FNCA／MEXTTechnical Material

Achievement
Sub-Project on Mutation Breeding of Ricefor Sustainable Agriculture( FY 2013 - 2017 )Mutation Breeding Project
Forum for Nuclear Cooperation in Asia (FNCA)
June, 2020

Forward
Mutation breeding is not a new technology but its continued application throughout theworld proves its usefulness. Mutation breeding is a simple and ubiquitously applicabletechnique, and is advantageous in improving one or a few specific traits of the preferredvariety. The rice is the most important staple food crop particularly in Asian countries.
The fist subproject on rice under the FNCA Mutation Breeding Project, namely“Composition or Quality Improvement in Rice”, was successfully implemented from
FY2007 to FY2012. Subsequently, the subproject “Mutation Breeding of Rice for
Sustainable Agriculture” was carried out from FY2013 to FY2017 with ten participating
countries. This report covers the research activities of the last subproject.
Sustainable agriculture is a very important issue that we have to address under the
global climate change. The last subproject was aimed to develop mutant varieties andpromising mutant lines that contribute to the sustainable agriculture. Most of theparticipating countries succeeded to obtain high-yielding mutant lines with enhanced
tolerance to biotic and/or abiotic stresses. Some mutant lines have already been released
and brought economic benefits. We have to say that sustainable agriculture cannot be
achieved in a short time; however, the last subproject was successful in that people
involved in this subproject started to consider what we should do and demonstrated thatthe mutation breeding can contribute to this issue.
I believe mutation breeding technology has been built on years of research experience.
I hope this report can be of any help to share the knowledge among the people involvedin this research field.
Finally, on behalf of the people involved in this subproject, I would like to extend my
gratitude to the Ministry of Education, Culture, Sports, Science and Technology, Japan,
and Nuclear Safety Research Association for their continuous support.
April, 2020
Yoshihiro HASE
Project Leader of Japan, Mutation Breeding Project, FNCA
Senior Principal Researcher,Takasaki Advanced Radiation Research Institute,
National Institutes for Quantum and Radiological Science and Technology (QST)

Contents
1. Bangladesh .......................................................................................................................1
Development of Rice Mutant Variety with Higher Yield and Improved
Agronomic Traits Through Carbon Ion Beam Irradiation.
2. Indonesia .........................................................................................................................11
Breeding of Rice Variety(s) for High Yielding and Early Maturity
Through a Wide Cross and Mutation.
3. Japan ...............................................................................................................................19
Trials on Cross and Mutation Breeding of Rice for Adaptability
to Nature Farming
4. Malaysia ..........................................................................................................................30
Mutation Breeding of Rice for Sustainable Agriculture in Malaysia.
5. Mongolia .........................................................................................................................59
Improvement of Wheat Productivity Through Mutation Techniques.
6. The Philippines ..............................................................................................................66
Improvement of Traditional Rice Varieties by Gamma Irradiationin the Philippines.
7. Thailand ..........................................................................................................................77
Application of Mutation Breeding of Submergence tolerance Rice for
Sustainable Agriculture, in Thailand.
8. Vietnam ...........................................................................................................................89
Mutation Breeding of Rice for the Sustainable Agriculture in Vietnam.

Development of Rice Mutant Variety with Higher Yield and Improved
Agronomic Traits through Carbon Ion Beam Irradiation
A. N. K. Mamun1, M. H. Kabir1, A. K. Azad2, M. A. Azam2, M. R. Islam1, M. T. Jahan1,
Md. Monirul Islam1, Pronabananda Das1 and Md. Monirul Islam1
1

Plant Biotechnology and Genetic Engineering Division, Institute of Food andRadiation Biology, Atomic Energy Research Establishment, Bangladesh Atomic
Energy Commission, Bangladesh,

2 Plant Breeding Division, Bangladesh Institute of Nuclear Agriculture, Bangladesh.

Summary
BINA dhan14 is becoming more popular to the farmers due to the advantage ofhigher yield, early maturity, long grain fine rice and less water requirement over parent
as it is cultivated in rain-fed condition. Advanced promising mutant line obtained from
BRRI dhan 29 was released as a variety called BINA dhan18 and BINA dhan19 wasdeveloped from NERICA10. These varieties were developed through carbon ion beamirradiation from the QST, Takasaki, Japan with kind cooperation of FNCA during theperiod of late 2013 to early 2018. The main criteria of these varieties are lodging resistantwith higher yield, long grain fine rice. BINA dhan14 is cultivated during Boro and Aus
season, BINA dhan18 is moderate duration with higher yield Boro rice and BINA dhan19
is highly drought tolerant and also cultivated round the year. BINA dhan18 is about 100
cm tall and requires 148-153 days (seed to seed) to mature. Maturity was observed 1315 days earlier than that of the parent variety BRRI dhan29. Thousand grain weight isabout 26.5 g, amylose and protein contents are 23.2% and 7.06%, respectively. The
average yield of 7.25 t/ha and the highest yield of 10.5 t/ha was recorded in favorable
environmental condition. The average yield of BINA dhan19 is about 3.84 t/ha and the
maximum yield is 5.0 t/ha. The plant of BINA dhan19 is about 80-90 cm tall and does
not get lodged. During severe drought the plant became almost stop growing but whenrain is started then it starts growing vigorously and gave almost equal yield likefavorable condition. It matures within 90-105 days and thousand grain weights is about
23 g. The grain contains 7.32% protein and 23.8% amylose. Efforts are continuing
towards indigenous rice cultivars of Lombur, B-11 and Hori dhan to develop new mutant
variety with higher yield, improved agronomic traits and tolerant to abiotic stress.

1Introduction
The continuous increase of population in the country demands more food. In turn,it requires more land for rice cultivation as rice is the staple food for 166 million people
in Bangladesh and also the most important food crop in Asia and rest of the world(AIS, 2008). We are facing severe threat of climate change challenges in crop sector.
In Bangladesh, rice is grown three seasons in a year i.e., Aman season (July to
December), Boro season (December to May) and Aus season (March to June). Riceprovides 75% of total calories and 51% of protein in our population. About 60% of thetotal agricultural labor force is engaged for rice production (AIS, 2008). It covers
77.96% of total cropped area in Bangladesh (AIS, 2007) and contributing 14.6% of ournational GDP (BBS, 2004). It is a diploid species, with a small genome in comparison toother cultivated cereals (Moin et al., 2017). The average rice yield in the country is
about 2.5 t/ha. It is very low yield comparing to other rice growing countries in theworld (Islam, 1989). The low yield performance of rice in the country is the effect of
the cultivation of low yield potential genotypes, drought, flash-flood and soil salinity.
Salinity is also one of the significant abiotic constraints to rice cultivation in the
coastal areas of Bangladesh. Salt stress brings about huge losses in worldwide
agricultural productivity (Moradi et al., 2003; Yu et al., 2016). In the world, about 400
million hectares of land are affected by high soil salinity and about 1 million hectaresof land are affected by high salinity in the coastal regions in Bangladesh (Karim et al.,1990). Salinity hindrances almost every aspect of the physiology and biochemistry ofplants and significantly reduces yield. A number of indigenous cultivars are prone tolodging but had some degree of tolerant to salinity and submergence. Its grains are
medium bold and contains 10.3% protein and 28.6% amylose (Kamruzzaman andAsaduzzaman, 2004). Mutation Breeding Project of FNCA aimed for breeding
technology with major crops using irradiation either by gamma-ray or by ion-beam.
Mutations are the major source of genetic variability and artificial mutations can be
induced by mutagens (Wei et al., 2013; Oladosu et al., 2016). Many reports are available
in induced mutation of rice for genetic improvements with higher yield and otheragronomic traits (Azam and Uddin, 1999; Hidema et al., 2003). Recently, ion beamirradiation has been appeared to utilize an effective means of inducing mutations.
The biological effects of ion beams have also been investigated and observed to beshown a high relative biological effectiveness (RBE) in lethality, mutation and also totransfers high energy to the target compared to low linear energy transfer (LET)
radiation such as Gamma-rays, X-rays and electrons (Blakely, 1992). It has been also
demonstrated that ion beams induce mutations at high frequency and induce novel2

mutants in Arabidopsis (Hase et al., 2000, Shikazono et al., 2003, Tanaka et al., 1997,
2002). The use of ion beams for inducing mutations in rice (Oryza satva L.) breeding
has also been attempted (Hayashi et al. 2007; Hidema et al. 2003; Rakwal et al. 2008).
It is generally and widely accepted that in mutation breeding induced traits become
fixed during M 2 to M 4 generations (Azad et al., 2010; Hamid et al., 2006;
Shamsuzzaman et al., 1998; Azam and Uddin, 1999). Indigenous genotypes have been
indicated as the tremendous sources of genes for new alleles (Evenson and Gollin, 1997;
Guevarra et al., 2001; Hoisington et al., 1999; Jackson, 1999; Tanksley and McCouch,1997). Based on the information mentioned above, the study was undertaken todevelop new mutant variety with higher yield and yield contributing traits among the
local rice genotypes in Bangladesh using carbon ion beam irradiation. In rice, mutants
were developed from immature embryos (Ookawa et al., 2014), calli obtained from seeds(Serrat et al., 2014) and cell suspension cultures (Chen et al., 2013). Seeds are, however,easier to handle and do not require a specialized structure, and is therefore, the mostwidely used material (Da Luz et al., 2016; Oladosu et al., 2016). Screening of existinglandraces as well as creation of new mutant variety with improved salt tolerant traits
is also a major goal for future study. In this study, we aimed to describe the possibility
to get mutants with higher yield, photoperiod insensitive, and shorter plant height fromlocal T. aman rice genotypes Ashfal and also to get mutant with higher yield and earlymaturity from BRRI dhan29 and it is extended to obtain mutant from NERICA10 in riceby ion beam irradiation.
Materials and Methods
Hulled rice seeds of Ashfal, BRRI dhan29 and NERICA10 were exposed to 26.7
MeV/n carbon ions with different doses of 0, 10, 20, 40, 60, 80, 100, 120,140, 160, 180
and 200 Gy at Japan Atomic Energy Agency (currently QST, Takasaki, Gunma, Japan).
One hundred and fifty seeds were used for each irradiation dose. The irradiated seeds
were allowed to germinate in petri-dishes. Germinated seeds were sown in seed bed to
grow seedlings. Seedlings were transplanted in the field. Single seedlings weretransplanted into each hill. Seedlings were transplanted at the PBGED experimental
field, IFRB, AERE, Savar, Dhaka, and also at the experimental field of BINA,
Mymensingh. Plot size was 5.0 x 2.0 m2 and distance between rows and plants weremaintained 20 cm and 15 cm respectively. The experiments followed RCB design withthree replications. Recommended dose of fertilizers, cultural and intercultural
practices were done as and when required. Porous pipe was depth into 15 cm of soil tomaintain irrigation in rice field. All

fertilizers were applied during final land3

preparation excluding urea. Urea was applied at three equal installments at 7, 30 and
55 days after transplanting. Three hand weeding were made and the plots were kept
saturated with irrigation till maturity during Boro season. Heights were measured
randomly from ten mutant plants of each irradiated dose at the time of harvesting.
Number of days to heading was recorded as the number of days required from sowing
time when 50% of plants of each line headed. Days of maturity was recorded as thenumber of days required from sowing time when 90% of plants of each line appearedwith yellowish grains. These two data were recorded through visual observation byvisiting the plots every alternate day. Number of effective tillers per plant and panicle
length was measured at the time of maturity with randomly selected five competitiveplants. Filled and unfilled grains/panicle was recorded from five hills per plot at harvest.
Grain yield was recorded from 1 m2 area which was later converted to yield at ton/ha at
14% moisture. Moisture data was recorded with a grain moisture meter.
Results
Table 1 showed that BINA dhan18 was cultivated at seven different zones in
Bangladesh to observe the growth uniformity and yield performance. Table 2 indicatedthat data of yield attributing traits of mutant (RM(2)-40(C)-1-1-10) and parent (BRRI
dhan29) are non-significant. Maturity period and yield was found significantly
different between mutant and parent and also among the location cultivated in
Bangladesh (Table 3). It was observed that highest (7.8 t/ha) yield with short (145days) duration was observed at mutant grown at Pakkhifanda, Rangpur area over theparent. Table 3 also indicated that mutant yielded higher and required short duration
to mature over parent at every location grown in Bangladesh. Table 4 proved thatmutant produced long grain fine rice as grain length was recorded 9.0 (7.4) mm overparent 8.0 (6.5) mm.

4

Table 1. Multi location trial of BINA dhan18 at different sub-station of BINA.

Location

Date of seed sowing

Age ofDate oftransplanting

seedlings(days)

BINA farm, Mymensingh

02 December 201309 January 201439

Maijbari, Mymensingh

02 December 201310 January 201440

02 December 201314 January 201444

02 December 201314 January 201444

01 January 2014

12 February 201443

01 January 2014

13 February 201444

02 December 201331 January 2014

61

BINA sub-station farm,
MaguraFarmer’s Field, Magura
BINA sub-station farm,
Rangpur
Pakkhifanda, Rangpur
BINA sub-station farm,BarisalTable 2. Yield attributing traits of mutant (RM(2)-40(C)-1-1-10) over parent (BRRI
dhan29).
Mutant/variety

Plant

Effective

PanicleFilledUnfilledheighttiller (no.)

lengthgrains/grains/(cm)

paniclepanicle

(no.)

(no.)

(cm)
RM(2)-40(C)-1-1-10

101

11.42

24.7

113

33

BRRI dhan29

100

11.87

24.5

111

33

LSD(0.05)

NS

NS

NS

NS

NS

NS- not significant

5

Table 3. Comparison of maturity period and yield of mutant (RM(2)-40(C)-1-1-10) withthe parent variety (BRRI dhan29) at different sub-station of BINA.
Location

Maturity period (days)
RM(2)-40(C)Yield (t/ha)

BRRI dhan29-1-1-10

RM(2)-40(C)-1-1-10

BRRI dhan29

BINA farm, Mymensingh

150

165

6.9

6.5

Maijbari, Mymensingh

152

165

5.8

6.3

157

168

7.6

5.7

155

168

5.8

5.5

155

165

7.5

7.1

145

160

7.8

7.2

155

170

5.1

4.9

Farmer’s Field, Barisal

151

162

5.2

5.2

LSD (0.05)--

Average

153

165

BINA sub-station farm,
MaguraFarmer’s Field, Magura
BINA sub-station farm,
Rangpur
Pakkhifanda, Rangpur
BINA sub-station farm,Barisal

0.36
6.46

6.05

Table 4. Comparison of grain characters of the mutant (RM(1)-40- (C)-1-1-10) and theparent variety (BRRI dhan29).
Mutant/variety
RM(1)-40(C)-1-1-

1000-grain

Grain length

Grain

Length:weight (g)(mm)

breadth (mm)breadth ratio

26.0

9.0 (7.4)

3.0 (2.5)

3.0 (2.96)

20.6

8.0 (6.5)

2.5 (2.2)

3.2 (2.95)

10
BRRI dhan29

Numbers in the parentheses indicate the values of dehusked grain.

6

Control (NERICA10)

BINAdhan19 (Mutant)Discussion
On the basis of lethality, ion beams induced higher frequencies of mutation than
gamma rays. In view of this, seeds of Ashfal rice, BRRI dhan29 and NERICA10 were
irradiated with different doses of carbon ion beams and were grown in Boro and Aman
season. These results showed that photoperiod sensitivity was altered by carbon ion
irradiation in case of BINA dhan14 whilst it was developed from highly photoperiod
sensitive cultivar Ashfal. It was also found that plant heights of the three studied
land races at M 2 progenies were significantly shortened than that of their parents.
This is also observed consistent with the fact that the fertile M 2 plants had shorter
plant height than their parents. This might be due to the fact that the fertile mutant
lines shifted from vegetative phase to reproductive phase earlier than parents and it
is caused by ion beam irradiation. It has been reported that one dominant and one
recessive gene are responsible for photoperiod sensitivity in rice (Yu and Yao, 1968;
Yokoo and Fujimaki, 1971; Yokoo et al., 1980). It could be happened that carbon ion
beams irradiation inactivated or down regulated the activity of the dominant gene in
the irradiated seeds which makes the mutant lines homozygous monogenic recessive
for photoperiod insensitivity. BINA dhan18 showed higher yield and early maturity
compared to parent of BRRI dhan29 which could be due to the useful effect of carbon
ion beam. BINA dhan19 also showed higher yield, early maturity and highly droughttolerant compared to parent of NERICA10 which also thought to the useful effectof carbon ion beam. It was obvious from the study that BINA dhan14 was developed
using 200 Gy and BINA dhan18 and also BINA dhan19 was developed using 40 Gy
7

respectively through carbon ion beam irradiation. The rest of the doses of theparticular genotypes under investigation were omitted from these evaluations
because they were prone to lodging for weak culm although they had shortened plants.
Moreover, these progenies had shorter panicle with smaller grains and lower numberof seed grains and also their inferior performance. Although the grain characteristics
such as size and shape altered in case of most of the mutants but grain weight almost
similar as the parent. Hence, it could be concluded that it is possible to induce fixedmutants in indica type rice Ashfal, BRRI dhan29 and NERICA10 through 40-200Gy
doses of carbon ion beams irradiation. This result is also in agreement with the resultsof Honda et al. (2006) who reported genetically fixed mutants in M 1 generation ofsweet pepper for dwarf height and yellow pericarp. Therefore, all of these suggestthat heavy ion beams irradiation has unique properties that induce fixed mutation.
Acknowledgements
This work was carried out under the Forum for Nuclear Cooperation in Asia
(FNCA) and IAEA/RCA Mutation Breeding Project. Special thanks to Dr. Atsushi
Tanaka, Dr. Hase, National institutes for Quantum and Radiological Science andTechnology (QST); Ms. Aki Koike, Nuclear Safety Research Association (NSRA); Dr.
Liu Luxiang, RCA Coordinator of China for their kind help and cooperation.
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10

Breeding of Rice Variety(s) for High Yielding and Early Maturity Through a
Wide Cross and Mutation
Sobrizal
National Nuclear Energy Agency, Center for the Application of Isotopes and Radiation,
Jl. Cinere Pasar Jumat P. O. Box 7002 JKSKL, Jakarta 12070 Indonesia.
Abstract
To fulfill the Indonesian domestic demand in which the annual population growth isstill more than 1.4%, Indonesian rice production should be increased. This can beachieved by increasing rice productivity and harvesting index through growing highyielding and early maturity of rice varieties. In previous research, breeding lines thathave wide genetic diversity have been constructed through Indica-Japonica cross of
IR36/Koshihikari. To gather the desirable characters in one lines some of breeding lines
were crossed each other and some improved lines were obtained. To remove undesirable
characters in improved lines some of these limes were irradiated by gamma ray and 12
promising mutant lines with high yield and early maturities were selected. The growthdurations of these mutant lines were ranging from 93.7 to 99.3 days from sowing toharvesting, significantly shorter than those of original line SKI 88, national leading
variety Ciherang and the early maturity national leading variety INPARI 13. Yield of
most mutant lines were not significantly difference with that of original line, SKI 88
and national leading variety, Ciherang, but significantly higher than that of INPARI 13.
To fulfill the requirement of variety release in Indonesia, other multi-location yieldtrials and other examinations such as pests, diseases as well as other grain quality
examinations should be conducted.
Key words: rice, high yield, early maturity, wide cross, mutation

Introduction
Rice is a staple food of majority Indonesian population. In the mid-1980s Indonesia
firstly achieved self-sufficiency for rice, however the growth of rice production has
slowed down since the 1990s (BAPENAS, 2002). The decline in rice production isfrequently attributed to loss of rice fields caused by conversion to non-agricultural uses,
11and to the declining productivity. To fulfill the domestic demand in which the annual
population growth is still more than 1.4 %, Indonesian rice production should be
increased. This can be achieved by increasing rice productivity and harvesting index
through growing high yielding and early maturity of rice varieties.
Since the release of IR8 varieties in 1966 (Cantrell and Hettel, 2004), the potential
for paddy production has not been significantly increased (Sobrizal and Ismachin, 2006).
The formation of new varieties is only able to maintain the potential of IR8 by
improving the growth duration, grain quality, and its adaptability to environmental
stress. The narrowness of the genetic diversity of the released rice varieties contributes
to the occurrence of slowed down of growth yield potential. Many released rice varieties
are related to each other, so the diversity is less and yield potentials are almost same
(Susanto et al., 2003). In 1982 the national average production was 4.04 t / ha, while in2016 it was 5.24 t / ha (BPS, 2017), so the 34-year increase was only 1.20 t / ha. In order
to increase the yield potential for future, different genetic resources are needed in the
new varieties assembly program.
In previous research, breeding lines that have wide genetic diversity have been
constructed through Indica-Japonica cross of IR36/Koshihikari (Sobrizal, 2008). Intra
sub-specific crossing with large genetic distances between the two parents allow for theoccurrence of transgressive segregation so that it will produce a progeny with arelatively large genetic diversity. Transgressive segregation is the emergence of anindividual in a segregated population in which the individual phenotype exceeds that ofboth parental phenotypes and is commonly found in progeny derived from inter andintra-specific crosses (Vega and Frey, 2004). Great genetic diversity will provide
flexibility in selection, including selection towards increased yield potential. The
selected plants are purified to become pure lines, and can be used as breeding materials
in subsequent breeding programs. By using these unique germplasms as well as
mutations 12 rice promising lines with high yield and early maturities have been
constructed. The objective of this research is to develop high yielding and earlymaturities of rice varieties.
Materials and Methods
Construction of Breeding Materials
To increase the genetic variability of breeding materials, IR36 and Koshihikari
were crossed, and developing of pure lines from this cross was conducted as described in
Sobrizal 2013. To gather desirable characters in a line some of selected pure lines were12

crossed as shown in Fig. 1. From these crosses selected pure lines (SKI 64, SKI 88, SKI
153, and SKI 276) were used as further breeding materials. These lines showed good
performance with high yield potency, but long growth durations. To reduce their growthdurations, 50 gram seeds of each line were irradiated by 200 Gy of gamma ray at Centerfor the Application of Isotopes and Radiation Technology, Pasar Jumat, Jakarta.
Irradiated M1 seeds were sown, and twenty day-old seedlings were transplanted to
paddy field by planting a single seedling per hole at experimental field, Sawangan,
Depok with 20 cm spacing between plants.
Five hundred M1 plants were harvested individually to obtain the M2 seeds.
Harvesting was conducted by collecting only one main panicle in each M1 plant. M2
seeds were sown, and 20 M2 plants derived from each M1 plant were transplanted todevelop M2 lines. Selections of early maturity plants were conducted in each M2 lines
and selected plants were harvested individually.
Selected early maturity plants were purified up to M5 generations, then, selected
pure lines were designated as RSKI #. RSKI lines were subjected to yield trials.
Breeding scheme of plant materials used in this study can be shown in Fig. 1.

Fig. 1. Breeding scheme of plant materials used in this study.
Yield trials
Yield trials were conducted using 12 selected early maturity mutant lines, checkvarieties of original line SKI 88, national leading variety Ciherang and national leading
early maturity variety INPARI 13. Seedlings were transplanted to plot with plot size of
4 x 5 m and planting spacing of 25 x 25 cm. Experiments were arranged in RBD with 3
replications.

13

Results and Discussion
In this study, all the F1 plants derived from crosses of KI lines as in Fig. 1 grew welland selection was conducted in F2 population with emphasis on agronomical characters.
Selected plants were purified and selected pure lines were designated as SKI #. Among
selected pure lines, the seeds of SKI 64, SKI 88, SKI 153, and SKI 276 lines were
irradiated by 200 Gy of gamma ray. As much as 500 M1 plants were harvested
individually to obtain M2 seeds. Selection for early maturity was conducted in M2 lines
and 105 selected lines were purified up to M6 generations. From field observation of 105
lines in rainy growing season of 2012/2013, 12 homogeneous early maturity M7 mutant
lines were selected. These lines were evaluated for yield performance at Pusakanagara
Experimental Farm in dry growing season of 2013 and wet growing season of 2013/2014.
The results were shown in Table 1.
Table 1. Growth duration and yield of lines/varieties tested.

*) The numbers followed by different characters are significantly different for 5% level.

Table 1 showed that growth durations of mutant lines were ranging from 93.7 to
99.3 days from sowing to harvesting. They were significantly shorter than growthdurations of original line SKI 88, national leading variety Ciherang and the earlymaturity national leading variety INPARI 13. The growth duration of SKI 88 was 117.3
days, Ciherang was 118.7 days and INPARI 13 was 103.7 days.
14

Yield of mutant lines were ranging from 7.83 to 9.79 t/ha, while SKI 88 was 9.61
ton/ha, Ciherang was 9.45 ton/ha and INPARI 13 was 8.16 t/ha. The yields of ten
mutant lines (RSKI 64-1, RSKI 64-2, RSKI 88-1, RSKI 88-2, RSKI 88-3, RSKI 88-4,
RSKI 88-5, RSKI 88-7, RSKI 153-1, RSKI 276-1, RSKI 276-2) were not significantly
difference with that of original line, SKI 88 and national leading variety, Ciherang, but
significantly higher than that of INPARI 13. More over the growth durations of theseten mutant lines were significantly shorter than that of the national leading earlymaturity, INPARI 13. Performance of mutant RSKI 88-7 and its original plant SKI 88
shown in Fig. 2. Figure 2 shows that the panicles of mutant plants of RSKI 88-7 haveappeared while its original plant of SKI 88 has not appeared yet.

Fig. 2. Comparison of early maturity mutant line of RSKI 88-7 and its original line of
SKI 88.
To confirm the performance of growth duration and yield of selected lines, theselines were evaluated by growing them at farmer field in Musi Rawas District, South
Sumatera and the result sown in Table 2. Table 2 showed that the growth duration of all
selected early maturity lines were significantly shorter than that of original line andCiherang. Even all selected lines were significantly earlier than national early maturity
variety INPARI 13. The yields of some selected early maturity lines (RSKI 64-2, RSKI
88-1, RSKI 88-3, RSKI 88-5, RSKI 153-1, RSKI 276-1) were not significantly different
15

from that of national leading early maturity variety INPARI 13. The yields of otherselected early maturity lines were significantly lower than that of national leading earlymaturity variety INPARI 13.
Table 2. Result of yield trial of selected lines in Musi Rawas district.

*) The numbers followed by different characters are significantly different for 5% level.
To further evaluate the yield performances, selected early maturity lines were
subjected to yield trials in five locations. The highest average yield in five locations is8.58 t/ha for RSKI 64-2 and followed by 8.48 t/ha for RSKI 153-1 and 8.48 t/ha for RSKI
276-2, whereas the average yield of an original line SKI 88, a national leading variety
Ciherang, and a national leading early maturity variety were only 8.06 t/ha, 8.22 t/ha,
and 7.54 t/ha, respectively (Table 3). Multi-location yield trials to reach at least 16
locations as well as other examinations such as pest and diseases, grain quality should
be continued as a requirement of variety release in Indonesia.

16

Table 3. Yield of tested lines in 5 locations of yield trials.

*) The numbers followed by different characters are significantly different for 5% level.
Conclusions
Based on the results it can be concluded as follow;
1.

The growth durations of mutant lines were ranging from 93.7 to 99.3 days fromsowing to harvesting, they were significantly shorter than those of original line SKI
88, national leading variety Ciherang and the early maturity national leading
variety INPARI 13.

2.

Yield of most mutant lines were not significantly difference with that of originalline, SKI 88 and national leading variety, Ciherang, but significantly higher thanthat of INPARI 13.

3.

To fulfill the requirement of variety release in Indonesia, other multi-location yieldtrials and other examinations such as pests, diseases and other grain quality
examinations should be conducted.

Acknowledgement
Thanks to my colleagues at the Division of Irradiation and Instrumentation CAIRT– NNEA for their helps in seed irradiation, and my colleagues at Plant Breeding
Laboratory, CAIRT – NNEA, especially Mr. Carkum, SP., for his excellent technicalassistance.
17

References
BPS 2017. www.bps.go.id
BAPENAS/DEPARTEMAN PERTANIAN/USAID/DAI FOOD POLICY ADVISORY
TEAM.
2002.
Indonesianfoodpolicyprogram.
(Available
atwww.macrofoodpolicy.com)
CANTRELL, R. P., HETTEL, G. P. 2004. Research strategy for rice in the 21st century.
Proceeding of World Rice Research Conference, Tsukuba International Congress
Center (Epochal Tsukuba) Tsukuba, Ibaraki, Japan.
SOBRIZAL. 2013. Improvement of yield and grain quality of rice through a wide crossand mutation breeding. Achievement Sub-Project on Composition or Quality in
Rice (2007 – 2012), Mutation Breeding Project, Forum for Nuclear Cooperation in
Asia (FNCA) March, 2013
SOBRIZAL, ISMACHIN, M. Peluang mutasi induksi pada upaya pemecahan hambatan
peningkatan produksi padi. Jurnal Ilmiah Aplikasi Isotop dan Radiasi 2 (2006)
50.
SUSANTO, U., DERADJAD, A. A., SUPRIHATNO, B. Perkembangan pemuliaan padi
sawah di Indonesia. Jurnal Litbang Pertanian. 22 (2003) 125.
VEGA, U., FREY, K. J. Transgressive segregation in inter and intraspecific crosses ofbarley. Euphytica 34 (1980) 875.

18

Trials on Cross and Mutation Breeding of Rice for Adaptability
to Nature Farming
Hirokazu NAKAI*, Yoshiyuki NARA*, Chiharu SUZUKI*, Hiroyasu TABUCHI*,
Shigeki NOZAWA** and Yoshihiro HASE*** Institute for Agriculture, Medicine, and the Environment,** Takasaki Advanced Radiation Research Institute, National Institutes for Quantum
and Radiological Science and Technology (QST)
Summary
We have tried to develop rice varieties, through cross and mutation breeding foradaptability to nature farming in these past more than ten years. We succeeded inselecting breeding lines for adaptability to nature farming, in the hybrid populations
from the cross of representative native varieties of Japan, ‘Asahi’ and ‘Kamenoo’. Twobreeding lines, namely AKH2 and AKH4, which were of high yield with high eating
quality in nature farming, were selected from the cross breeding. However, the selected
breeding lines were too late for heading date to be adaptable to wide range of Japanisland other than southern part of Japan e.g. Kyushu district. We tried to improve thenegative trait for late heading of the breeding lines through the use of induced mutations
within five years. This paper refers to the breeding process and discuss on the merit ofmutation breeding and on how to use the technique for gaining adaptability to low input
sustainable agriculture.
Introduction
Conventional agricultural systems dependent on chemical fertilizer and agriculturalchemicals should be converted to low input sustainable agriculture with less amount ofchemicals in the situation of serious climate change covered in the world. It is reported
that more than 20% of all greenhouse gases, e.g. CO 2 , CH 4 , and N 2 O, might be emitted
from the field of agriculture (Nagano et al., 2012, Skinner, 2014). We have tried toperform cross breeding combined with use of induced mutations of rice for adaptability
to nature farming, aiming at developing the system of sustainable agriculture. Nature
farming is defined in this paper as a farming system that uses only residue of rice plants
as the nutrition with no agrochemicals and chemical fertilizer (Okada, 1953). We arereporting a case of the breeding performed in Yunomae-cho, Kumamoto-prefecture,
Japan. The AKH2 breeding line was finally selected for high yield and high eating
19

quality in the nature farming condition, from the hybrid population derives from thecross between Japanese native varieties, ‘Asahi’ and ‘Kamenoo’. However, the AKH2
breeding line was found to carry weak points for late heading and long culm. We havethereupon tried to improve the demerits of late heading and long culm through use of
induced mutations. Accordingly, some breeding lines with earlier heading date and/or
shorter culm length as well as higher yield in the low input conditions were selected. The
selected MAKH2 breeding line was noticed as promising new rice variety adaptable tonature farming or low input sustainable agriculture.
This paper refers to the breeding process and discuss on the merit of mutation
breeding and on how to use the technique for gaining adaptability to low input
sustainable agriculture.
Materials and methods
In 2005, Japanese native varieties, Asahi and Kamenoo were crossed at the Ohito
Experimental Farm located in Izunokuni-city, Shizuoka-prefecture located at almost
center of the main island of Japan. The 1,638 F 2 plants derived from 16 F 1 plants weregrown with planting space of 30 cm × 20 cm in the fields of nature farming of Ohito
Experimental Farm in 2007. All the harvested rice straws and 100 kg rice bran wereapplied to the rice fields as nutritional materials. The 140 plants were selected by eyemeasurement from all the F 2 plants in the fields. In 2008, 140 F 3 lines originating fromthe selected F 2 plants were grown and 36 F 3 lines were selected by eye measurement in
the field. The selected F 3 lines were measured for wight of grain and eating quality using
the measuring instrument (Kett Electric Laboratory, Tokyo, Japan).
Table 1 shows the breeding process performed from 2009 to 2017 in Yunome-town,
Kumamoto-prefecture in Kyushu-district, Japan. In 2009, seeds of the selected 36 F 3lines were sent to a farmer, Mr. SHIIBA, Takema in Yunomae-town, Kumamotoprefecture to be grown as F 4 lines in the fields of nature farming. In the selection tests,
any nutritional matters were never applied to the breeding field.
Ten or less breeding lines were used for adaptation and yield tests from 2013 to 2015,
after selection tests up to 2012. In the adaptation and yield tests, the selected 10 or lesslines were grown in randomized block design with three replications (60 plants weregrown per one line of one block). Grain yield and eating quality were measured as wellas main agricultural traits, e.g. culm length, panicle length, number of panicles per plantand so forth. Two breeding lines, AKH2 and AKH4, were finally selected for registeringcommercial cultivar patent. AKH2 was, in fact, registered as commercial cultivar‘Kumaminori’ in 2018.
20

Table 1. The process of cross breeding of native varieties, Asahi and Kamenoo, performedin the farmer’s fields in Yunomae-town, kumamoto-prefecture, Kyushu-district(2009~2017).
Year

2009

2010

2011

2012

2013

2014

2015

2016

2017

F4

F5

F6

F7

F8

F9

F 10

F 11

F 12

36

23

17

14

10

7

6

5

2

Hybridgeneration andnumber of lines
grown

However, both breeding lines carry a week point for late heading not to be adaptable
to wider range to the northern part in Japan. In such a situation, we tried to improvethe demerit of the breeding lines through the use of mutation breeding. In 2013, about
5,000 F 8 seeds of AKH2 and AKH4, were irradiated by gamma-rays of 200 Gy at Japan
Atomic Energy Agency (currently Takasaki Advanced Radiation Research Institute,
National Institutes for Quantum and Radiological Science and Technology). About 4,000
M 1 plants each of AKH2 and AKH4 were grown in the fields at the Ohito Experimental
Farm. Seed fertility was checked using randomly selected 50 M 1 plants. The selection
process for early heading of the mutants is shown in Table 2. As shown in the Table 2,
after 5 years since the irradiation time, two noticeable mutants were selected from the
population of AKH2 in 2018.
Table 2. Selection process for early heading of rice mutants from the irradiatedpopulations of AKH2 and AKH4.
Original
line
AKH2
AKH4

Year and generation
2013

2014

2015

2016

2017

2018

M1

M2

M3

M4

M5

M6

4,428N*

15,840N

119L**

11L

6L

2L

14,400N

105L

14L

6L

0L

48%***
3,936N
45%****N: number of plants. **L: number of lines.
*** average seed fertility for 50 M 1 plants.

21

Results
Fig.1 shows grain yield and eating quality of the breeding lines, AKH2 and AKH4,
which were selected from cross breeding of Asahi and Kamenoo. In the figure,‘Hinohikari’ was put as a control because of it being representative leading variety in
Kyushu district. As seen in the figure, grain yield of the AKH2 was higher than AKH4
and Hinohikari. As for eating quality, significant difference was not found among the two

700

75.0

650

74.0
73.0

600

72.0

550

71.0

500

70.0

450

69.0
68.0

400

67.0

350
300

eating quality

grain yield（㎏/10a）breeding lines and Hinohikari.

66.0
AKH2

AKH4

grain yield(kg/10a)

Hinohikari

65.0

eating quality

Fig.1. Grain yield and eating quality of the breeding lines, AKH2 and AKH4, incomparison with control (Hinohikari) in the nature farming (from results for adaptationand yield tests in 2014).

We requested some farmers, who were concerned with nature farming in the Kyushu
district, to grow the three lines using farm machines. Fig.2 shows one of the results fromthe farmers on grain yield and eating quality. As seen in the figure, the AKH2 showed
the highest yield. Eating quality of rice was clearly higher in the two breeding lines thanin Hinohikari, suggesting that eating quality is to be higher in the selected breedinglines than in the popular leading variety, Hinohikari in the Kyushu district. We finallyselected the AKH2 breeding line for registering commercial variety in 2018.

22

68

440

67

420
400

66

380

65

360
340

64

320
300

AKH2

AKH4 Hinohikari

AKH2

2016

AKH4 Hinohikari

Eating quality

Grain yield（kg/10a）

460

63

2017

Grain yield（kg/10a）

Eating quality

Fig. 2. Grain yield and eating quality of breeding lines, AKH2 and AKH4 selected fromthe cross breeding in case of being grown by planting machine by a farmer in Kumamoto
prefecture in 2016 and 2017.
Fig.3 shows heading date and culm length of the breeding lines of AKH2 and AKH4
comparing with Ashahi and Kamenoo (parent varieties) and Hinohikari (arepresentative leading variety in Kyushu area). As seen in the figure, heading date of
the both breeding lines was almost same as one of the parent varieties, Asahi, which wassignificantly later in heading date than Kamenoo and Hinohikari. It was noted thatheading date of the selected breeding lines was significantly later than that of the local
leading variety, Hinohikari. It may be inconvenient for farmers in the local area to growthe breeding lines because of it being late in heading date. Culm length of both breedinglines was almost same as that of the parent varieties, Asahi and Kamenoo. However, it
was found that culm length of the breeding lines was significantly longer than that of
Hinohikari. We tried to improve the weak point for late heading of the breeding lines to
get wider adaptability to grow in northern part of Japan.
Fig.4 shows the frequency distribution for heading date of M 3 mutant lines
originating from irradiation of gamma-rays to F 8 seeds of the breeding lines of AKH2
and AKH4. As seen in the figure, the earliest heading date of the mutant originating
AKH4 was put in 21~31th in July. Heading date of total 15 mutant lines from both thebreeding lines was put in 1 ~10th in September, whereas heading dates of AKH2 andAKH4 were respectively 8th and 6th in September. Heading date of the mutant lines put
in 1~10th in September seen in the figure was earlier than that of both the breedinglines, AKH2 and 4. Anyhow, mutant lines put in 1~10th in September were not so23

practically effective as breeding materials for early heading.

9/18
9/13

100

9/8

80

9/3
8/29

60

8/24

40

8/19

20
0

heading dateculm length (cm)

120

8/14
AKH2

AKH4

asahi

kamenoo hinohikari

8/9

culm length (cm)heading dateFig. 3. Heading date and culm length of the breeding lines, AKH2 and AKH4 selectedfrom the cross combination of Asahi and Kamenoo (2014).

12

Frequency

10
8
6
4
2
0

2131July

110Aug.

1120Aug.

2131Aug.

110Sep.

Heading dateFig. 4. Frequency distribution of heading date of the mutant lines originating from
irradiation of gamma-ray of 20 krad to F 8 seeds of AKH4 (white bar in the figure) andAKH2 (black bar in the figure).

24

Fig.5 shows culm length and heading date of 6 mutant lines selected as breeding
materials for the relevant breeding purpose, which were originating from irradiation of
gamma-rays to F 8 seeds of AKH2. As seen in the figure, heading date of all the mutant
lines selected was significantly earlier than that of control, AKH2. Heading date (12thin August) of the mutant line, MAKH2, was about one month earlier than AKH2. In caseof MAKH2-7, heading date was about one week earlier than the control (AKH2). Culm
length of the mutant lines was significantly lower than that of the control, excepting one
case of MAKH2-7. We, after all, selected two mutant lines, MAKH2-6 and MAKH2-7, of
which heading date are earlier than control, AKH2, with same or more grain yield as thecontrol.
Fig.6 shows plant type of the selected M5 mutant lines, MAKH2-7 and MAKH2-6
comparing with control, AKH2. It was found that MAKH2-7 carried much higher numberof panicles comparing with the control, though the culm length was as high as the control.
We have finally selected the MAKH2-7 mutant line as a candidate for registering new

100
90
80
70
60
50
40
30
20
10
0

9/22
9/12
9/2
8/23
8/13
8/3
7/24
7/14

Fig.5. Culm length and heading date of M 4 mutant lines (2017) originating from
irradiation of gamma-rays of 20 krad to F 8 seeds of AKH2.

25

Heading dateCulm length（cm）

rice cultivar for adaptability to nature farming or low input sustainable agriculture.

Fig.6. Plant type of the selected mutant lines, MAKH2-7 and MAKH2-6 and control,
AKH2.

Discussion
We succeeded in development of new rice variety, Kumaminori, through cross ofnative varieties, Asahi and Kamenoo, which shows higher grain yield and better eating
quality in the nature farming or low input sustainable agriculture. The new variety has
been released to farmers to grow in Kyushu district, Japan. However, heading date of
the variety is too late to be adaptable to wider range toward northern part of Japan. It
is noted that farmers are not to be suffering from lodging due to long culm length of the
new variety, because of its strong culm. Farmers are, in general, used to be sufferingfrom lodging of rice in Kyushu district, where is a route of typhoon in autumn season. It
was practically proofed for strong culm of the new variety, Kumaminori that plants ofthe variety were not fell down when the typhoon came in 2008, while the modern leading
variety, Hinohikari was done (Fig.7).
We have also succeeded in improving late heading date of the breeding line (AKH2)
or new variety, Kumaminori within only five years through use of induced mutations. It
is, in general, to take more than ten years in case of cross breeding for completing
breeding purpose. In fact, we have taken more than 13 years to develop the new variety,Kumaminori.

26

Fig.7. Comparison of scenery of the rice fields of Kumaminori in nature farming and
Hinohikari in conventional one when attacked by the typhoon in 2018.

The other merit of mutation breeding is to improve a target character without
altering other desirable traits of the original variety. It is supposed that the selectedmutant line, MAKH2-7 is to be inherited with desirable characters from the original
variety, Kumaminori, e. g. high grain yield and eating quality in the condition of nature
farming or low input sustainable agriculture. It was especially noted that the original
variety, Kumaminori (AKH2) has been proved from epidemiological survey to carry ahealing function to atopy and allergic disease. It was found earlier by Hiroshi
HASEGAWA that the Yukihikari variety developed in Hokkaido was to carry healing
function to atopy or allergic disease, whereas the representative leading variety,
Kirara397 in Hokkaido tended to cause the disease (Miura et al., 2003; Hasegawa, 2019).
Representative native variety of Japan, Kamenoo and Ahahi were found to carryhealing function to atopy or allergic disease (Sakuma and Komatsu, 2018, The Japan
Agricultural News, 13th December 2019). The varietal difference of rice for ability toreact to the disease was earlier found to be controlled by gene systems (Monnma, 2005;
Sonoyama et al., 2009). The results of epidemiological tests suggest that Kumaminori or
the AKH2 breeding line could be inherited healing function to allergic disease from27

mother varieties, Asahi and Kamenoo. We would expect that the MAKH2-7 mutant line
would carry the healing function. Epidemiological tests for allergic disease of the mutant
line are in progress. We are now taking a step for registration of the mutant line,
MAKH2-7 for commercial variety.
We have found through the breeding of rice adaptable to nature farming that native
varieties could be effective materials for breeding of rice for nutritious foods, as well as
for adaptability to low input sustainable agriculture. We also would pay attention to theidea proposed by Indonesia and the Philippines in the sub-project “Mutation Breeding ofrice for Sustainable Agriculture (2013~2017)” that native varieties can be effective
breeding materials for mutation breeding of rice adaptable to low input sustainableagriculture (Nakai, 2018).
In conclusion, we propose that mutation breeding would be useful tool fordevelopment of rice varieties for adaptability to low input sustainable agriculture. In
addition, we want to suggest that the native varieties be used as effective breeding
materials for mutation breeding of rice for adaptability to low input sustainableagriculture, contributing to rehabilitation of the earth environment and human health.
Acknowledgment
We would thank to Mr. Takema SHIIBA for offering rice fields of the breeding and forkind support on the works. We also would like to give our thanks to Nature Farming andCulture Agency of MOA for financial support for the breeding project. We want to express
our gratitude to Ms. Aki KOIKE of NSRA for kind support on the trials.
References
NAGANO, Hirohiko; KATO, Shigeru; OHKUBO, Shinnji and INOBUSHI. Emission ofcarbon dioxide, methan, and nitrous oxide from short- and long-term organic farming
Andosols in central Japan, Soil Science and Plant Nutrition 58, 793-801, 2012.
SKINNER, Colin, GATTINGER, Andreas;, MULLER, Adrian;, MADER, Paul,FILESSBACH, Andreas; STOLZE, Mattias; RUSER, Reiner and NIGGLI, Urs.
Greenhouse gas fluxes from agricultural soil under organic and non-organic
management-Aglobal meta-analysis, Science of The Total Environment, 15, 553-563,
2014.
OKADA, Mokichi. Shizenn Nouhou Kaisetu (in Japanese) (“A commentary on Nature
Farming” translated by the author), Eikou Shuppann Co. Ltd., 1953.
MIURA, Takako; MIURA, Shunnsuke; Matsuda, Michio, HASEGAWA, Hiroshi and
YANAGIHARA, Tetsuji. Examination of different reaction between rice strains on
28

rice-allergy patients with atopic dermatitis, Arerugi no Rinnshou 23-14, 44-50, 2013
(in Japanese).
Hiroshi Hasegawa, Yukihikari to komearerugi (http://hasekuri.a.la9.jp/yukihikari.htm,
2019/03/20) (in Japanese).
SAKUMA, Masaru and KOMATSU Miyuki. Turuokasi no Sizennnouhou Jissenn ni yoru
Kennkouna Machidukuri(in Japanese) (Performance of nature farming for promoting
healthy town in Turuoka-city, translated by the author) Koukai Sinnpojiumu
Yuukinougyou ・ Sizennnouho

karahirogaru

Machidukuri

in

Tokyo

(in

apanese)( Symposium on organic・nature farming held in Tokyo, translated by theauthor) 17 February 2018.
The Japan Agricultural News. 13th December 2019.
MONNMA, Michiko. Characteristics of protein profile of low allergenic rice variety,“Yukihikari”, Rep. Nat’l. Food Res. Inst, No69, 7-12, 2005.
SONOYAMA, Kei; OGASAWARA, Toru; GOTO, Haruka; YOSHIDA, Tomoyo;
TAKEMURA, Naoki; FUJIWARA, Reiko; WATANABE, Jun; ITO, Hiroyuki; MORITA,
Tatsuya; TOKUNAGA, Yoshinari and YANAGIHARA, Tetsuji. Comparison of gut
microbiota and allergic reactions in BALB/c mice fed different cultivar of rice, British,
Journal of Nutrition 103, 218-226, 2009.
NAKAI, Hirokazu. Project Review on FNCA Mutation Breeding Project in 2017, in FNCA
18th Coordinator Meeting 2018.

29

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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58

Improvement of Wheat Productivity Through Mutation Techniques
N. Bayarsukh, Ts Dolgor, B. Ganbaatar
Institute of Plant and Agricultural Science (IPAS), Mongolia
Abstract
The harsh climatic conditions of Mongolia and specific characteristic /late warming
soil in the spring and early cold in the autumn/ of the high altitude agricultural zone/late warming soil in the spring and early cold in the autumn/ cause the need for shortmaturity varieties. Breeding works are also in the process of selecting new varieties.
Breeding of new varieties is moving in this direction as well. In recent years, physicaland chemical mutagens have been widely used in the breeding of new wheat varieties
and lines with good characters. Early maturity variety of wheat (Triticum aestivum L.)
developed by induced mutation. New wheat mutant varieties have a high yield capacityand early maturity comparing most of local varieties, selected from hybridization. The
mutants had been matured from two to ten days earlier than other varieties.
Introduction
The wheat is dominant crop and cultivated in about 80% of arable land in Mongolia
including over 70% in the Central crop production zone, over 20% in the Western zone
and 5-8% in the Eastern zone.
Due to the negative impact of climate change the drought frequency increased,
carrying capacity of pasture land declined and plant species composition reduced by 2-4
times and yield declined 4-6 times, soil erosion and degradation increased by 7-25 times,
soil humus content declined by 37-52% and annually 0.5-1.5 t/ha organic matterdecomposed, yield potential of existing crop varieties reduced and pest and disease
distribution, frequency and damage increased.
Also, the Western and Khangai mountain cultivation areas located above at the
1500-1900 m above sea level and daily average temperature ranges between 13-18°C in
the warmest season of June and July, also the crop duration ranges between 75-110 days.
These harsh conditions of the Western and Khangai mountain area allow growing
cereals with very short duration.
Existing commercial wheat varieties have not good grain quality, drought tolerance,and disease and pests resistance and cannot sustain stable yield under climate change.
Therefore, more new varieties with high stable yield potential and quality are needed inthese mountain areas.
59

The induced-mutation considered useful efficient tool for the improvement specificplant traits like yield, stress tolerance, disease resistance, quality and increase breeding
efficiency. Thus, development of early maturity, drought and heat tolerant wheat
varieties with potential stable yield under changing climate condition throughapplication of mutation techniques has needed for stable food production in Mongolia.
Materials and Methods
Mutation induction in wheat
The studies carried out at the Institute of Plant and Agricultural Sciences during
2013-2017. We evaluated 10334 mutant lines M 1 -M 3 generation in the initial materials
field, including 3131 mutant lines originated chemical mutagen treatment. Table 1.
Table 1. Mutants in the initial materials field
Year

Generation /lines

Total

M1

M2

M3

2013

394

720

302

1416

2014

1025

750

477

2252

2015

460

1170

660

2290

2016

180

786

1440

2406

2017

-

840

1130

1970

Total

2059

4266

4009

10334

The yield trail of mutant varieties
In the yield trail the 12 registered wheat varieties selected for the test. Three ofthem mutant and nine are conventional varieties originated from domestic and Russian
breeding. Origin of mutant Darkhan-141 variety is gamma ray 18000 rad, but origin of
Darkhan-172 mutant variety has derived from chemical mutagen Sodium azide. The
varieties grown in the black fallow, with three replications and a plot size was 50 m2.
The crop sown at a rate of 3.5 million seeds per hectare. The soil of the experimental site
is Darkhan Uul province (49°28’N, 105°54’E), with pH 6.22, percentage of organic matter
1.35, available N-NO 3 3.5 mg/kg, P 2 O 5 3.9 mg/kg-1 and K 2 O 17.6 mg/ kg-1. The plots
harvested with a Wintersteiger harvester. Yield data adjusted to 14% moisture content
and the following grain quality parameters were determined. Grain from each plot wasdried and a composite 500 g sub-sample taken for quality (duplicate / triplicate) analysis.
Protein (percent of dry matter) was calculated by multiplying the corresponding total
60

nitrogen (by Kjeldahl) content by factor 5.7 (ICC 105/2) using a Kjeldahl auto analyzer.
The wet gluten quantity extracted from whole meal flour in an automated gluten washer(Single head gluten tester MJ-IIA).
Results and discussion
Experiment on initial materials of wheat mutation breeding
In 2013-2017, the 10334 mutant progenies studied and evaluated by agromorphological traits and stress tolerance. About 1808 advanced mutant lines selected
and used for wheat hybridization breeding material in the wheat breeding program.
Table 2.
Table 2. Average yield and yield components of mutants in the M 3 generation
Plantheight,cm

Plant
numberper m2

Product
able stem
per m2Seed
numberper spikeSeedweightper spike

Yield,
t/haParents

77.6

166

428

35.7

1.3

4.38

Mutant lines

72.3

182

440

36.7

1.4

4.43

Variance, ±

-5.3

+16

+12

+1.0

+0.1

+0.15

Study of mutants in the yield trails
Maturity: Early and late maturing mutants have frequently induced throughmutagenesis and identified easily. Early maturity in cereal crops is one of the usefulcharacteristics for cultivation in cool temperate regions, offering the opportunity toflower frost-free conditions, harvest prior to frost and in drought –prone regions, theability to produce a viable crop prior to drought conditions.
The heading time and maturity of wheat controlled by the plant’s ability to sense via
temperature and day length. The growth duration of wheat varieties in the yield trail
fluctuated from 78-91 days. The Khalkh gol-1 variety matured very early 78 days, but
Darkhan-144 late matured within 91 days.
The mutant variety Darkhan-196 matured in 2-12 days earlier than other varieties.
Mutant variety Darkhan-172 matured in 2-10 days earlier than other varieties. Also,these varieties matured in 1-3 days earlier than control Darkhan-131 variety which aresuitable in the mountain agricultural area. The mutant variety Darkhan-141 has growthduration of 84 days which is in 1-7 earlier than Darkhan -34, Altaiskaya -100, Altaiskaya-50, Arvin, and Darkhan -144 varieties. Table 3.
61

Yield and yield components: Stable and high yield potential in specific environmental
conditions is the most important objective of Mongolian plant breeding program. Yield iscomplex trait and influenced by breeding objectives such as plant architecture, maturity,
nitrogen utilization efficiency and resistance to biotic and abiotic stresses.
The grain yield of wheat varieties in the yield trail varied from 1.94 to 2.08 t/ha
which are higher than the average yield of control varieties of 1.67 to 1.88 t/ha. The
highest grain yield (2.08 t/ha) has the variety Arvin, however lowest was the Khalkh gol1 variety (1.67 t/ha). The mutant variety Darkhan-141 has grain yield of 1.94 t/ha, which
is 0.02 t/ha higher than mean yield of varieties. The mutant variety Darkhan-172 andDarkhan-196 have same grain yield (2.02 t/ha), which are higher (0.10 t/ha) than meanof varieties. These three mutants included in high yielding varieties. Table 3.
The thousand kernel weight of wheat varieties fluctuated from 30.5 g to 38.0 g. The
highest was the Darkhan-172 mutant of 38.0 g, but lowest was Khalkh gol-1 variety-30.5
g. Thousand kernel weight of mutant variety Darkhan-196 was 31.7 g and Darkhan-141
was 34.6 g.
Table 3. Growth duration and yield of wheat varieties

No

Variety name

Maturity,
days

Seed
number per
spike1000 kernel
weight, g

Yield, t/ha

1.

Khalkh gol-1

78

30

30.5

1.67 d2.

Darkhan-196 mutant

79

29

31.7

2.02 ab3.

Karagandinskaya-22

81

31

36.4

1.79 d4.

Darkhan -172 mutant

81

30

38.0

2.02 ab5.

Orkhon

84

30

33.2

1.88 c6.

Darkhan -34

85

32

35.1

2.04 ab7.

Darkhan -141 mutant

84

29

34.6

1.94 b

8.

Altaiskaya -99

83

31

34.7

1.86 c9.

Altaiskaya -100

86

30

34.1

1.99 b10.

Altaiskaya -50

86

26

36.2

1.72 d11.

Darkhan -166

86

33

36.0

2.08 a12.

Darkhan -144

91

33

36.9

2.03 ab

Mean

84

30

34.8

1.92

62

Grain quality of wheat varieties: Quality parameters of new mutant varieties meetrequirements of the national standard for food wheat MNS 0097:2010. The protein
content of varieties varied 11.9-13.7%. The Altaiskaya-100 variety grain contained
lowest protein 11.9%, however the Darkhan-196 mutant variety contains highest 13.7%.
The medium maturity mutant variety Darkhan-141 has good grain quality comparing toother varieties. Protein content of Darkhan-141 mutant variety was 13.0%, it is 0.2-1.1%
higher than other varieties, wet gluten (31%) 2.2- 5.7% higher and flour yield (73.0%)
1.7-6.4% higher than other varieties. Bread making quality score of mutant varieties
Darkhan-172 and Darkhan-141 were highest (4.3-4.4).
Table 4. Comparing of grain quality of mutants and local varieties
No

Variety nameProtein,

Gluten,Flour yield,

Breadmakingscore

1.

Khalkh gol-1

13.0

29.4

68.5

4.1

2.

Darkhan-196 mutant

13.7

29.5

68.7

3.8

3.

Karagandinskaya-22

12.3

28.3

69.3

3.9

4.

Darkhan -172 mutant

12.5

28.5

68.0

4.4

5.

Orkhon

12.3

28.2

66.8

4.1

6.

Darkhan -34

12.8

28.1

69.4

4.0

7.

Darkhan -141 mutant

13.0

31.8

73.0

4.3

8.

Altaiskaya -99

12.5

29.6

71.3

3.8

9.

Altaiskaya -100

11.9

27.1

70.9

3.5

10.

Altaiskaya -50

12.2

26.1

67.0

3.7

11.

Arvin

12.3

29.2

66.6

4.3

12.

Darkhan -144

13.1

32.2

68.6

4.3

Fertilizer use effeciency of mutant varieties
We have studied organic and mineral fertilizers influences for mutant variety’s yieldand grain quality. Analysis of soil samples from planting depth (0-20 cm) indicated low
level of available nitrogen and phosphorus, medium in potassium, indicating that thenutrient may be a limiting factor for wheat production in the area. The upper 20 cm(arable layer) contains low (0.86%) organic matter (C), increasing to 1.22% in the subsoil(soil layer 20-40 cm). During the growth stage of wheat the soil nutrients in different
treatments had different trend over time. We observed that the application of fertilizers
influenced positively to the soil fertility. The use of fertilizers at sowing time, are63

effective on improvement of seed germination and seedling growth of wheat mutant
varieties, especially for Darkhan 141. Field germination of selected varieties increased
from 6.3 % to 14.3% under fertilization.
These results showed that interactive effect of varieties and fertilizers had
significant effect (P<0.05). The highest increased grain yield was recorded in NPK
treatment. Application of N-P-K at the rate of 30-20-20 kg/ha to Darkhan 141 had
maximum increased (1.22 t/ha or 83.6% compare to control) grain yield. The lowest grain
yield (on an average 1.23 t/ha) was recorded in Darkhan 34 standart variety. Darkhan172 mutant had 43.3% additional yield under the complex mineral fertilizer application.
Table 5.
Table 5. Effect of fertilizers on yield of wheat mutant varieties, 2014-2016
Treatments

Darkhan-34

Darkhan 141

Darkhan 172t/haAdditional
yield, %t/haAdditional
yield, %t/haAdditional
yield, %Control

1.15

-

1.46

-

2.03

-

N

1.05

-

1.64

12.3

2.30

13.3

NP

1.27

10.4

2.16

47.9

2.90

42.8

NPK

-

-

2.68

83.6

2.91

43.3

Gumat

1.44

25.2

1.62

11.0

1.98

-

Mean

1.23

17.8

1.91

38.7

2.42

33.1

The fertilization effected positively on grain protein content and increased 0.42.79 %. The highest percentage of grain protein (16.3%) and gluten content (36.9) wereobtained from combined nitrogen and phosphorus (NP) fertilizer. Table 6.
Table 6. Effect of fertilizers on grain quality of mutant varieties, 2014-2016
Treatments

Darkhan 34

Darkhan141

Darkhan 172

Protein, %Gluten, %Protein, %Gluten, %Protein, %Gluten, %Control

12.5

29.2

11.9

29.5

13.0

29.6

N

14.1

30.9

12.6

30.8

13.2

34.0

NP

16.3

36.9

13.0

34.4

12.9

30.1

NPK

-

-

14.2

31.4

13.5

32.8

Gumat

11.9

23.8

11.5

24.6

-

-

64

Conclusion
The experiment results showed developing new varieties with high yield earlymaturity and good grain quality through mutation induction. Mutant varieties has very
high fertilizer use efficiency.
The new mutant variety Darkhan-172 is registered as promising new early wheat
variety for growing in the western and mountain areas in Mongolia.
Acknowledgments•

This work was supported by FNCA “Mutation breeding” project.

•

All members of FNCA project for sharing information and experience.

•Prof. Hirokazu Nakai for supervising rice mutation breeding.

References
1. Dolgor.Ts, Results of mutation breeding and future PSARTI Research volume 27,
2009,
2. Maslov.A.B, Mutagenesis in the wide hybridization and polyploidy breeding andgenetic experiments Moscow, 1983
3. Maluszynski.M, Micke.A, Sigurbjornsson.B, Szarejko.I and Fuglewicz.A, The uses ofmutants for breeding and for hybrid barley, Barley Genetics V: 969-977, 1987
4. Maluszynski, M.; Nichterlein, K.; Van Zanten, L.; Ahloowalia, B. S. Officiallyreleased mutant varieties-The FAO/IAEA Database, Mutation Breeding Review No.
12 December 2000. Joint FAO/IAEA Division, Vienna, Austria, ISSN 1011-2618.
5. Shu.Q.Y, Forster.B.P, Nakagawa.H.
Plant mutation breeding and biotechnology, Joint FAO/IAEA Program Malta, 2012
6. Dolgor.Ts, Bayarsukh.N. Mutation breeding achievements in Mongolia, March 2017;
The National nuclear energy project coordinator’s meeting, Ulaanbaatar, Mongolia.

65Improvement of Traditional Rice Varieties by Gamma Irradiationin the Philippines
Fernando B. Aurigue1, Ana Maria S. Veluz, Arvin O. Dimaanoand Adelaida C. Barrida2
Project Leader: 1 July 2017 onwards; 2 2013-2015 (retired)Department of Science and Technology-Philippine Nuclear Research InstituteCommonwealth Avenue, 1101 Diliman, Quezon City, Metro Manila, Philippines
Abstract
Two Philippine traditional rice varieties, namely: Umangan and Native Borie, wereused to develop mutant lines that are suitable for organic farming or sustainableagricultural practices of rice production.
Seeds were subjected to acute gamma ray treatment and grown as M 1 generation
together with the non-irradiated materials as control. Comparisons were made between
plants treated with 200 Gy and 300 Gy, and also with the non-irradiated plants. Early
flowering plants with reduced height, more tillers, and higher number of filled grains
per panicle were observed from the segregating M 2 generation but no selection was made.
M 3 generation was grown from bulked seeds and putative mutant plants with thedesired agronomic characteristics were selected based on the results of statisticalanalysis. The M 4 generation did not show any line that is better than the control in anyof the parameters measured.
Apparently, plants with desirable mutated traits were noticed but not isolated earlyenough. Continuous bulking of seeds and late selection for the desired characteristics
showed the importance of following the prescribed methodology. Failure to develop
putative mutant lines during the given period (2013-2017) is attributed to the stalling of
the project for 1½ years due to lack of personnel, change of workforce, leadership, andpriorities in the Philippine government, and seasonal cultivation of rice in theexperimental area. With the lessons learned, the current Director of the Institute andProject Leader on mutation breeding of rice have strengthened their partnership or
collaboration with the Philippine Rice Research Institute and state universities andcolleges that conduct rice improvement programs to use the gamma irradiation facility
for developing mutant varieties of rice and other crop plants.
Keywords: gamma irradiation, mutant, rice, traditional rice variety, varietal
improvement
66

Introduction
Rice (Oryza sativa L.) is the Philippines’ most important staple crop. Rice productionis also the main source of livelihood of more than 2.5 million households or about 2.1
million farmers (FAO, 2018). It is very important to the food supply and economy ofFilipinos. The country is the eight largest rice producer in the world, accounting to 2.8%of global rice production (FAO, 2018) but the Philippines is the third largest rice importerin 2015, having imported 1.8 million metric tons that year, and continues to be one of
the largest importer of rice in the world market to satisfy the domestic demand (Exconde,2018). The major reason for this imbalance is that the population of the Philippines hasgone up to 104.9 million in 2017 and Filipinos have been consuming more milled ricethan what is being produced domestically. Per-capita rice consumption was 109.875 kg
in 2015 to 2016 (Ordinario & Arcalas, 2018). Domestic consumption of milled rice ranged
from 12.85 metric tons in 2013 to 13.25 metric tons in 2017 (USDA, 2019).
According to Crop Statistics of the Philippines, during the period of 2014 to 2018,rice production in the country grew at an average annual rate of 0.3%, from 18.97 million
metric tons in 2014 to 19.28 million metric tons in 2017. Likewise, the total area
harvested increased from 4.74 million hectares in 2014 to 4.81 million hectares in 2017
or at an average annual rate of 0.4% (PSA, 2018). However, the national average yield
for all ecosystems remains low at about 3.87 metric tons per hectare at a relatively highproduction cost of US$0.24 (PhP12.00) per Kg.
To sustain rice production, other means of increasing productivity must be employed.
Organic farming is becoming popular in the Philippines, especially with the organic ricethat has increasing demand and commands higher selling price. Additionally, healthconscious-Filipinos are looking into options of consuming organically-grown rice andtraditional rice varieties (TRVs). Growing number of farmers are planting TRVs mainly
because they are more adapted to the ecosystem and do not need much agrochemical
inputs as compared to hybrid rice which are highly dependent on agricultural chemicals
such as synthetic fertilizers and pesticides (herbicides, fungicides and insecticides).
Another means is to develop mutant lines from TRVs that are highly adapted toexisting ecosystems but do not require as much inputs as the more popular and highly
promoted hybrid rice varieties. Mutant varieties that are suited to organic agricultural
practices are more sustainable for year-round production. The objective of the project isto develop mutant lines with improved agronomic characteristics through gamma
irradiation of seeds of two select TRVs.

67

Materials and Methods
Seeds of two TRVs were obtained from the Philippine Rice Research Institute(PhilRice), Department of Agriculture (DA) in the Science City of Muñoz, Nueva Ecija,
Central Luzon. These two rice varieties, namely: 1.) Umangan; and 2.) Native Borie wereselected according to their popularity or extent of use in Luzon island, Philippines.
Based on results of previous studies on radio-sensitivity of rice seeds to acute gamma
radiation, the seeds were treated with 200 Gy and 300 Gy of gamma rays as therecommended dose levels, while non-irradiated seeds served as the control. Irradiatedand control seeds were sown in beds and protected from birds and rodents. Soil samples
were collected and analyzed for elemental analysis prior to land preparation. Dependingon the results for nitrogen, phosphorus and potassium, the recommended amount oforganic fertilizer was incorporated evenly in the soil 3 days before transplanting.
Seedlings were soaked in biofertilizer for 30 minutes before transplanting. A
Randomized Complete Block Design (RCBD) with at least three replicates was followed
every cropping season. During the second year or at M 2 , radiation-modified kappacarrageenan (RMKC) solution, which has been developed as a plant growth promoter at
PNRI from another project, was applied as foliar spray at 100 ppm. Foliar application
was done every week for 1 month. On the third year or at M 3 , the use of RMKC wasmodified by increasing the concentration to 200 ppm but applying only three times: 14days after transplanting (DAT), 35 DAT, and 60 DAT. At M 4 , the recommended
application rate followed was 200 ppm at 15 DAT, 30 DAT, and 45 DAT.
Proper cultural management was employed during the whole cropping season in all
generations of planting. The activities included the maintenance of proper water level at
different stages of plant growth, manual control of golden apple snail, installation of nets
to prevent birds from eating the grains and trapping of field rats. The same
methodologies were followed throughout the conduct of the project (up to M 4 only).
Sowing of seeds of putative mutant plants selected from M 3 generation was delayedfor 1½ years. This is largely attributed to a chain of unprecedented events starting off
with health trepidation leading to an early retirement in December 2015 for Ms. Barrida
as Project Leader. By June 2016, a new President of the Philippines was installed,bringing new national and institutional priorities, and a status quo in the organizational
set-up of some projects. Finally, by July 2017, Mr. Aurigue received his designation as
the new Project Leader, thereafter resuming the planting, evaluation and selection of
putative mutant lines as planned for the project.
Seeds from selected M 3 plants were sown on paddy soil placed in black polyethylene
plastic (PEP) bags on July 31, 2017 for M 4 generation plants. The seedlings were68

transplanted into two rice paddies on August 17 and 18, 2017 following RCBD with three
replications. Data for vegetative traits were gathered on November 6, 2017. Necessary
cultural practices were employed until harvesting on November 28, 2017. Data gatheringon yield parameters were conducted afterwards when the intact seeds have been sundried properly with less than 12% moisture content.
Results and Discussion
The M 1 generation of both Umangan and Native Borie flowered earlier (80 DAT)
than the control plants. For Umangan, high number of grains per panicle was obtained
from 200 Gy with 184.5, followed by 300 Gy with 133.0 (Table 1). For Native Borie, hightillering was observed at 200 Gy with 25.7 followed by the control with 21.3 (Table 2).
Table 1. Agronomic characteristics of non-irradiated and irradiated Umangan at M 1
generation
Dose

Plant
height(cm)*

Numberof tillers
per
Plant*

Length ofpanicle
(cm)*

Total
number ofgrains per
panicleNumberof filled
grains per
panicle*Numberof unfilled
grains per
panicle*Control

97.55

18.4

22.23

116.7 b

82.6

34.1

200 Gy101.46

17.5

22.76

184.5 a

101.1

83.4

300 Gy101.40

19.7

23.43

133.0 ab

102.3

30.3

* not significant
Means followed by similar letter within the same column are not significantly different
at 5% level DMRT
Table 2. Agronomic characteristics of non-irradiated and irradiated Native Borie at M 1
generation
Dose

Plant
height(cm)*

Numberof tillers
per plant

Length ofpanicle
(cm)*

Total
number ofgrains per
panicle*Numberof filled
grains per
panicle*Numberof unfilled
grains per
panicle*Control

122.3321.3 b

23.00

188.3

142.7

45.6

200 Gy121.6125.7 a

26.86

184.3

123.3

61.0

300 Gy121.0517.3 c

25.05

174.3

122.0

52.3

* not significant
Means followed by similar letter within the same column are not significantly different
at 5% level DMRT69

In the M 2 generation, results for Umangan also showed early flowering at 200 Gy
with 70 DAT followed by 300 Gy with 77 DAT. At 200 Gy, there was 7.15% reduction inplant height. Increase in the number of grains per panicle was noted at 300 Gy (Table 3
and Figure 1). Meanwhile, for Native Borie, early flowering at 85 DAT was observed forboth 200 Gy and 300 Gy. The control plants took 100 DAT to flower. There was anincrease in number of tillers per plant as 16 tillers were counted for 200 Gy and 17 tillers
for 300 Gy (Table 4).
Table 3. Agronomic characteristics of non-irradiated and irradiated Umangan at M 2
generation
NumberofUnfilled
Grainsper
Panicle100seedWeight(g)

Dose

Numberof DaystoFlowerPlantheight(cm)

Numberof Tillers
per
Plant

LengthofPanicle(cm)

Numberof Filled
Grainsper
PanicleControl

80

82.93

17.0

22.16

86.0

24

4.24

200 Gy

70

77.00

15.0

21.46

93.0

18

4.51

300 Gy

77

80.26

18.0

21.53

99.0

21

4.47

Table 4. Agronomic characteristics of non-irradiated and irradiated Native Borie at M 2
generation
Plantheight(cm)

Numberof Tillers
per
Plant

LengthofPanicle(cm)

Numberof Filled
Grainsper
PanicleNumberofUnfilled
Grainsper
Panicle100seedWeight(g)

100

106.33

11.0

25.66

121.0

45

4.11

200 Gy85106.66

16.0

26.66

119.0

48

3.91

300 Gy

85

97.53

17.0

25.33

112.0

47

3.70

Dose

Numberof DaystoFlowerControl

70Figure 1. Control and M 2 plants derived from Umangan irradiated with 300 Gy of acute
gamma radiation.
In the M 3 generation, the number of days to flowering, number of tillers per plant,
and the total number of filled grains per panicle were significantly affected by thetreatments for Umangan (Table 5). At 200 Gy, plants flowered 14 days earlier than thecontrol, whereas at 300 Gy plants flowered 11 days earlier than the control. Meanwhile,the number of tillers per plant increased from 11 to 14 for plants derived from both 200
Gy and 300 Gy. The number of grains per panicle obtained at 200 Gy (152 seeds) and at
300 Gy (132 seeds) is comparable to those of the control plants (147 seeds).
In Native Borie, the plant height, number of days to flower, and number of tillers
per plant were significantly affected by the treatments at M 3 (Table 6). Plants derived
from 200 Gy and 300 Gy flowered 7 days earlier than the control. Reduction in plantheight was about 10.46% and 6.46% less than the control at 300 Gy and 200 Gy,
respectively. The number of tillers per plant increased by four at 300 Gy (18 tillers) andby two at 200 Gy (16 tillers) compared to the control (14 tillers). Figure 2 shows foliar
application of RMKC solution during tillering stage.

71

Table 5. Agronomic characteristics of non-irradiated and irradiated Umangan at M 3
generation
Plantheight(cm)*

Numberof tillers
per
plant

Lengthofpanicle(cm)*

Numberof filled
grainsperpanicleNumberofunfilled
grainsperpanicle*100-seedweight
(g)*

92 b

94.66

11.0 b

23.43

121.0 ab26

4.37

200 Gy

78 a

95.20

14.0 a23.05

128.0 a24

4.44

300 Gy

81 a

96.43

14.0 a23.10

111.0 b

21

4.49

Dose

Numberof daystoflowerControl* not significant
Means followed by similar letter within the same column are not significantly different
at 5% level DMRT
Table 6. Agronomic characteristics of non-irradiated and irradiated Native Borie at M 3
generation
Plantheight(cm)

Numberof
Tillersper
Plant

LengthofPanicle(cm)*

Numberof Filled
Grainsper
Panicle*NumberofUnfilled
Grainsper
Panicle*100-seed
Weight(g)*

106 b

109.06 a

14.0 b

27.27

122.33

36.33

3.99

200 Gy99 a105.11 ab

16.0 ab26.93

122.67

49.66

3.84

300 Gy99 a98.73 b18.0 a26.76

112.67

32.66

4.16

Dose

Numberof daystoFlowerControl* not significant
Means followed by similar letter within the same column are not significantly different
at 5% level DMRT72

Figure 2.

UmanganNative Borie
Spraying of radiation-modified kappa-carrageenan solution on two
traditional rice varieties at M 3 generation during tillering stage.

In the M 4 generation, there was no significant differences between means for eachparameter observed (Table 7). The selected putative mutant lines were similar to theirrespective control parent. Likewise, results of statistical analysis imply that the yield of
selected putative mutant lines was not different from their respective control parent(Table 8). Some of the putative mutant lines growing in the field are shown in Figure 3.

Figure 3. Different putative mutant lines at M 4 generation.

73

Table 7.

Vegetative characteristics of non-irradiated and putative mutant lines of

traditional rice varieties at M 4 generation
Plant
Total
HeightNumberLine(cm)
of
80 DAT
Tillers

Numberof
Productive
Tillers

Numberof
Unproductive
Tillers

Percent
Productive
Tillers

Umangan (Control)

104.50

10.4

9.2

1.2

88.5

Umangan (300 Gy) 4

101.60

9.9

7.9

2.0

79.8

Umangan (200 Gy) 8

97.40

8.0

7.5

0.5

93.8

Umangan (300 Gy) 23

103.00

9.6

8.5

1.1

88.5

Umangan (300 Gy) 28

98.40

8.6

7.3

1.3

84.9

Umangan (200 Gy) 39

103.73

10.2

9.0

1.2

88.2

Native Borie (Control)

122.60

8.1

7.0

1.1

86.4

Native Borie

111.97

10.9

8.0

2.9

73.4

130.80

8.8

8.4

0.4

95.4

112.43

12.5

12.3

0.2

98.4

110.10

11.5

11.5

0.0

100.0

111.03

10.2

10.2

0.0

100.0

(300 Gy) 27-1
Native Borie(300 Gy) 27-2
Native Borie(300 Gy) 34
Native Borie(300 Gy) JF-1
Native Borie(200 Gy) JF-2
Table 8. Yield parameters of non-irradiated and putative mutant lines of traditional
rice varieties at M 4 generation*Line

PanicleLength(cm)

NumberofBranchesper
PanicleNumberofFilledGrainsNumberofUnfilled
Grains100-Seed
Weight
(g)

Yield
(g)

Umangan (Control)

21.88 ab5.87 ab45.03 bc

71.93

2.17 b54.90 abcd

Umangan (300 Gy) 4

21.21 b5.80 ab38.03 bc

69.87

2.13 b51.07 bcd

Umangan (200 Gy) 8

21.34 b5.83 ab42.10 bc

69.87

2.17 b

51.63 bcd

Umangan (300 Gy) 23

21.03 b5.70 ab36.40 bc

69.50

2.13 b

42.30 cd

Umangan (300 Gy) 28

20.58 b5.23 b

24.93 c59.90

2.10 b25.70 dUmangan (200 Gy) 39

22.88

6.00 ab57.13 abc

74.23

2.67 b

67.20 abcab74

Native Borie (Control)

25.60 a6.90 a86.27 a107.839.20 a94.40 aNative Borie

25.32 a6.40 ab69.27 ab104.203.03 b86.27 ab22.72 ab5.90 ab55.33 abc

72.07

2.30 b57.83 abcd

Native Borie

24.31

6.37 ab62.20 abc

88.10

2.80 b80.93 abc

(300 Gy) 34

ab

Native Borie

23.95

6.20 ab61.20 abc

86.27

2.77 b

76.60 abc

(300 Gy) JF-1

ab

Native Borie

23.52

6.00 ab59.97 abc

82.20

2.70 b

72.47 abc

(200 Gy) JF-2

ab

F-test

**

**

**

ns

*

**

(300 Gy) 27-1
Native Borie(300 Gy) 27-2

*Mean of 10 samples
Means followed by the same and/or sharing the same letters within a column do notdiffer significantly at p≤0.01 (**) or p≤0.05 (*)
ns = not significant
With such findings, no desirable mutant line was developed by gamma irradiation.
After reviewing the data generated from M 1 to M 3 , and upon investigation of theprocedures done in handling the generation advancement, it was found out that seeds
derived from the same gamma irradiation dose level were apparently bulked for eachvariety and no selection of putative mutant plants was conducted prior to M 3 generation.
Summary and Conclusion
The potential to develop mutant lines from two Philippine TRVs, namely: Umangan
and Native Borie, that are suitable for organic farming or sustainable agricultural
practices of rice production was not tapped at the right time or generation.
Plants derived from seeds subjected to 200 Gy and 300 Gy of acute gamma ray
showed desirable characteristics at M 1 and M 2 generations but selection of putative
mutant plants was done at M 3 generation which was grown from bulked seeds. The M 4
generation did not show any line that is better than the control in any of the parameters
measured.
Due to unforeseen or unexpected events from 2013 to 2017, the project was stalled
for 1½ years following the early retirement of the Project Leader without immediate
replacement, change in national and institutional leadership and priorities in the75

Philippine government, and planting of rice limited to just once a year. Now that no
mutant line was obtained for this particular project, partnership or collaboration with
PhilRice and state universities and colleges was strengthened to ensure that riceimprovement programs through mutation induction by gamma irradiation will be
continued until new mutant varieties of rice and other crop plants are developed,
registered, and utilized by farmers.
Acknowledgment
The authors are grateful to PhilRice-DA for providing seeds of TRVs. The staff of
PNRI Cobalt-60 Multi-purpose Irradiation Facility are acknowledged for treating theseeds with gamma radiation. Science Aides who prepared the paddy, sowed the seeds
and transplanted the seedlings, maintained the experimental field, harvested the grains,
and assisted in data gathering are all appreciated. They are Mr. Eduardo C. Costimiano,
Ms. Rosalie M. Semana, Ms. Dolores M. Lazo (2015), and Mr. John Faustus C. Vidal(2017). Ms. Mary Jane C. Manrique, who assisted in the conduct of the experiments in
2013-2014, is also acknowledged.
References
Exconde, B. 2018. On Rice Production and Consumption Patterns in the Philippines.
The Lobbyist. 16 February 2018.
[FAO] Food and Agriculture Organization of the United Nations Regional Rice Initiative.
www.fao.org accessed on 19 January 2020.
Ordinario, C. U. and Arcalas, J. E. Y.

2018.

Snapshot of rice – consumption dataremains grainy as Pinoys grapple with supply, prices.

The Broader Look.

Business Mirror. 30 August 2018.
[PSA] Philippine Statistics Authority. 2019. Accessed on 19 January 2020.
Simeon, L. M. 2019. PhilRice seeks competitive rice yield. The Philippine Star.
[USDA] United States Department of Agriculture. 2019. indexmundi.com accessed on
19 January 2020.

76

Application of Mutation Breeding of Submergence tolerance Rice for
Sustainable Agriculture, in Thailand
Kanchana Klakhaeng1, Udompan Promnart2, Peera Doungsoongnern2,
Kanokporn Boonsirichai3
1 Rice Research and Development Division, Rice Department, Thailand,
2 Prachin Buri Rice Research Center, Bansang, Prachinburi, Thailand,
3 Thailand Institute of Nuclear Technology, Ongkharak, Nakhon Nayok, Thailand.

Abstract
The world is facing a food and energy crisis of unprecedented proportions and also
climate change, especially the devastating flood in Thailand, which affected at least 25of the country’s 77 provinces and affected 9 million citizens in 2011. Normally in plant
genetics and breeding research, mutation is a source of variation. Spontaneous mutation
rate is very low, induced mutation have contributed to the discovery and identificationof gene function following the completion of genome sequencing project in rice. Therefore,
radiation can cause genetics change in living organism to increase significantly. The aimsof this project are to improve rice variety with enhanced to tolerance to flood and/or
wetland conditions with good grain quality. RD31, a non-glutinous photoperiod–
insensitive rice variety was irradiated with 0.44 kGy electron beam due to its several
preferable agricultural traits such as high yield and moderately resistance to brownplanthopper, bacterial leaf blight, brown spot disease and dirty panicle disease. M 1
plants were planted in dry season 2014. Five hundred panicles were harvested from themain tiller of each M 1 plant. M 2 plants were grown as 1 panicle/row for 500 rows or
10,000 plants in total. M 3 lines were screened for submergence and 317 lines survived
with the score of 5-7. M 4 were screened for blast resistance. The result showed that all
mutant lines were highly resistance to blast. Some of these lines were screened forsubmergence and found that 91 submergence tolerant mutant lines were identified.
Grain quality were tested and revealed that 16 mutant lines showed 23.22-24.44 %amylose content which were lower than RD31 the original (27.52%). Physical propertyof all these mutant showed clear chalkiness of endosperm. M 5 mutant lines were grownfor observation yield trials and also physical and chemical properties were examined. M 6
mutant lines were grown for intrastation yield trial which were screened forsubmergence tolerant during 2018. Further work will be carried out before released the
new rice variety.
77

Keywords: Rice, Electron Beam, Flood Tolerance
Introduction
Rice is the principle staple crop of Asia and any deterioration of rice production
systems through climate change would seriously impact food security in the world.
Climate change has many facets, including changes in long-term trends in temperatureand rainfall regimes.

The impact of these changing conditions on agriculture are

already being seen. Although climate change is a global phenomenon. It will manifest
itself as locally variable impacts. Modern rice varieties are not adapted to these
conditions and their yield is severely reduced because of high mortality, suppressed
tillering ability, reduced panicle size, and high sterility. These are probably the reasons
why farmers in affected areas still rely on low-yielding local landraces.
In the past, conventional breeding was used to develop new varieties, but thosevarieties left breeders unsatisfied still.

Mutation breeding can be used to obtainsuitable rice varieties which can lead to sustainable agriculture. Therefore, rice can befurther improved. The rice improvement by mutation breeding through ion beam andgamma rays irradiation is very useful for creating genetic diversity. The irradiated rice
population and its subsequent generations serve as the source of genetic variation for
selection or screening according to our objectives.

The aims of this project are to

improve rice varieties with enhanced to tolerance to flood and/or wetland conditions.
Materials and Methods
Two hundred gram of RD31 recommended rice variety was irradiated with 440 grayelectron-beam in 2014. M 1 plants were grown by broadcasting. Five hundred panicles
from each main clum were collected as M 2 seeds. M 2 plants were planted as 1 panicle/row
for 500 rows or 10,000 plants in total. Agricultural traits, including plant height, numberof panicle per hill and maturity of these M 2 plants were observed. Seeds from six plants
were collected as M 3 seeds. M 3 plants were screened for submergence tolerance(Appendix 1) and M 4 plants were screened for blast resistance (Appendix 2). The
remaining were screened for submergence tolerance, and were examined for grain
quality. M 5 –M 6 plants were planted for yield trial observation.
Results
RD31, a non-glutinous and photoperiod–insensitive rice cultivar, is one of thecertified cultivars for irrigated areas in the central plain of Thailand. It has a relatively
78high yield and moderate resistance to brown plant hopper, bacterial leaf blight, brownspot disease and dirty panicle disease with a good grain quality.
M 1 plants were planted by broadcasting in dry season 2014. Five hundred
panicles were harvested from the main tiller of each M 1 plant to obtain M 2 seeds. M 2plants were grown as 1 panicle/row for 500 rows or 10,000 plants in total in dry season
2014. (Figure 1). Plant height, number of panicle per hill and maturity of these M 2 plants
were observed and recorded (Table 1).

Figure 1. Electron beam irradiated M 2 plants of RD31.
Table 1. Plant height of RD31 and M 2 populations.
Plant groups

Plant height (cm)Height (cm)

No. of plants

121

20

-Dwarf40-54--Semidwarf

55-1002-Intermediate-tall

101-120

279

-Tall

121-155

218

RD31
M 2 of RD31

The plant height of RD31 was 121 cm. It was classified according to Loresto andChang (1978) as tall class. Loresto and Chang (1978) used the following criteria toclassified plant height in the F 2 populations : 1) tall (121-155 cm), 2) intermediate-tall(101-120 cm), 3) semi-dwarf (55-100 cm), and dwarf (40-54 cm). While Fernandez et al.,classified dwarfs into four categories : 1) tall (more than 130 cm), 2) intermediate (110130 cm), 3) semi-dwarf (80-110 cm), and dwarf (40-80 cm). Upon electron-beamirradiation, the height of M 2 plants ranged from 99-143 cm (Table1 and Figure 2),79covering all three height classes, including semi-dwarf, intermediate-tall and tall. The
observed frequency were 2, 279 and 218, respectively.

Figure 2. Distribution of M 2 plant height according to the classification of Loresto andChang, (1978).
Panicle number of each hill was counted as it was a key component of the yield. Yield
components refer to the structures of the rice plant that directly translate into yield. The
relationship among growth duration, plant stature and grain yield was well explained morethan 1,400 years ago (Liu, 1986). Yield components is important for fixing up the character,which plays an ultimate role in influencing the yield. Many reports identified as yield of crops
depend on yield components including, the number of panicles per given area, the number of
spikelets (potential ) grain per panicle, the 1,000 grain weight, and the percent of fill grains
per panicle. Upon electron-beam irradiation, panicle number of each hill was counted. Yield
components were determined during each rice cycles, first at the vegetative phase (tiller
number), second at the reproductive phase (number of fertile tillers and size of panicle sinks)
and finally at the grain filling phase (spikelet filling rate). The results showed that M 2
population provided as 5-9 panicle/hill in 176 plants and 10-15 panicle/hill in 323 plants(Table 2).

80

Table 2. Panicle/hill of RD31 and M 2 plants.
Plants

Panicle/hill
Number of panicles

Number of plants

RD31
M 2 of RD31

10
<5

0

5-9

176

10-15

323

Normally, maturity of RD31 depends on planting method which is 118 days fortransplanting and 111 days for boardcasting. The M 2 plants were grown by transplanting andmaturity dates were varying. Approximately 500 M 2 plants with shortening maturity period
were selected. Three hundred and thirty-three of them had 110-115 days for maturity; 166
plants with 116-120 days; one plant with 121 days (Table 3). Shortened maturity period was
the preferable trait to cope with climate change. The central plain of Thailand frequently
encounters a water shortage during the dry season which is follow by a heavy flood during
the rainy season. Cultivars with a shortened maturity period are highly desirable because
harvesting can be done early before the flooding period. Therefore, two crop will be possible
per year before and after the flooding period.

Table 3. Days to maturity of RD31 and M 2 plants.
Plant groups
RD31

Maturity (days)
Days to maturity

Number of plants

123

20

110-115333116-120

166

121

1

M 2 of RD31

About 3,000 M 3 lines were screened for submergence (Appendix 1). The plants were
submerged under a flash flood condition for 14 days. Only 317 lines survived the flood withthe score of 5-7, which were selected for planting in the following season (Table 4, Figure3
and 4).

81

Table 4. Number of survived M 3 plants with score.
Score

Number of plants

1

-

3

-

5

95

7

222

9

2,680

Figure 3. Submergence tolerant mutants lines from field screening.

Figure 4. Protection of 317 mutant lines from bird damage by nets.
M 4 were screened for blast resistance (Appendex 2). The result showed that all mutant
lines were highly resistance to blast (Table 5). Some of these lines were screened forsubmergence tolerance and found that 91 submergence tolerant mutant lines were identified.
Among the biotic factor disease is the most important factor which results in crop losses of $582billion every year (Asghar et al., 2007). Rice blast caused by Pyricularia oryzae Cavara, is
one of the most destructive and wide spread diseases (Jin et al., 2000). This disease has caused
significant yield losses in many rice growing countries e.g. 75% losses of grains in India(Padmanabhan, 1965), 50% in Philippines (Awoder and Esuruoso, 1975) and 40% loss in
Neigeria (Ou, 1985). In Brazil rice blast is considered to be one of the major yield constrains
in both irrigated and upland ecosystems (Prabhu and Morais, 1986). However, the use ofresistant cultivar is the most economical and environment friendly method for the
management of rice blast.
Table 5. Number of M 4 lines which were screened for blast resistance.
Score

Site 1 (PCR)

Site 2 (KHS)

0

305

317

1

11

-

2

1

-

7

-

-

9

-

-

Grain quality were tested and revealed that 16 mutant lines showed 23.22-24.44 %amylose content which were lower than RD31 the original (27.52%) (Table 6). Physicalproperty of all these mutant showed clear chalkiness of endosperm (Table 7).
Table 6. M 4 lines which were examined for percentage of amylose content.
% amylose content

Classification

No. of plants

10-19

Low amylose

-

20-25

Intermediate amylose

16

26-34

High amylose

301

Table 7. Physical property of M 4 lines, chalkiness.
Score

Description

No. of plants

0

None

-

1

Small (less than 10%)158

2Medium (11-20%)

147

3Medium (11-20%)

149

4

Large (more than 20%)-5Large (more than 20%)-83

M 5 mutant lines were grown for observation yield trials during dry season 2017 andphysical and chemical properties were examined as shown in Table 8.
M 6 mutant lines were grown for intrastation yield trial during wet season 2018 whichwere screened blast resistance and screened for submergence tolerant during 30th July 2018
and de-submerge on 13th August 2018 as shown in Table 9.
Further work such as interstation yield trials, farmers field yield trials and adoption fromthe farmers, nitrogen used efficiency, physical and chemical properties, screening for blast
resistant and screening for submergence tolerant will be carried out for more information
before released the new rice variety.

Table 8. Observation yield trials and physical and chemical properties of mutant lines during
dry season 2017.
No.SourceIdentity

Mat.

Ht.

#16 WS(days) (cm)1121
3 RD31-B-390-3-B- 113
123
2
5 RD31-B-483-6-B- 113
121
3
6 RD31-B-389-3-B- 110
124
4
8 RD31-B-479-1-B- 111
126
5
9 RD31-B-393-2-B- 113
113
6 13 RD31-B-472-6-B- 113
123
7 14 RD31-B-415-4-B- 112
8 43 RD31-B-388-5-B- 113
118
124
9 44 RD31-B-298-5-B- 112
10 46 RD31-B-395-4-B- 113
115
113
11 47 RD31-B-297-2-B- 112
121
12 49 RD31-B-331-5-B- 112
128
13 50 RD31-B-368-6-B- 112
14 71 RD31-B-368-4-B- 111
116
120
15 74 RD31-B-369-5-B- 113
16 75 RD31-B-385-3-B- 113
127
123
17 76 RD31-B-312-6-B- 113
115
18 77 RD31-B-449-1-B- 113
19 88 RD31-B-290-6-B- 112
121
20 96 RD31-B-276-4-B- 113
123
122
21 123 RD31-B-314-4-B- 114
22 134 RD31-B-277-5-B- 114
120
RD31
118
125
ck.
ck.
RD49
111
110

Grain yield/ kg/rai Grain size Brown rice size Clk.
ave.
608
406
546
319
478
350
98
487
354
132
113
418
234
138
150
395
290
212
525
488
551
437
420
409

Sh.
4.05
3.93
4.21
4.08
3.91
3.94
3.95
3.94
4.03
4.16
4.19
3.92
3.90
3.93
4.06
4.21
4.11
4.04
4.02
3.98
4.16
4.01
4.09
4.30

84

Sh.
3.23
3.22
3.32
3.40
3.28
3.22
3.22
3.21
3.40
3.26
3.27
3.41
3.25
3.27
3.33
3.41
3.26
3.26
3.28
3.41
3.29
3.35
3.48
3.63

1.88
1.28
0.87
1.59
1.39
2.06
1.87
1.38
2.16
1.94
2.03
1.45
1.40
0.77
1.96
1.56
1.00
1.80
1.94
2.11
0.91
1.88
0.74
0.63

Amy. Gel Con. Gel
AlkD El.
(%)
temp. (oC)Ratio
28.23 38 H 74.5-79 3 H 1.46
27.90 38 H 74.5-79 3 H 1.48
27.65 38 H 74.5-79 3 H 1.57
27.25 38 H 74.5-79 3 H 1.58
27.58 38 H 74.5-79 3 H 1.54
28.13 38 H 74.5-79 3 H 1.54
28.16 38 H 74.5-79 3 H 1.47
27.68 38 H 74.5-79 3 H 1.57
27.87 39 H 74.5-79 3 H 1.65
27.38 38 H 74.5-79 3 H 1.64
28.23 38 H 74.5-79 3 H 1.61
29.09 38 H 74.5-79 3 H 1.58
28.00 39 H 74.5-79 3 H 1.62
27.58 38 H 74.5-79 3 H 1.63
27.87 38 H 74.5-79 3 H 1.62
27.84 38 H 74.5-79 3 H 1.55
27.90 38 H 74.5-79 3 H 1.53
28.29 38 H 74.5-79 3 H 1.58
27.55 38 H 74.5-79 3 H 1.55
27.87 38 H 74.5-79 3 H 1.65
28.06 38 H 74.5-79 3 H 1.61
28.03 38 H 74.5-79 3 H 1.49
28.13 38 H 74.5-79 3 H 1.68
27.06 38 H
70-74 6 I/L 1.67

Table 9. Intra station yield trial of M6 mutant lines.
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
ck.
ck.

Source Identity
Ht. (cm) % recov Mat. Ht. (cm) Grain yield/ kg/rai#16 WS
before Sub(days) Mat.
ave.
3
RD31-B-390-3-B-B-B
70
80
132
129
760
5
RD31-B-483-6-B-B-B
69
70
133
130
619
6
RD31-B-389-3-B-B-B
73
70
131
134
615
8
RD31-B-479-1-B-B-B
67
80
132
133
813
9
RD31-B-393-2-B-B-B
73
70
133
134
650
13 RD31-B-472-6-B-B-B
72
70
132
131
698
14 RD31-B-415-4-B-B-B
71
70
131
129
679
43 RD31-B-388-5-B-B-B
71
80
132
131
854
44 RD31-B-298-5-B-B-B
69
65
132
134
662
46 RD31-B-395-4-B-B-B
68
70
132
130
692
47 RD31-B-297-2-B-B-B
71
65
132
128
514
49 RD31-B-331-5-B-B-B
70
65
133
131
522
50 RD31-B-368-6-B-B-B
71
80
134
130
865
71 RD31-B-368-4-B-B-B
72
70
133
129
623
74 RD31-B-369-5-B-B-B
70
70
134
132
632
75 RD31-B-385-3-B-B-B
68
70
133
131
589
76 RD31-B-312-6-B-B-B
72
70
134
132
535
77 RD31-B-449-1-B-B-B
71
70
134
129
661
88 RD31-B-290-6-B-B-B
72
70
134
131
673
96 RD31-B-276-4-B-B-B
70
80
134
133
711
123 RD31-B-314-4-B-B-B
71
65
130
129
522
134 RD31-B-277-5-B-B-B
71
65
131
129
488
RD31
73
30
134
133
151
RD49
74
20
135
127
79

Bl'PCR
2018

R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R

Disscussion
IRRI released the first high-yielding modern rice cultivar, the IR8, for the tropicalirrigated lowlands in 1966 (Chandler, 1969). Since then, IRRI has developed many highyielding rice varieties such as IR36, IR60 and IR72 (Peng and Khush, 2003). New plant type(NPT) was developed at IRRI in the late 1980s. The NPT was designed based on the resultsof simulation modeling. The proposed NPT has a low tilling, few unproductive tillers, 200-250
grains per panicle, a plant height of 90-100 cm, thick and sturdy clumps, dark green and erectleaves, vigorous root system with 100-130 days for maturity (Peng et al., 1994)
Under flooding condition, the oxygen and carbon dioxide level are reduced.
Photosysthesis and cellular respiration are also affected. Xu and Mackill (1996) reported amajor gene that control the ability of rice to tolerate submergence condition, which was named

Sub-1. Then, several studies have shown that plants with Sub-1 gene could survive under afully submerged condition for two weeks. Quantitative trait locus (QTL) analysis revealed
that submergence tolerance is controlled by a single major QTL on chromosome 9 along with85

a number of other minor QTLs (Xu and Mackill. 1996; Nandi et al.,1997; Toojinda et al.,2003).
Acknowledgements
The authors would like to thank IAEA and FNCA for technical cooperation and technicalassistance. The project was fund by Rice Department and Thailand Institute of Nuclear
Technology.

Appendix 1. Screening for submergence tolerance.
Seeds were germinated and subsequently planted in the row in the experimental field for
75 cm long and 25 cm wide. FR13A variety was used as tolerant check, IR42 variety as
susceptible check and the original parental variety were planted after every tenth row forcomparison.
Fourteen days after germination, water level was raised to 10 cm. In the 30th days, thewater level was suddenly increased to 150 cm and maintained for 14 days as a flash flood
condition. All were observed once a week for their performance under flooding condition.
After 14 days of flooding or susceptible check died, the water was drained out, to allow theremaining plants recover for 14 days. Then, visual scoring for recovering ability was carriedout according to Standard Evaluation System for Rice (IRRI, 1996)(Table 1a).
Table 1a. Standard Evaluation System for Rice, scoring submergence tolerance.
Index

Description

Scale

1

Minor visible symptom of injury

100

3

Some visible symptom of injury

95-99

5

Moderate injury

75-94

7

Sever injury

50-54

9

Partial to complete death

0-49

Appendix 2. Screening for blast resistant.
Seeds of each mutant lines were soaked and after two days sprouted seeds of each lines
were sown in a single row on a raised bed of the disease nursery plot. The length of each row
sown was 50 cm and after every two test rows/lines there was a row of a susceptible of KDML
105 The row were kept 10 cm apart. The disease nursery was also bordered around with twolines of susceptible. The nursery was watered on 5th day of transplanting and the chopped
leaves were spread over the entries of the nursery when the seedling were 10 days old. At
86three week stage, the test entries of the nursery were also sprayed with already preparedinoculum of P. oryzae as described by Khan et al ., (2001) by adjusting the spore suspension
to 1x106 spores/ml the nursery was continuously sprayed with tap water to maintain thehumidity. Normal agronomic practice were followed and data of disease were recorded three
weeks after spray inoculation by using rating scale of IRRI (1996).

Appendix 2b. 0-9 grade disease rating scale used for screening for blast nursery.
Grade

Disease severity

Host response

0

No lesion observed

Highly Resistant

1

Small brown specks of pin point size

Resistant2

Small roundish to slightly elongated, necrotic gray spots,

Moderately

about 1-2 mm in diameter, with a distinct brown margin.

Resistant

Lesion are mostly found on the lower leaves
3
4
56

Lesion type same as in 2, but significant number of lesions

Moderately

on the upper leaves

Resistant

Typical susceptible blast lesions; 3 mm or longer infecting

Moderately

less than 4% of leaf area.

Susceptible

Typical susceptible blast lesions of 3 mm or longer infecting

Moderately

4- 10% of the leaf area.

Susceptible

Typical susceptible blast lesions of 3 mm or longer infecting

Susceptible

11-25% of the leaf area.
7

Typical susceptible blast lesions of 3 mm or longer infecting

Susceptible

26-50% of the leaf area.
8

Typical susceptible blast lesions of 3 mm or longer infecting

Highly Susceptible

51-75% of the leaf area many leaves are dead9

Typical susceptible blast lesion of 3 mm or longer infecting

Highly Susceptible

more than 75% leaf area affected
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88

Mutation Breeding of Rice for the Sustainable Agriculture in Vietnam
Le Huy Ham, Vo Thi Minh Tuyen, Nguyen Thi Hong
Agriculture Genetics Institute, Vietnam
Introduction
In Vietnam, rice plays an important role for national food security and politicalstability. Rice also has a direct effect on social security because it is consumed by nearly
89 million of the total population and an important source of income for more than 90
million people living in agricultural and rural areas. Rice is the country’s main crop,
accounting for more than 90% of total cereal productivity. Rice production in 2015
reached 45 million tons, and it is 0.3% higher than in 2014. Vietnam is one of the top 5
countries that studies reveal will be severely impacted by climate change. A rise in sea
level of 1 meter will inundate about 5,000 square kilometers (km2) of the Red River Delta
and 15,000–20,000km2 of the Mekong River Delta, reducing total rice production in
Vietnam by about 5 million tons. Bad harvests, natural calamities, floods, and pests anddiseases will also occur more often. Ensuring domestic food security has thus become anational objective that requires long-term strategies and policies, especially for theprotection of the agricultural land area. Vietnam does not only seek to achieve domestic
food security but it also plays an important role in the international rice market, andconsequently, in the food security of the international community.
Despite of a decrease in rice land area, rice production has been rising due to rapid
yield growth. This in turn has been driven by irrigation and land development, together
with technological change. In 2009, Vietnam had nearly 7.4 million hectares (ha) of ricelands, which declined to 2 million ha in 2015 (Ministry of Agriculture and Rural
Development, 2015). However, rice productivity increased from 39,5 million tons in 2009
to more than 45 million tons in 2015. This was a remarkable achievement due to theapplication of advanced science and technology such as the introduction of different rice
varieties, new production models, and an efficient irrigation system. Post harvest losses
have also been reduced greatly due to the mechanization of rice harvesting and drying,and soil improvement.
Mutation Breeding in Vietnam
At present, about 15% of rice production area is covered annually by mutant
89varieties in Vietnam. Great achievement by the use of nuclear techniques and related
biotechniques. More than 55 mutant varieties were developed, in which most of varieties
are cereal crops, especially rice.
The used materials were local lines (Cuom, Chiem bau, Tam thom, Nang thom, Nang
huong, Te etc.) and the varieties presently used in the crop productivity (C4-63, A8,
CR203, Khang dan, IR64, IR50404, Bac thom). The treatment methods were dry seeds
with different radiation doses (Gamma ray, X-ray with 80, 100, 150, 200, 250 Gy, usefuldoses were 100, 120, 200 Gy); the others were germinated seeds with radiation doses of20, 30, 40, 80 Gy, useful doses were 30, 40, 60 Gy).
Since the 1990s, the Agricultural Genetics Institute (AGI), Vietnam Academy ofAgricultural Sciences (VAAS), has been engaged in R&D activities and contributed muchto the technology transfer to the agricultural sector of Vietnam. Mutation breeding is oneof the major fields of institute in crop improvement, in which the biggest
accomplishments have been achieved in the development of mutation varieties for crops
production. Before 2000, objective of mutation breeding is often quantity of yield variety.
One very interesting example from AGI is DT10 mutant variety which has been certified
as national variety in 1990 due to the high performance, good tolerant to harmful
condition. This variety is available in rice more than 25 years and now this is still
disseminating in some specific location. This variety covers the area around 1.0 mil. ha
in agriculture production. After success of mutant variety DT10, scientists from AGI
released series of mutant varieties (20 mutant varieties) and satisfied production
demand.
In the South Vietnam, mutation induction has been carried out mostly in Cuulong
Rice Research Institute and Institute of Agriculture South Science (IASS). These
institutions have also succeeded in the mutation breeding by the use of nuclear
techniques. Over the past decade, the institutes have developed eight excellent mutant
varieties with high yielding, improved quality, disease resistance, tolerance to pest andlodging resistance. One of these excellent mutant varieties (VND9s-20) which has beencertified as national variety in 1999 became one of the top five varieties for riceproduction. It covered 300 thousand ha per year in south of Vietnam, due to its highyielding, good quality and tolerance to brown plant hopper. Not only has this variety
been widely cultivated in the low lands, but also expanded to the high lands and remotemountain areas, where poor farmers are benefiting from growing it.
The great support from the Vietnamese Government makes the application of
nuclear techniques in food and agriculture so successful. Due to the excellent
performance of the mutant rice varieties mentioned above, some of these mutant
90

varieties were adopted for the national strategy program of “Eradicate hunger andalleviate poverty” in different areas, particularly for central highland region, where there
are many ethnic minorities living in remote mountain areas. Most of the eight mutant
varieties have shown out-standing performance under local production conditions.
Release of new mutant rice for large scales of production
Recently, beside of yield varieties, scientists has been concentrating creation of newvariety having good performance on quality (aroma, protein, amylose content, componentof quality), as well as tolerance to harmful condition of environment such as salinity, coldor high temperature, drought, lodging variety and so on. Mutation breeding is powerfultool for rice breeding improvement.
During 2008 - 2017, groups of rice breeders continue effective collaborations for pureline selections under field trials of rice mutants and their crossings with high quality
and tolerance in Red River Delta (North Vietnam) and Mekong River Delta (South
Vietnam). There are ten outstanding new varieties have been certified as new varieties
and ongoing projects to enlarge mutation varieties in different provinces (Table 1).
Khang dan mutant variety has been transferred the trading rightcopyright from
AGI to the seed company since 2008 after it was certified as national variety. With someadvantages, the area covered with this variety is around 300 thousands ha/year in northVietnam. In 2012, the seed company is rewarded the national prize for Khang dan
mutant variety due to the leading area for agriculture production of this variety.
ĐB5, ĐB6 mutant varieties have high yield, good tolerant to pest and disease. OM
2496 has high productivity, aroma and salinity tolerance. Nam dinh 5 variety has Quality,
aroma and high yield. PD2 is glutinous rice and photo sensitiveness. It has high yield
and aroma. P6 mutant has short growth duration (85-90 days in summer season) andhigh temperature tolerant. Mutant rice varieties DT39 Quelam, CNC11 have improvedin productivity for high yield, better quality and tolerant to bacterial leaf blight.
Moreover, DT39 gave high nutrition content such as iron, zinc, Kali and Maggie incomparison to origin variety.
Mutant rice varieties DT39 Quelam and CNC11 has been transferred the trading
rightcopyright from AGI to the seed company.

91

Table 1. Leading mutant varieties in rice production in Vietnam (duration 2008-2015).
No
1

Variety

Year ofcertify

Dominant characters

Area ofproduction/year (ha)Khangdan

2008

High yield, good tolerant to pest and disease

300,000

ĐB5

2008

High yield, good tolerant to pest and disease

5,000

ĐB6

2008

High yield, good tolerant to pest and disease

5,000

OM 24962009

High productivity, Aroma, Salinity tolerance

3000

PD2

2010

Glutinous rice, photo sensitiveness, high

10,000mutant2
3
4
5

yield, aroma
6P6 mutant

2011

Short growth duration 85-90 days in summer

15,000season, high temperature tolerant7

Nam dinh 52012

Quality, aroma, high yield

3,500

8

ĐB15

2012

High yield, good tolerant to pest and disease

600

9DT39 Que2013

Quality, high protein, high yield, resistant to800

leaf blight

lam
10

CNC11

2015

Quality, high protein, high yield, resistant to300

leaf blight

Applications of gamma rays irradiation and marker assisted selection for improving ofrice varieties for the sustainable agriculture.
Climate change creates adverse conditions for rice production in rice growing
regions. For adaptation to mentioned challenges, cultivation of varieties resistant tobiotic and abiotic stresses is required.
In present, the application of irradiated techniques and biotechnology in rice
breeding has focused on some major traits, such as high yield, short duration, goodquality, bacterial leaf blight resistance, blast resistance and salt tolerance.
92

In 2013, dry seeds of three varieties were irradiated with 300 grey of Cobalt-60
gamma rays. Among them, BT62.1 was resistant to bacterial leaf blight (carrying Xa7,

Xa21 genes), P5.3 was resistant to blast (carrying Piz gene), and BT3.1 was salt tolerantvariety (carrying saltol gene). All of these varieties have low yielding. Purpose ofirradiation is improvement of yield. M1 and M2 plants of these varieties were evaluatedfor mutant characteristics. We expect to get the mutant lines not only have resistance todiseases but also have high yield, short duration, and good quality.
In 2014, M3 and M4 progenies of these varieties were used for marker assisted
selection (MAS) and inoculation in the green house to select elite lines resistant tobacterial leaf blight resistance, blast resistance and salt tolerance. Twenty M4 mutant
lines (from M4.1 to M4.20) retained as short duration, good quality, bacterial leaf blightresistance, blast resistance or salt tolerance as original varieties but they have thehigher yield than the original ones.
In 2015, these lines are evaluated the agronomic and biological traits as well as
resistance characters in field condition (narrow scale). The results showed that: 6
promising mutant lines were selected (2 mutant lines showed high resistance to the BB
pathogen, 2 lines resisted the BL pathogen and 2 lines showed high salt tolerance in thefield. The promising mutant line, DT80 carrying saltol gene and can withstand salinityof 0,6%. DT80 was evaluated in salinity field of the Nam Dinh province in spring season2015.
In 2016, DT80 mutant line was evaluated the agronomic and biological traits as wellas tolerant characters in field condition (narrow scale) and send the National testing
Center. Beside of this activity, promising lines should be evaluated in different local
conditions for evaluation of wide adaptability of new variety and evaluate the responseof the farmer. The result of this evaluation indicated that DT80 mutant variety have
improved in productivity for high yield (Potential yield: 6,8 - 7 ton/ha), better quality(amyloze contents: 14%), short growth duration (105-110 days in summer season),medium plant height (112 cm), Wide adaptation and easily cultivation. This new mutant
variety were evaluated with some major pests and disease and the evaluation in the fieldshowed that DT80 were affected by these biotic and abiotic stresses in the majority ofharmful levels from 1-3 points.
DT80 was sent to The National Testing Center for Crops to test for Value ofCultivation and Use of crop (VCU) 3 seasons and Distinctness, Uniformity and Stabilityof crop (DUS) 2 seasons.
DT80 mutant variety has been transferred the trading right from AGI to the Thanh
hoa seed company in November, 2016. In 2017 it registered as new variety by MARD.
93

Results of irradiation ion beam on rice breeding
Carbon ion beam have been recently considered as potential mutagens. A
characteristic feature of ion beams is their ability to deposit high energy on a target,
densely and locally. Rice seeds were treated with Carbon ion radiation (40, 60, 80 and
100 Gy) by AVF-Cyclotron at the Japan Atomic Energy Research Institute (JAERI),
Takasaki, Japan and were planted and selected at the Agricultural Genetics Institute.
There are some results of carbon ion Beam irradiation up to now.

Table 2. Overview irradiation data.
Varieties

Time

Dose

Bac thom and Khang dan

6/201040Gy and 60Gy

CMBT and BLBT

3/201340Gy and 60Gy

DT80, DT82, DT86, T5, TBR2 and RVT6 1/2015

60Gy and 80Gy

BC/6 and P12

6/2015

40Gy, 60Gy, 80Gy and 100Gy

ĐB2, KN6, BH96/2016

40Gy, 60Gy, 80Gy and 100Gy

Results of carbon ion beam irradiation on Bac thom and Khang dan
There are many types of variations in M2 generation from irradiated Bacthom andKhang dan. Four promising mutants were selected from Khang dan in which two lines
came from dose of 40Gy and two other mutant lines were derived from dose 60 Gy. Mostof the mutant lines express higher yield than the original variety and better resistant topests and diseases. However, the objective of the project is to increase yield and to obtain
good quality rice variety, therefore the research is concentrated in Bacthom variety.
Selection process was conducted according to better agronomic traits; short growduration, high yield and purity of mutant lines in the M4 and M5 generations. The
mutant lines were grown and evaluated similar to the M5 and M6 but in the expanded
testing area and perform in some different locations to assess purity, growth anddevelopment capacity, as well as yield and resistance to pests and diseases. The
promising lines will be chosen for further research. Result indicated in the table 1 and 2
below. In M7 generation of Bacthom mutant variety showed that grow duration of somemutant lines (M7-2, M7-6 and M7-8) were similar to control; meanwhile some mutant
line indicated short grow duration than the origin such as M7-4; M7-3. In M7 population,
purity of 3 mutant lines M7-3, M7-4 and M7-5 were in level 1 better than the origin inlevel 5 as well as M7-2, M7-6 and M7-8. However, purity level of mutant line can be94

improved by selection process in further generation. Panicle length is also one ofcomponent of yield performance of variety. Most of mutant lines have panicle length thatis longer than the control of Bacthom variety. That mean of most mutant lines may havehigher yield compare to original Bacthom variety.
Table 3. Biological and agricultural characteristics of 6 Bacthom mutant lines in M7(summer season 2013).
Lines

GrowthPlant

Flowering

Plant

PaniclePuritydurationheightduration

hardnesslength(points)

(day)

(cm)

(points)(points)(cm)

BT (Cont.)

107

108.0

5

3

21.5

5

M7-2

107

115.6

5

3

25.8

5

M7-3

104

114.4

5

1

23.6

1

M7-4

102

117.2

5

3

24.5

1

M7-5

105

108.2

5

1

22.1

1

M7-6

106

109.6

5

3

22.4

5

M7-8

108

102.8

5

3

24.2

5

•

Plant hardness: 1-Very hard,

3-Hard, 5-Medium hard,

7-Weak hard,

9-

Weak hard.
•

Flowering duration: 1-Focus (≤ 3 days), 5-Medium (4-7 days), 7-Long (≥7 days)•

Purity: 1-High (different trait ratio <0,25%), 5-Medium (different trait ratio:

0,25-1%), 9-Low (different trait ratio > 1%).
With regards to the grain yield component of mutant lines in the M7 generation
showed a significant difference compare with the control for full seed per panicle thehighest were obtained at M7-4 and M7-8 lines and showed significant difference fromother lines (table 2). This resulting in potential yield of M7-4 and M7-8 were higher thanthat of original and other lines with 9.63 tons/ha and 12.48 tons/ha. These other mutant
lines performed grain yield higher than original variety except M7-6 which was 5.34
ton/ha. However, one elite mutant to be selected will not only depend on the yield
performance character but also on the cooking quality and aroma characteristic of thevariety.

95

Table 4. The grain yield components of 6 mutant lines of mutant Bacthom in M7(Summer season 2013).
No. ofLinespanicle/hill

No. of seeds

No. of full

Weight ofPotential

seed

1000 seeds

yield/panicle(gr)(ton/ha)/panicle

BT (Cont.)

5.1

140.5

130.9

19.6

5.50

M7-2

5.3

177.6

159.3

22.1

7.84

M7-3

5.2

182.2

160.4

19.8

6.94

M7-4

5.8

212.4

197.7

20.0

9.63

M7-5

6.0

132.7

119.3

19.5

5.86

M7-6

5.5

127.3

118.5

19.5

5.34

M7-8

6.2

257.9

236.1

20.3

12.48

Cooking quality is main objective of the breeding improvement. Six mutant lines of
Bacthom were tested in room condition for aroma, softness, stickiness, and taste toevaluate the quality of selected mutant lines in comparison to original variety.. The
result of this experiment indicated that M7-2; M7-3; M7-6 and M7-8 have cooking
qualities similar to the original variety Bacthom, meanwhile M7-4 and M7-8 indicateddry and hard when cooked even M7-4 and M7-8 but have highest grain yield.
Results of carbon ion beam irradiation on CMBT and BLBTIon beam irradiation 3/2013: The variable new mutant lines were screened andevaluated in M2 generation. (selected lines with short growing period, medium plantheight, tillering ability, a number of seeds per panicle is higher than that of variety
control. Specially, selected lines reveals small tillering angle, this is characteristic need
to be improved from original lines). Selection process was conducted according to better
agronomic traits; short grow duration, high yield and purity of mutant lines in the M3
and M4 generations.
Screening of mutant lines in M5 and M6 generation. In this period, agrobiologicalcharacteristics and tolerance traits is being tested in the field conditions (narrow scale).
MAS was applied to identify the target genes. The prospective lines in M5 generation to
be continued in varietal testing system for assessment of the agro -biologicalcharacteristics: field grain yield and grain quality towards approval as national varieties.
Five mutant lines (two from CMBT, three from BLBT) with high potential yield and goodagronomic traits were selected96Based on agronomic traits in fields, test in greenhouse and MAS in lab, 14 promisingmutant lines in M5 generation were selected: 5 mutant lines from CMBT and 9 mutant
lines from BLBT.
The results showed that 4 promissing lines (BL.1,2,6,7) have a growth duration isshorter and number of fulled seed is much higher than that of control. So that these lines
obtained the higher yield than control variety (Table 9).
The results showed that 2 promissing lines (CM.3,4) have a growth duration isshorter and number of fulled seed is much higher than that of control. So that these lines
obtained the higher yield (7.5 ton/ha) than control variety (Table 5).
In 2015 spring season, the total of 6 promissing mutant lines were obtained. Theseline will be selected in 2016 for evaluating the agronomic traits and resistance to pests
and diseases.
Table 5. Agronomic traits and yield potential of BB mutant lines in M5 from BLBT
(Spring season, 2015).
No

Mutantlines

Durationof growth(days)Height ofplant (cm)Number of

PanicleNumberpanicle/exertion of fulled

plant

(cm)

seed

Weightof 1000
grains(gr)

Potential
yield(ton/ha)

Cont.

BLBT

130

109

6.5

2.3

184.7

22.3

5.7

1

BL.1

129

95

8.0

3.0

219.0

19.8

6.9

2

BL.2

128

95

8.0

3.2

236.7

20.1

7.2

3

BL.3

132

95

5.0

4.2

167.7

19.5

6.5

4

BL.4

130

95

7.0

3.0

192.3

20.2

5.9

5

BL.5

127

100

7.0

0.8

180.0

21.2

5.6

6

BL.6

125

100

7.0

1.7

257.7

21.5

7.5

7

BL.7

130

105

6.5

0.6

276.9

21.0

7.1

8

BL.8

129

100

5.8

0.9

242.0

21.4

6.1

9

BL.9

125

102

6.0

0.5

261.3

21.4

6.4

97

Table 6. Agronomic traits and grain yield components of Salinity tolerance mutant
lines in M5 from CMBT (Spring season, 2015).

No

Mutantlines

Duration Height of Number ofPanicleNumberof growthplant

panicle/exertion of fulled(days)

(cm)

plant

(cm)

seed

Weight of1000
grains(gr)

Potential
yeild(ton/ha)

Cont.

CMBT

128

111

6.0

2.5

159.5

22.2

5.5

1

CM.1

128

105

8.0

3.5

183.0

20.3

5.9

2

CM.2

130

105

8.0

1.3

183.3

20.1

6.4

3

CM.3

129

100

8.0

-0.5

248.0

20.8

7.5

4

CM.4

130

100

8.0

-0.7

251.0

20.5

7.1

5

CM.5

129

100

7.0

5.7

145.3

21.3

6.7

Table 7. Agronomic traits and grain yield components of promising mutant lines(summer season, 2016).
No

Mutantlines

Durationof growth(days)Height ofplant (cm)Number of

PanicleNumberpanicle/exertion of fulled

plant

(cm)

seed

Weightof 1000
grains(gr)

Potential
yeild(ton/ha)

Cont.

BLBT

105

110

5.5

2.3

145.7

22.5

5.3

1

BL.1

102

98

5.8

3.0

179.0

20.0

6.5

2

BL.2

108

100

6.1

3.2

186.7

20.1

6.8

6

BL.6

105

102

6.8

2.7

217.5

21.3

7.5

7

BL.7

103

110

6.5

0.3

176.9

21.0

6.1

3

CM.3

107

108

7.0

0

178.0

20.5

6.5

4

CM.4

105

109

6.5

0.2

175.0

20.6

6.1

Cont.

CMBT

128

109

6.0

2.5

159.5

22.0

5.5

98

The results of evaluating the agronomic trait in the field (Table 11) showed thatthese lines have a shorter duration, with higher production in comparison with control.
Three mutant lines: BL7, CM.3, CM.4 were high yield, but their panicle exertion is very
short (0-0.3cm). This reason make these lines can increase higher empty seed ratio for
reducing the yield. Out of which, the promissing BL6 line revealed hard stem hardness,
can resistance with abiotic and biotic stresses. Next, these lines will be adapted indifferent rice ecosystem to production expanding and sent to The National Testing
Center for Crops to test for Value of Cultivation and Use of crop.
Table 8. Summary results of carbon ion beam irradiation (From 1/2015).

Variety

DT80

DT82

DT86

T5

TBR2

RVT6
Total

M4

M5

(mutant(Promissing

lines)

line)

25

5

0

25

37

8

0

89%

10

15

12

8

80Gy

91%

20

18

5

0

60Gy

91%

26

20

8

0

80Gy

91%

30

18

9

4

100Gy

95%

18

24

9

6

60Gy

87%

9

16

12

7

80Gy

88%

15

21

5

0

60Gy

87%

29

32

9

0

80Gy

88%

5

0

0

0

60Gy

85%

31

25

5

3

80Gy

92%

45

18

6

3

278

269

93

31

Dose

M1

M2

M3

Sterility

variation

variation

60Gy

83%

15

80Gy

86%

60Gy

99

Results of carbon ion beam irradiation from 2015 to 2017
The variable new mutant lines were screened and evaluated from M2 to M5
generation. We selected lines with short growing period, medium plant height, tillering
ability, a number of seeds per panicle is higher than that of variety control. The resultsin table 7 showed that: 278 mutant individual in M2 generation were selected in 2015.
In 2016, 269 mutant individual in M3 generation and 93 mutant lines in M4 generation
were selected. In 2017, based on agronomic traits in fields, 31 promising mutant lines in
M5 generation were selected: 18 promising lines from 60 Gy, 7 promising lines from 80
Gy and 6 lines at 100 Gy.
In 2016, 56 mutant individual in M2 generation were selected with short growing
period, medium plant height, tillering ability, a number of seeds per panicle is higher
than that of variety control. In 2017, Set of 102 mutant lines were obtained in M3
generation, 39 mutant lines in M4 generation were selected. The results showed that
(Table 8): 17 mutant lines were selected from 60 Gy, 7 mutant lines from 80 Gy and 15
mutant lines at 100 Gy.
Table 9. Summary results of carbon ion beam irradiation (From 6/2015).
Time
irradiation

Variety

6/2015
BC/6

P12

M4

Dose

M1

M2

M3

Sterility

variation

variation

40Gy

75%

0

0

0

60Gy

91%

15

26

17

80Gy

91%

5

12

0

100Gy

95%

3

9

0

40Gy

68%

5

0

0

60Gy

91%

12

18

0

80Gy

91%

10

23

7

100Gy

95%

6

14

15

56

102

39

Total

100

(mutant
lines)

Table 10. Summary results of carbon ion beam irradiation (From 6/2016).
Variety

BH9

KN6

ĐB2

Dose

Germination*

M1

M2

Sterility

variation

40Gy

100%

75%

60Gy

100%

91%

31

80Gy

99%

91%

23

100Gy

97%

95%

14

40Gy

100%

65%

60Gy

98%

91%

11

80Gy

100%

91%

22

100Gy

100%

95%

24

60Gy

100%

91%

80Gy

91%

100Gy

95%

20

From irradiated time 6/2016: 145 mutant individual in M2 generation were selected
in 2017. Among that: 44 mutant lines were selected from 60Gy, 45 mutant lines from
80Gy and 58 mutant lines from 100Gy.
The promising mutant line with some improved traits such as: light aroma, softer
endosperm, good tillering ability, big and longer seed size, more grain number per
panicle....comparing to original variety. Thus, our research was focused on database of 5
target genes regulated these features as in table below:
Through sequence and BLASTN, total of 24 point mutants were identified in target
genes (as in table).

101

Table 11. Results of study to clarify mutation related to high yield and good quality
Material of study.
No.
1

Name

Features

Original variety (DT82)

BLB resistance, low yield, tillering ability (4-5
tillers), plant height (113-115cm), dark green
leaf, small seed, dark yellow husk seed2

Ppromising line (60Gy)

BLB resistance, improved yield, better tillering(8-10 tillers), plant height (120 cm), light green
leaf, better quality, big and long seed, lightyellow husk seed

Table 12. Target gene information.
No.
1
2

Gene name
Os08g0424500
Os06g0133000

Regulation
BetaineGene features
aldehyde

Chr.8(21703754:21707360),

dehydrogenase, Rice fragrance

including 13 exons

Glutinous endosperm

Chr.6(1929936:1935488),

including 15 exons
3
4

5Os03g0171600
Os07g0261200

Os03g0203200

Leaf senescence, seed size andChr.3(4284298:4286750)

grain number

including 1 exon

Grain number, plant height, andChr.7 (9173610:91755560)

heading date 7

including 2 exons

Shoot branching

Chr.3(5895977:5897579),

including 2 exons102

Table 13. Identified point mutations in target genes.
Gene

Number of mutant

Os08g0424500
Os06g0133000

6

Location of mutants
Exon 1 (40, 41, 42, 47, 48); exon 12 (121)
Exon 3 (33, 34, 35, 70); exon 4 (15); exon 9

6

(115)

Os03g0171600

5426, 615, 621, 1017, 1263

Os07g0261200

4Exon 1 (73, 253); exon 2 (32, 36)

Os03g0203200

3

Exon 2 (24, 98, 401)

Total

24

Conclusion-

The dose of 60 Gy is effective for screening good trait mutants-

To be need further trial to assess the efficiency of dose 80Gy and 100Gy-

Mutations conducted from ion beam were almost point mutations

Work plan- Testing promising lines in different ecological zones- Continue screening mutation lines to select the good and stable lines- Repeat the irradiation with range of doses to performed the most effective dose formutation breeding.

Conclusion
Successful story of mutation plant breeding in Vietnam is contributed from politicalsupport by Vietnamese government for mutation plant breeding research such as
international organization such as FAO/IAEA, through project IAEA/VIE/05/13/14 from1997-2003 and Ministry of science and Technology Foundation was supported by
Program: Applied research and development of energy technologies; code: KC05.09/1115 from 2012-2015. Beside of this from 2008, Vietnam became official member of Forumfor Nuclear Cooperation in Asia (FNCA). Scientists from different institutions havechance to go abroad and attend training course, scientific symposium and workshop, or
to exchange information and experiences with scientists working in mutation breeding.
It’s clear that mutation induction nowadays is one of powerful tool for crops improvement103

in Vietnam. Mutant rice varieties from institutions contribute to increase of riceperformance and productivity in recent years. This is confirmed that Vietnam
Government has been concentrating research and development of the activities on
nuclear techniques in agriculture. Beside of that farmers also benefit from scientificresearch activities.

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