ± 1.90

MINT 109.42 ± 3.24

26.0 ± 0.56

27 ± 6111.63 ± 2.20(cm)

(Note : Data from Sekinchan mutant plot – Using farmers practice)Part 3: Evaluation and characterization of advanced rice mutant lines of rice MR219-4(NMR151 / PBR 0159) and MR219-9 (NMR 152/ PBR 0156) under drought conditions
Leaf rolling and leaf drying are used as an indication of tolerance in drought studies.
In this study, leaf rolling and leaf drying were recorded under water stress condition.
Table 8 shows the results of ANOVA for leaf rolling and leaf drying for the evaluatedlines. The results showed that there were significant differences among the evaluatedlines for leaf rolling and leaf drying. The mean values for leaf rolling and leaf drying ofeach line are presented in Table 8.
Table 8: Mean value of leaf rolling and leaf drying traits of evaluated lines
Line

Leaf rolling mean score

Leaf drying mean score

MR219-4

3

1

MR219-9

1

1

MR211

7

7

MR219

5

3

ARN 1

0

1

LSD (0.05)

0.29

1.70

47

An early sign of soil water declining is leaf rolling which is a simple expression ofleaf wilting. Fischer et al. (2003) have suggested leaf rolling as a criterion for scoring
drought tolerance in rice cultivars. Therefore, leaf rolling is useful for quick screening
hundreds of lines (Shyful A.A.R et al., 2017). The method of screening is by scoring theplants on a scale 0 to 9 (Table 8) according to Standard Evaluation System adopted forrice (IRRI 1996). A drought resistant check variety, ARN 1 had the best score of 0 for leaf
rolling. Meanwhile, MR211, the susceptible check variety had a score of 7 indicating
susceptibility to drought. MR219 had a score of 5 which was considered as moderately
susceptible. Thus, in this study, MR219-4 can be considered as moderately resistant andMR219-9 as resistant to drought.
According to Fischer et al., (2003), typically leaf drying begins at the tip of the leaf,
which is usually under greater water deficit than the basal part that closer to the stem.
Leaf drying was observed visually by scoring the plants on a scale 0 to 9 (Table 8) basedon Standard Evaluation System adopted for rice (IRRI 1996). Leaf water deficiency canbe further reduced beyond the point of turgor loss in which reaching the point of tissue
death (Fischer et al., 2003). According to Kadioglu and Terzi (2007), low score of leaf
drying can be advantageous in terms of less damage under water stress. In this study,
lower score in MR219-9 and MR219-4 which had scored 1 indicating that they were lessdamage from water stress.
There were significant differences between treatments for number of tillers, days toflowering, plant height, flag leaf area and days to maturity (Table 9). Lines showedsignificant differences (p<0.05) for number of tillers, days to flowering, flag leaf area,
panicle length and days to maturity (Table 9). There was significant interaction between
treatments and lines for number of tillers, days to flowering, flag leaf area, panicle lengthand days to maturity.
Table 10 shows that there were significant differences (p<0.05) between treatments
for grain weight per plant, grain yield per plant, 100-grain weight, dried plant weightand harvest index. The lines were significantly different (p<0.05) for grain weight perplant, grain yield per plant, 100-grain weight, dried plant weight, biomass and harvest
index (Table 10). There was significant interaction between treatments and lines forgrain weight per plant, grain yield per plant, 100-grain weight, dried plant weight,
biomass and harvest index.

48

Table 9: Analysis of variance for morphological traits
Mean squares
SourceDf

NumberDays to

Plant

Flag

PanicleDays toof tillers

floweringheightleaf

lengthmaturity

(cm)

area

(cm)

(cm2)
Treatments (T)

1

0.07

0.0034*17391

532.12*30.63

0.0111*

Replications/T

4

0.06

0.0001

9296

93.56

11.69

0.0001

Lines (L)

4

0.18*

0.1440*6385

168.40*29.25*

0.0820*

T×L

4

0.13*

0.0016*1113

169.13*7.96

0.0004*(R/T) × L

16

0.03

0.0003

9496

45.01

19.85

0.0002

Plants/Plot

60

0.02

0.0001

9061

24.81

7.38

0.0001(R/T)*significant at level p<0.05

Table 10: Analysis of variance for agronomic traits
Mean squares
Source

Df

Grain

Grain

100-

Dried

Biomass

Harvestweight

yield

grain

plant

(g)

index

per

per

weightweightplant (g)

plant

(g)

(g)

(g)
Treatments (T)1

267.67*79.88*

0.41*

596.86*65.13

0.040*

Replications/T

4

20.42

9.80

0.10

173.80186.630.010

Lines (L)4

104.95*58.33*

2.16*

636.93*1214.72*

0.009*

T×L

4

124.87*55.91*

0.21

350.76*864.66*

0.005(R/T) × L

16

21.29

7.65

0.71

170.03173.51

0.006

Plants/Plot

60

20.59

11.94

2.25

110.17167.49

0.003

(R/T)

*significant at level p<0.05

49Part 4: Evaluation of Nitrogen uptake in advanced mutant lines of MR219-4 (NMR151 /PBR 0159) and MR219-9 (NMR 152/ PBR 0156)
The advanced mutant line MR219-9 (NMR152/PBR0156) showed comparable
growth, yield and N uptake under both flooded and non-flooded conditions (Table 11).
The yield and yield components are not significantly different from the parent (MR219)
but total N uptake was lower than MR219 regardless of water regime. The field trial
showed that MR219-9 (NMR152/PBR0156) has a better total N content which iscomparable to the aerobic rice check variety (MRIA 1) and this indicates that thisadvance mutant line MR219-9 is a potential aerobic rice variety. However, further
research is needed for the development of a comprehensive agronomic management
procedure of MR219-9 (NMR152/PBR0156) rice mutant line.

Table 11: Total N uptake and 15N of MR219-9/NMR152/PBR0156
under flooded and non-flooded condition

Part 5: Screening of Foliar blast disease in selected potential mutant lines
As for disease screening the data revealed that, there were only slight diseaseprogress from 21 DAS to 28 DAS except for control (MR219) (Figure 10). At 21 DAS, all
the test lines were scored within the range 3-4, which showed moderately resistant (MR)while at 28 DAS, the test lines scored 4-5, which signified moderately resistant (MR) to
moderately susceptible (MS) but the control check MR219 scored 7, which wassusceptible. Thus, this study indicated that NM2 (ML3), NM3 (ML10) and NM5 (NMR
151) could be slightly better candidate for foliar blast resistant as compared to NM1(ML30) and NM4 (NMR 152).

50

(Source : MARDI)

Figure 10: a) Observation disease reaction after 28 days (DAS),b) Folia blast scale index,c) Test lines rows along with both side bombardment rows,d) Test lines seeds sown in rows between the two inoculum beds.

Part 6: Evaluation on effects of MR219 mutant rice on glycaemic responses in BALB/c
mice
Mean comparisons and blood glucose response among the treatments
The ANOVA revealed highly significant differences among the treatments, samplingtime and also in the interaction of treatments with sampling time. Most of blood sugar
level of food samples reached its maximum reading at 60 min after feeding andsignificantly dropped at 120 min (Table 12). The mice were recorded to have the highest
blood glucose level at this particular time where the carbohydrates of the food were fully
digested and reacted in blood glucose change. The extent and duration of the blood
glucose response depend on the absorption rate which in turn depends on factors suchas gastric emptying as well as the rate of hydrolysis and diffusion of nutrients in the gut(FAO-WHO, 1998).
Table 13 shows the mean values for glucose reading among the treatments. Basedon the results, the highest blood glucose reading was recorded in mice fed with standard
51

glucose (8.11 mmol/L). Three food samples namely; ML18, ML21, and the parental line
MR219 caused a pre-diagnosis of diabetes on the BALB/c mice. The glucose reading of
the blood collected from mice fed with those three food samples were 5.96, 6.34 and 6.21
mmol/L, respectively. Meanwhile, two food samples namely; ML3 and ML30 showednormal glucose level with the values of 5.47 and 5.49 mmol/L, respectively. The low
glucose reading was observed in blood collected from mice that were fed with the checkvariety MRQ74 and saline water (4.87 and 4.78 mmol/L, respectively).
Table 12: Glucose readings after given test samples

Table 13: Mean glucose reading and glycemic index of treatments

The glycaemic index (GI) is the incremental area under the blood glucose response
curve, and is shown in Figure 11. The higher the amylose content of each food samples,the incremental of the area under curve became smaller and resulted in a low GI.
Although the relationship between rice starch and GI is complex, amylose content isgenerally accepted as being the principal determinant of GI in rice (Larsen et al., 2000).
52

Here, ML3 and ML30 both had moderate GI rating with a value of 65.00% and 66.00%,
respectively. Amylose content of ML3 was the highest among other mutant lines andparental variety, resulted in low GI as compared to others. Meanwhile, ML21 had thehighest GI value (75.00%) because of its low amylose content (20.70%). Amylose is atightly packed structure. Thus, it is more resistant to digestion. It breaks down more
slowly, releasing glucose more gradually into the bloodstream and lowers the insulin
demand. High amylose rice is less sticky, and have a much lower glycaemic load, which
could be beneficial for diabetic patients. As a conclusion, high amylose content foodshould have a low GI and vice versa. Some researchers reported that low amylose content
rice has high GI than intermediate and high amylose content rice (Panlasigui, 1989;
Juliano et al., 1989a; 1989b). However, in the present work, the parental variety MR219
amylose content was higher than ML30 but it had high GI value as compared to ML30.
This scenario might be due to different response of some variety or rice mutant lines
crossed the sampling time or due to a delayed enzymatic hydrolysis as mentioned byO'dea et al. (1980).

Figure 11: Blood glucose response curves of tested foods at 30minutes intervals

Conclusion
In summary, the data obtained from the farmers revealed that NMR 152 hadconsistently produced between 9-10 t/ha in granary area as compared to 6 t/ha producedby other varieties within the same planting areas. Field trials also showed that the
production cost was reduced by 10%, mainly due to the reduction in fertilizer andpesticide usage. At the same time, the yield could be increased between 40 - 60%,53

depending on the planting areas. Ion beam irradiation at 60 Gy significantly induced thegenetic variability in physicochemical characteristics and nutritional compositions. The
estimation of glycemic index revealed that two mutant lines could be consumed bydiabetic patients. The two mutant lines (ML3 and ML30) were recorded to have normal
glucose reading which was identified to have a moderate GI of 65 and 66, respectively.
As low and moderate GI foods are recommended for diabetic patients, these two mutants(ML3 and ML30) have a high potential for their consumption. The findings also raise thevalue of knowing the GI in our food for awareness. Further study should be carried out
on ML3 and ML30 by testing these mutant lines on diabetic patients. As for mutationinduction from variety Pongsu Seribu2 using ion beam, data from the M6 generation
showed that the highest yield was significantly observed in MINT 4 (10.04 ± 3.38 t/ha)
and followed by MINT 3 (9.52 ± 2.80 t/ha), MINT 10 (9.42 ± 3.24 t/ha), MINT 5 (8.68 ±2.99), MINT 9 (8.46 ± 0.43).
In conclusion, mutation breeding is still one of the promising techniques for thedevelopment of new and novel varieties in combination with advanced molecular
genetics that can bring plant mutation breeding into new era. Apart from that, thecurrent FNCA project fits well in addressing the climate change issue. The mutant andorganic input will help in mitigating production issues (low yield, less water resources,
soil fertility) in rice cultivation affected by environment impact as a result of climatechange.

Acknowledgements
The authors would like to thank Ministry of Science, Technology & Innovation(MOSTI) and Malaysian Nuclear Agency for providing the fund to carry out this SubProject on Composition or Quality of Rice coordinated by FNCA of Japan. The authors
are grateful to the staff at the ion-beam facility at Takasaki Advanced Radiation
Research Institute, QST, Japan. Special thanks go to Ms. Aki Koike from NSRA for her
considerable patience, liaison and organizational skills that were very instrumental forthe success of this project. The author also would like to acknowledge Dato Mansor bin
Haji Md Noor from HMN (M) Sdn Bhd for his technical assistance during LVT trial plot
and commercialization process. A special thanks also goes to Mr Mohammad Zaidi
Shaari from BAYER (M) Sdn Bhd for his assistance in the coating technology.

54

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