FNCA／MEXTTechnical Material

Achievement
Sub-Project on Composition or Quality in Rice
( 2007 – 2012 )Mutation Breeding Project
Forum for Nuclear Cooperation in Asia (FNCA)
March, 20130

This report was prepared by the Nuclear Safety Research Association (NSRA) as a part of activities on
Forum for Nuclear Cooperation in Asia (FNCA), organized by MEXT.

Foreword

Rice is the most important crop not only in Asia but also in the world. In fact, 90 % of all the
production of rice in the world is produced in Asia. One report also states that almost 60 percent of
all the population in the world eats rice, whereas about 30 percent eats wheat. Farmers or the
relevant scientists, especially in every Asian rice- producing country, have made great efforts
directed at increasing rice yield. In addition, people have recently been interested in the quality orcomposition of rice, in view of an aspiration for health and life. Considering these si tuations, in
FY2007 we took up rice as materials to perform mutation breeding in the sub-project on“Composition or Quality Improvement in Rice”, to be finished in FY2012.
It must be noted that important agronomic traits, e.g. grain yield and resistance t o various
environmental stresses, were kept in mind to be improved in this sub-project, aiming mainly at
improving the quality or composition of rice. The contents of amylose, protein, fiber, vitamin A,
anthocyanin, resistant starch and phytin were treated in terms of the quality or composition of rice.
The breeding purposes were completed by every member country depending on their own needs. It
was our primary concern on whether or not the results obtained from the project could be realized
for technology transfer to society as well as those being of scientific worth.
I am pleased to state that we have obtained a lot of fruits worthwhile from both the practical andfundamental points of view in the sub- project. It is noted that a new mutant variety, DT 39, hasbeen certified by the Ministry of Agriculture, Vietnam, to be released in January 2013. This variety
of rice is of high yield with high protein content as well as resistance to bacterial leaf blight, sheathblight and brown plant hopper.

Attention must also be paid to the fact that the variety wasdeveloped just within the term of the sub-project for 5 years, while it might have taken at least 10
years when using the conventional breeding method.
This book may prove that many mutant lines have been selected, in every member country, for highyield and/or resistance to various environmental stresses, e.g. diseases, insects, drought and so forth,
as well as the improved quality or composition of rice. I want to say that some of the mutant lines
obtained will be officially released as new commercial varieties in the near future.
In 2009, a workshop for training on how to use ion beams was held in TIARA, JAEA. With theworkshop as a start, a system for the utilization of ion beams has been established f or the member
countries to perform the mutation breeding of rice efficiently through the use of ion beams as well
as gamma-rays. The fruits of the trials for the utilization of ion beams may also be found in this
book.

i

It is worth emphasizing that, during the performance in a series of FNCA mutation breeding
sub-projects, strong merits of mutation breeding have been confirmed especially on the following
three points: (1) to improve target traits without altering the other ones, (2) to develop new genes,(3) to effectively improve target traits on plants of vegetative propagation, e.g. fruits. This must bea good chance to now recognize the strong points of mutation breeding to be promoted for moving
forward the establishment of sustainable agriculture, in the crucial situations of earth environments
and food problems. I would be extremely appreciative, if the breeders or researchers concerning
agriculture could be encouraged by the fruitful results of the sub -project presented in this book so
as to move forward in the future.
March 2013
Hirokazu NAKAI
Project Leader of Japan, Mutation Breeding Project, FNCA
Professor Emeritus, Former Vice President,
Shizuoka UniversityiiContent

1. Bangladesh ········································································ 1
1.1
1.2
1.3
1.4
1.5
1.6
1.7

Summary
Introduction
Materials and Methods
Results
Discussion
Acknowledgements
References

2. China ············································································· 11
2.1
2.2
2.3
2.4
2.5
2.6
2.7

Introduction
Objective of the Project
Materials and Methods
Results and Discussion
Conclusion
Acknowledgement
References

3. Indonesia ········································································· 21
3.1
3.2
3.3
3.4
3.5
3.6
3.7

Abstract
Introduction
Materials and Methods
Results and Discussion
Conclusion
Acknowledgement
References

4. Japan ············································································· 31
4.1
4.2
4.3
4.4

Introduction
Materials and Methods
Results and Discussion
Conclusioniii

5. Korea ············································································· 37
5.1
5.2
5.3
5.4

Introduction
Materials and Methods
Results and Discussion
References

6. Malaysia ·········································································· 45
6.1
6.2
6.3
6.4
6.5
6.6

Introduction
Materials and Methods
Results and Discussion
Conclusions
Acknowledgement
References

7. The Philippines ································································· 61
7.1 Abstract
7.2 Introduction
7.3 Objectives
7.4 Economic Impact
7.5 Methodology
7.6 Results and Discussion
7.7 Future Plans
7.8 Conclusion
7.9 Acknowledgement
7.10 References

8. Thailand ···································································· 71
8.1
8.2
8.3
8.4

Introduction
Materials and Methods
Results
Conclusion

9. Vietnam ·········································································· 81
9.1
9.2
9.3
9.4
9.5
9.6

Introduction
Materials and Methods
Results and Discussion
Conclusion
Acknowledgement
Referencesiv

1. Bangladesh

High Yielding mutants with shorter life cycle selected in rice
irradiated with carbon ion beam
A. N. K. Mamun, A. K. Azad 1, M. H. Kabir, P. K. Roy, M. R. Islam and M. T. Jahan,
M. A. Azam1, M. L. Hakim, & G. Ahmed
Biotechnology and Genetic Engineering Division, Institute of Food and Radiation Biology,
Atomic Energy Research Establishment, Bangladesh Atomic Energy Commission,
1

Plant Breeding Division, Bangladesh Institute of Nuclear Agriculture, Bangladesh

1.1 Summary
To investigate the possibility of induced fixed and stable mutants in M 1 generation of rice in which,
one hundred and fifty seeds of the highly photoperiod sensitive and tall indica type rice cv. Ashfal
and popular variety BRRI dhan29 were irradiated with different doses of carbon ion beams from theRadiation Applied Biology Division, Quantum Beam Science Division, Japan Atomic Energy Agency,
Japan. In case of BRRI dhan29 and Mutant of BRRI dhan29, it was grown during Boro season, 2012
and in the case of Ashfal M 1 was grown during long day length at Boro (December-May) season andM2 during short day length at Aman season (July–December) in 2009 whilst M 3 once again grown
during Boro season in 2010. Nine M 1 hills in which, 2 from each of 40, 120 and 160Gy, and 1 fromeach of 60, 80 and 200 Gy were headed even under long day in Boro season despite none of theparent plants of Ashfal headed during Boro season. Of the heading of M 1 plants that had the plantheight as the parent that could not produce any seed. Interestingly only the shorter plant height thatcapable to produced seeds. The M2 progenies took 85 days and 110 days for heading and maturity
respectively, during short days in Aman season. In contrast, the parent Ashfal headed after 7
November and matured at 152 days. In M 3 generation, the progenies bred true as in M 2 for heading,maturity and plant height. It was further confirmed that the Ashfal parent and the mutants had similar
1000-grain weight. Interestingly in case of mutant of BRRI dhan29 showed higher yield and earlymaturity compared to the parent of BRRI dhan29. Finally, it could be concluded that it is possible toinduce stable mutant(s) with photoperiod insensitive and shorter plant height and also higher yieldand early maturity traits in rice treated by 40 to 200Gy dose range of carbon ion beams in M 1 ofindiac rice cv. Ashfal and popular rice variety in Bangladesh BRRI dhan29.
Key words: rice, indica type, ion beams, mutation, photoperiod sensitivity, fixed M 1 mutant.
1.2 Introduction
Rice (Oryza sativa L.) is a short day plant (Vergara and Chang, 1985). Almost all rice cultivars
matured in a shorter time when they were grown under a short photoperiod (about 10 h) than under along photoperiod (about 14 h). The degree of photoperiod sensitivity greatly varies among cultivars.
In Bangladesh, rice is being grown in three seasons in a year; Aman season (July to December), Boroseason (December to May) and Aus season (March to June). Ashfal is a local T.aman (July to
December) cultivar being cultivated in the coastal south western part of Ban gladesh from time
immemorial. It is highly photoperiod sensitive with long duration and tall height cultivar. It is prone
to lodging 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 and Asaduzzaman, 2004). Ashfal-2-

cannot be grown in Boro (long day) season as it is highly photosensitive. It often becomes lodged
even in Aman season due to its tall culm which cause yield losses to a considerable extent.
Recently, ion beam irradiation has been appeared to utilized an effective means of inducing
mutations. The biological effects of ion beams have also been investigated and observed to be showna high relative biological effectiveness (RBE) in lethality, mutation and al so to transfers high energy
to the target compared to low linear energy transfer (LET) radiation such as Gamma -rays, X-rays andelectrons (Blakely,1992). It has been also demonstrated that ion beams induce mutations at highfrequency and induce novel 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 andwidely accepted that in mutation breeding induced traits become fixed during M 2 -M4 generations(Azad et al., 2010; Hamid et al., 2006; Shamsuzzaman et al., 1998; Azam and Uddin, 1999). But it
has been reported that it is possible to isolate fixed mutants even in M 1 generation of heavy ion
irradiated sweet pepper (Honda et al., 2006).
In this study, we aimed to describe the possibility to get fixed M 1 mutant(s) with photoperiod
insensitive, short day-length and shorter plant height from local T.aman Ashfal and also to get higher
yield and early maturity from BRRI dhan29 in rice by ion beam irradiation.

1.3 Materials and Methods
Dehusked seeds of Ashfal rice and BRRI dhan29 were exposed to 26.7 MeV/n carbon ions withdifferent doses of 0, 10, 20, 40, 60, 80, 100, 120, 160 and 200 Gy in January 2009 at Japan Atomic
Energy Agency (Takasaki, Gunma, Japan). One hundred and fifty seeds were used for each
irradiation dose. The irradiated seeds were allowed to germinate in petri dishes. Seedlings weretransplanted on February 11, 2009. Single seedlings were transplanted into each hill. Spacings weremaintained between rows and hills are of 20 cm and 15 cm respectively. All plants were grown undernatural environment at the Bangladesh Institute of Nuclear Agriculture (BINA) head quarters’ farm
Mymensingh, Bangladesh. The day length at the time of heading in Boro (December to May) season
is between 12 - 13 h whilst that in Aman season (July- December) is 10 - 11 h. Fertilizers wereapplied at a rate of N-90 Kg, P-18 Kg, K-63 Kg, S-10 Kg and Zn-1.60 Kg/ha in the forms of urea,
tripple super phosphate, muriate of potash, gypsum and zinc sulphate equal ly in all plots. All
fertilizers were applied during final land 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. Heights were measured randomly from ten M 1 plantsof each irradiated dose at 3 weeks after transplanting. M 2 seeds were harvested individually fromthree fertile M 1 plants at 5 months after transplanting. The M 2 seeds were spread on a seed bed on
July 16, 2009. Survived seedlings were transplanted on August 17, 2009 and they were grown during
Aman season. 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 the number of days
required from sowing time when 90% of plants of each line appeared with yellowish grains. These
two data were recorded through visual observation by visiting the plots every alternate day. Plant
heights, number of effective tillers per plant and panicle length were measured at the time of maturity
with randomly selected 5 competitive plants. M 3 seeds were harvested separately from each M 2 plant.
-3-M3 plants were grown as plant progeny during next Boro (long day) season (December 2009 to May
2010). Data were recorded as the same as described in M 2. Grain characteristics such as length (L)and width (W) of unhusked and husked grain were recorded in October 2012. The mutants derived
from 140 Gy dose were excluded previously that are not included.

1.4 Results
Seeds of Ashfal irradiated with carbon ions were grown during Boro (long day) season in 2009.
Survival rate and plant height was determined to examine the effect of carbon ion irradiation over theplant growth cycle. Survival rate of the irradiated seeds were significantly decreased at 60Gy andremains gradually at higher doses (Table 1). Seedling height also gradually decreased with theapplication of increased rate of irradiation dose. The height of the seedlings were reduced almost half
compared to unirradiated seeds and it was started at 80Gy and gradually goes to at higher doses.
Since the cultivar Ashfal is a highly photoperiod sensitive in which, none of the control plants headed
during the Boro season. However, we have found nine M 1 plants headed under the same growing
conditions (Table 1).
Table 1. Effects of carbon ion irradiation on survival rate, seedling height and heading in highly
photosensitive rice cultivar Ashfal grown in Boro season
Dose

NumberNumber ofSurvival

Seedling

NumberNumber of(Gy)of seeds

survived

rate (%)

height* (cm)of headed

fertilesown

plantsplantsplants

0

150

147

98.0

23.5 ± 0.4

0



10

150

122

81.3

23.0 ± 0.7

0



20

150

141

94.0

21.7 ± 0.4

0



40

150

121

80.7

19.2 ± 0.8

2

1

60

150

94

66.0

15.5 ± 0.2

1

0

80

150

15

10.0

13.5 ± 1.5

1

0

100

150

9

6.0

12.5 ± 0.5

0



120

150

6

4.0

11.6 ± 1.3

2

0

160

150

11

7.3

12.9 ± 1.1

2

1

200

150

3

2.0

12.3 ± 2.0

11* Mean  SE of seedling heights at 3 weeks after transplanting were shown.

It was confirmed that photoperiod sensitivity was genetically altered in the fertile M 1 plants in which,
M2 seeds were harvested from them and grown during next Aman (short day) season in 2009.
Seedlings were raised in seed bed with 301, 650 and 246 M 2 seeds which were derived from the 40,
160 and 200Gy treated with carbon ion beam respectively. Germination rate was appeared very low
which probably due to damage of soft embryo tissue caused by ion beam or the effect of seed
dormancy occurred or the effect of both. Of which 9, 126 and 18 M 2 seedlings were transplanted to
the field. The parent Ashfal headed within 122 days and matured within 152 days after sowing in thefield. In contrast, M 2 progenies derived from 40 and 200Gy doses of carbon ion beams and 200Gy
has taken almost 85 and 110 days to heading and maturity respectively. The number of effective-4-

tillers per plant was observed 13 in Ashfal whilst it ranged from 4 to 13 in M 2 progenies (Table 2).
The panicle length was found similar to Ashfal in all M 2 progenies studied. These facts suggests thatthe photoperiod insensitive phenotype of the three fertile M 1 plants was inherited to all M 2 progenies
without any segregations.
Table 2. Characteristics of M 2 progenies grown in Aman season and M 3 progenies grown in Boro season
M 2 generation
grown in aman season, 2009
Mutant line

M 3 generation
grown in boro season, 2010

Plant
height(cm)

Number ofeffective
tillers/plant

Paniclelength(cm)

Plant
Height*(cm)

Number ofeffective
tillers/plant*

Paniclelength*(cm)

RM(1)-40(C)-1-1

75

10

21

75.6 ± 1.0

10.0 ± 0.6

22.6 ± 0.8

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

73

11

23

77.8 ± 1.7

8.2 ± 0.7

22.8 ± 0.5

RM(1)-40(C)-1-3

77

5

23

79.0 ± 0.7

9.0 ± 0.6

22.4 ± 0.5

RM(1)-40(C)-1-4

73

10

19

77.0 ± 1.8

8.4 ± 1.0

22.8 ± 0.6

RM(1)-40(C)-1-5

76

7

24

76.8 ± 2.0

9.2 ± 0.7

22.8 ± 0.2

RM(1)-40(C)-1-6

72

7

21

74.0 ± 1.3

9.0 ± 0.8

22.8 ± 0.5

RM(1)-40(C)-1-7

74

7

21

81.0 ± 1.7

11.8 ± 0.6

23.0 ± 1.7

RM(1)-40(C)-1-8

81

6

22







RM(1)-40(C)-1-9

91

13

28

83.4 ± 5.1

11.6 ± 1.4

26.2 ± 1.6

RM(1)-200(C)-1-1

68

13

26

70.6 ± 2.0

9.2 ± 1.1

23.0 ± 0.4

RM(1)-200(C)-1-2

64

10

22

65.9 ± 0.7

6.0± 0.7

21.6 ± 0.7

RM(1)-200(C)-1-3

68

6

23

67.4 ± 2.4

7.8 ± 0.9

23.2 ± 0.7

RM(1)-200(C)-1-4

67

8

22

69.4 ± 4.8

8.6 ± 0.9

24.6 ± 1.4

RM(1)-200(C)-1-5

66

5

22

66.0 ± 1.4

6.2 ± 0.5

22.6 ± 0.9

RM(1)-200(C)-1-6

69

8

21

67.8 ± 1.2

7.8 ± 0.6

22.4 ± 0.7

RM(1)-200(C)-1-7

61

6

20

69.8 ± 1.6

6.6

± 0.7

22.4 ± 0.5

RM(1)-200(C)-1-8

62

9

22

67.6 ± 2.0

8.6 ± 0.5

22.6 ± 0.4

RM(1)-200(C)-1-9

71

7

22

70.2 ± 0.7

7.6 ± 0.4

22.0 ± 0.4

RM(1)-200(C)-1-10

56

5

19

69.4 ± 1.6

9.8 ± 0.6

22.6 ± 0.5

RM(1)-200(C)-1-11

58

7

22

69.4 ± 1.1

7.4 ± 0.2

22.2 ± 0.5

RM(1)-200(C)-1-12

64

6

21

69.2 ± 1.2

9.6 ± 1.7

21.8 ± 0.7

RM(1)-200(C)-1-13

75

6

23

69.4 ± 1.0

11.0 ± 0.9

22.6 ± 0.2

RM(1)-200(C)-1-14

66

9

21

69.6 ± 1.0

8.8 ± 0.5

23.2 ± 0.9

RM(1)-200(C)-1-15

63

7

21

67.0 ± 0.8

8.4 ± 0.9

22.6 ± 0.8

RM(1)-200(C)-1-16

56

4

16

70.6 ± 1.9

10.6 ± 1.0

22.6 ± 0.7

RM(1)-200(C)-1-17

61

8

21

65.0 ± 1.5

10.2

± 0.8

22.8 ± 0.5

RM(1)-200(C)-1-18

48

4

20

70.6 ± 2.0

9.2 ± 1.1

23.0 ± 0.4

Ashfal (parent)

148

13

24







*Mean  SE of 5 plants, RM indicates Rice Mutant; 40(C) and 200 (C) indicate irradiation with 40-5-For further confirmation of photoperiod insensitivity of mutant lines, M 3 plants derived from theeach M2 plants were grown during next Boro (long day) season in 2010. Interestingly none of theparental Ashfal was found to be headed in this season whilst all mutant lines headed almost 90 days
after sowing and matured within 119 days after sowing. Days to head ing and maturity were foundslightly longer than in the case of M 2 generation where that were grown during Aman season. It wasobserved that in M 2 generation, the plant height of mutant lines were markedly shorten than that of
Ashfal (Figure 1).The mutant lines from 40Gy were slightly shorten in plant height than that ofmutant lines from 200Gy and it was found consistent with the plant height of each mutant line
observed in M 2 generation.
Length (L) and width (W) of unhusked and husked grain and their ratio altered significantly inmutants over the parent Ashfal (Table 3). Length of unhusked and husked grain increased
significantly in all the mutants except the line RM(1)-160(C)-1. In RM(1)-160(C)-1, it was found to
decreased significantly. In contrast, width of unhusked and husked grain decreased significantly inall mutants whilst ratios of length and width (L/W) of unhusked and husked grain increased
significantly. Finally, 1000-grain weight remained unchanged in most of the mutants except 3.
Table 3 .Grain characteristics of eight mutants along with parent Ashfal

Mutant/variety

Grain length, L
(mm)

Grain
breadth(mm)

L: W

1000-Grain
weight (g)RM(1)-200 (C)-1-1

9.4 (7.0)

2.4 (2.0)

3.92 (3.50)

24.0

RM(1)-200 (C)-1-3

8.9 (7.0)

2.4 (2.0)

3.71(3.50)

23.9

RM(1)-200 (C)-1-9

9.1 (7.0)

2.3 (2.2)

3.96 (3.18)

22.5

RM(1)-200 (C)-1-10

9.4 (7.2)

2.4 (2.0)

3.92 (3.60)

24.0

RM(1)-200 (C)-1-13

8.5 (6.8)

2.3 (2.0)

3.70 (3.40)

23.8

RM(1)-200 (C)-1-17

9.3 (7.0)

2.4 (2.0)

3.88 (3.50)

24.2

RM(1)-200 (C)-1-18

9.4 (7.0)

2.4 (2.0)

3.92 (3.50)

22.6

RM(1)-160 (C)-1

7.5 (5.8)

2.8 (2.2)

2.68 (2.64)

20.5

Ashfal7.9 (6.2)

3.0 (2.6)

2.63 (2.38)

24.2

SE

0.2 (0.2)

0.1 (0.1)

0.18 (0.15)

0.4

Figures in parenthesis indicate grain characteristics from husked grain.

Preliminary yield trial with M 6 high yielding mutant lines of boro rice
Seedlings of 10 mutant lines derived from BRRI dhan29 by irradiated its seeds with carbon ion
beams in Boro season along with unirradiated BRRI dhan29 were transplanted on 22 January 2012 at
41 days after seed sowing. The experiment followed RCB design with 3 replications. Seedlings weretransplanted at 15cm distances within rows of 20cm apart. A unit plot size was 3.0m × 2.0m.
Recommended doses of fertilizers, cultural and intercultural operations were also followed as andwhen necessitated. Data on plant height, days to maturity; number of effective tiller, panicle length,
filled and unfilled grains panicle -1 and yield plot -1 were recorded. Maturity was recorded plot basis-6-while plant height, effective tiller number, panicle length, filled and unfilled grains/panicle wasrecorded from hills per plot at harvesting time. Grain yield was recorded from 1m 2 areas which waslatter converted to yield/ha at 14% moisture condition of grain. Moisture data was recorded with agrain moisture meter. Finally, the recorded data were subjected to proper statistical analyses
following the design used and are presented in Table 4.
It appears that the mutants and the check variety differed significantly for yield and yield attributing
traits except panicle length (Table 4). The mutants took 147 to 157 days to mature whilst their parent
BRRI dhan29 took 159 days. The mutant RM (2)-40 (C) -3-1-7 took the shortest period only 147
days for maturity. Filled grains was significantly the highest in the mutant RM(2) -50 ( C) -2-1-1 andit also had the highest grain yield at 14% moisture content of 8.58t/ha, which was 1.35 t/h more than
BRRI dhan29. This higher yield of this mutant was attributed to its considerably longer panicle
length and significantly higher number of filled grains/panicle (Table 4).This mutant together withthose with higher filled grains/panicle, longer panicle length, statistically as par yield and shorter
maturity period would be put into advance yield trial in the next Boro season.
Table 4. Yield and some yield attributing traits of 10 mutant lines derived from BRRI dhan29 with carbon
ion beam irradiation along with unirradiated BRRI dhan29 at BINA farm, Mymensingh

Mutant/variety

Days tomaturity

Plantheight(cm)

Effective
tiller(no.)

Paniclelength(cm)

Filledgrainspanicle -1(no.)

Unfilled
grainspanicle -1
(no.)

Grain
yield/ha(t)

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

151

112.27

6.93

26.53

139.13

27.00

7.02

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

149

96.30

8.20

27.00

119.33

23.87

6.93

RM(2)-40 (C) -1-1-7

149

95.53

8.60

25.87

113.60

26.13

5.80

RM(2)-40 (C) -4-2-5

149

91.67

11.40

25.40

109.13

10.87

6.90

RM(2)-40 (C) -4-2-7

159

115.00

8.80

25.73

94.33

18.07

6.53

RM(2)-40 (C) -4-2-8

157

117.60

6.47

28.20

140.60

30.80

6.34

RM(2)-40 (C) -1-1-5

149

112.87

7.93

25.33

136.67

21.07

7.30

RM(2)-40 (C) -3-1-7

147

108.80

7.33

25.80

116.47

21.40

7.10

RM(2)-40 (C) -4-2-2

157

103.07

9.33

25.20

103.73

24.73

6.88

RM(2)-50 (C) -2-1-1

155

115.40

7.80

27.00

169.00

22.87

8.58

BRRI dhan29

159

97.50

10.40

24.57

127.75

22.65

7.23

9.47

1.53

NS

31.42

6.76

0.72

LSD(0.05)
NS- not significant

1.5 Discussion
Heavy ion irradiation has been attaining an important to the mutation breeder as it produced raremutant(s). In view of this, seeds of Ashfal rice and BRRI dhan29 were irradiated with different
doses of carbon ion beams and was grown in Boro season. Three of nine headed plants were foundfertile and the rest six were exhibited sterile in case of Ashfal. Since the Ashfal is a highly
photoperiod sensitive cultivar and none of the control plants headed during Boro season. These-7-

results suggest that photoperiod

sensitivity was altered by carbon ion irradiation in a few M 1 plants.

Plant heights of M 2 progenies were found significantly shorten than Ashfal and this is also observed
consistent with the fact that the fertile M 1 plants had shorter plant height than Ashfal. This might bedue to the fact that the fertile mutant lines shifted from vegetative phase to reproductive phase earlier
than parental Ashfal and it is caused by ion beam irradiation. It has been reported that one dominant
and one recessive genes 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 M 1 plants homozygous monogenic recessive for photoperiod insensitivity. These facts leaded M 1plants to bred true in M 2 and M3 generations. The M 2 progenies from 160Gy were omitted from theseevaluations because they were prone to lodging for weak culm although they had shorten plants.
Moreover, theseprogenies had shorter panicle with smaller grains and lower number of seed grains.

The mutants derived from 40Gy dose also did not continue after M 3 generation because of theirinferior performance. The grain characteristics such as size and shape although most of the mutants
altered but grain weight mostly remained unchanged as the parent. This also confirmed that the M 1plants that mutated as photoperiod insensitive with shorten plant h eight were not contaminated withother variety. If it was contaminated then 1000-grain weight must differed significantly with Ashfal
and the contaminants. The mutant of BRRI dhan29 showed higher yield and early maturity comparedto parent of BRRI dhan29 which could be due to the useful effect of carbon ion beam.
Hence, it could be concluded that it is possible to induced fixed and stable mutant(s) in M 1 generationof indica type rice Ashfal and BRRI dhan29 through 40-200Gy doses of carbon ion beams irradiation.
This result is also in agreement with the results of Honda et al. (2006) who reported genetically fixedmutants in M 1 generation of sweet pepper for dwarf height and yellow pericarp. Therefore, all ofthese suggest heavy ion beams irradiation has unique properties that induce fixed mutation even in
M1 generation. In other mutational studies showed that mutants are usually detected in M 2 and M3
generations and thus need to handle numerous plants to screen. If mutants can be screened in M 1
generation, the number of plants that must be handle is dramatically decreased. Therefore, further
studies are needed to investigate whether other crop species could induce fixed mutant(s) in M 1
generation.
1.6

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, Japan Atomic Energy Agency(JAEA), Ms. Aki Koike, Nuclear Safety Research Association (NSRA) Dr. Liu Luxiang, RCA
Coordinator of China for their kind help and cooperation.

1.7 References
Azad MAK, Hamid MA, and Yasmine F (2010): Binachinabadam-4: a high yielding mutant variety ofgroundnut with medium pod size. Plant Mutation Reports 2(2):45-47.
Azam MA, Uddin I (1999). Binadhan-4, an improved rice variety bred through induced mutation.
Bangladesh J. Nuclear. Agric. 15: 59-65.
-8-

Blakely EA (1992).Cell inactivation by heavy charged particles.Rad. Environ. Biophys. 31:181-196.
Bonilla PS, Dvorak J, Mackill D, Deal K, Gregorio G (2002). RFLP and SSLP mapping of salinity
tolerance genes in chromosome 1 of rice (Oryza sativa L.) using recombinant inbred lines.
Philippines Agric. Sci. 85(1):64-74.
Gregorion GB, Senadhira DRD, Mendoza RD, Manigbas NL, Roxas JP, Guetra CQ (2002). Progressin breeding for salinity and associated abiotic stresses in rice. Field Crops Res. 76:91-101
Hamid MA, Azad MAK, Howelider MAR (2006). Development of three groundnut varieties withimproved quantitative and qualitative traits through induced mutation. Plant Mutation
Reports 1(2): 14-16.
Hase Y, Tanaka A, Baba T, Watanabe H (2000).FRL1 is required for petal and sepal development inArabidopsis. Plant J. 24:21-32.
Hayashi Y, Takehisa H, Kazama Y, Ichida H. Ryuto H, Fukunishi N, Abe T (2007).
beam irradiation on mutation induction in rice. In:

Effects of ion

Proc. Cyclotrons and their applicationsth

2007, 18 International Conference, pp. 237-239.
Hidema J, Yamoto, M, Kumagai T, Hase Y, Sakamoto A, Tanaka A (2003). Biologicaleffects ofcarbon ion on rice (Oryza sativa L.). JAERI- Review 2003-033:85-87.
Honda I., Kikuchi K, Matsuo S, Fukuda M,

Saito H.,

Ryuto H,Fukunishi N, Abe T (2006).

Heavy ion induced mutants in sweet pepper isolated by M 1 plant selection. Euphytica
152:61-66.
Islam MM (2004). Mapping salinity tolerance genes in rice (Oryza sativa L.) at reproductive stage.
Ph.D. Thesis, University of the Philippines Los Banos, College Laguna, Philippines.
Kamruzzaman M, Asaduzzaman M (2004).Cultivation of saline tolerant rice varieties in the coastal
area

to

cope

with

salinityinProtapkhali,paikgacha,

Khulna,Bangladesh.

enviroscope.iges.or.jp/contents/APEIS/inventory/0145.pdf
Niones JM (2004). Fine mapping of the salinity tolerance gene on chromosome 1 of rice (Oryza
sativa L.) using near isogenic line. M.Sc. thesis, University of the Philippines Los Banos,College Laguna, Philippines.
Rakwal R., Kimura S, Shibato J, Nojima K, Kim

YK, Nahm BH, Jwa NS, Endo S, Tanaka K,

Iwahashi H (2008). Growth retardation and death of rice plants irradiated with carbon ion
beams is preceded by very early dose- and time- dependent gene expression changes. Mol.
Cells 25(2):272-278.
-9-

Salil KB, Islam MM, Emon MR, Begum SN, Siddika A, Sultana S (2007). Identification of salt
tolerant rice cultivars via phenotypic and marker assisted procedures. Pakistan J. Biol.
Sci.10(24):4449-4454.
Shamsuzzaman KM, Hamid MA, Azad MAK, Shaikh MAQ (1998).Development of early maturingand high yielding cotton genotypes through induced mutations. Bangladesh J. Nuclear Agric.
14:1-8.
Shikazono N, Yokota Y, Kitamura S, Suzuki C, Watanabe H, Tano S,

Tanaka A(2003). Mutation

rate and novel tt mutants of Arabidopsis thaliana induced by carbon ions. Genetics
163:1449-1455.
Tanaka A, Tano S, Chantes T, Yolota Y, Shikazono N,

Watanabe H (1997). A new Arabidopsis

mutant induced by ion beams affects favonoid synthesis with spotted pigmentation in testa.
Genes Genet.Syst. 72:141-148.
Tanaka A, Sakamoto A, Ishigaki Y,Nikaido O, Sun G, Ase Y, Shikazono N, Tano S, Watanabe H(2002). An ultra violet-B- resistant mutant with enhanced DNA repair in Arabidopsis. Plant
Physiol. 129:64-71.
Vergara BS, Chang TT(1985). The flowering response of the rice plant to photoperiod. The
International Rice Research Institute, Los Banos, Laguna, Philippines.
Yokoo M, Fujimaki H (1971). Tight linkage of blast resistance with late maturity observed indifferent Indica varieties of rice. Japan J. Breed. 21: 35-39
Yokoo M, Kikuchi F, Nakane A, Fujimaki H (1980). Genetical analysis of heading data by aid ofclose linkage with blast resistance in rice. Bull. Nat. Inst.Agric.Sci. D31:95-126.
Yu CJ, Yao TT (1968). Genetische studien beim Reis. II. Die Koppelung des Langhullspelzengens
mit dem Photoperiodizitatsgen. Bot. Bull. Acad. Sin. 9:34-35.

- 10 -

2. China

FNCA Mutation Breeding Project
Sub-Project on Composition or Quality in Rice
J. Luo, P. Hu, S. Tang, G. Jiao and G. Shao
National Rice Improvement Center,
China-IRRI Joint Research Center on Rice Quality and Nutrition,
China National Rice Research Institute

2.1 Introduction
Rice (Oryza sativa L.) is one of the most important crops in the world and is a staple food crop for atleast 65% of the population of China. The performance of rice sector in terms of production and yield
had been very impressive in the most of last six decades. The wide adoption of semi -dwarf varieties
expedited the rice yield per unit from 2t/hm 2 in1960s to the level of 3.5t/hm 2 in 1970s. However, theincrease of rice production due to the development of semi-dwarf varieties still could not match therapid increase in population. Exploitation of heterosis in rice became the important choice for getting
more rice. Since 1949, the year of founding of the Peoples’ republic of China, the rice production has
continued to increase along with the rapid growth of the Chinese population. China's total population
reached to 1.37 billion people in 2011 and has increased 148.5% compared with the population in1949. Meanwhile rice yield per unit and total output increased 254.0% and 313.2% as 6.69 t/hm 2 and200.8 million tons respectively in 2011. However there is only 11.7% increase about the rice sowing
area during this period and the rice sowing area is 30.0 million hm 2 in 2011.
Hybrid rice has played an important role in demand of food supply in China. China has made a great
effort to increase its rice yield through exploiting the genetic resources of rice. The performance ofrice sector in terms of production and yield had been very impressive in the most of last five decades.
Hybrid rice that has a yield advantage of 10-20% over the conventional varieties was developed andcommercially grown in 1976, which resulted in the increase of yield to over 6.0 t/hm 2. The area
under hybrid rice had been increased from 0.14 million hm 2 in 1976 to about 18 million hm 2 in recent
years, more than 60% of the total rice planting areas in China (Table 1). In some regions, like Jiangxi
and Sichuan province, more than 90% of their rice varieties belong to hybrid rice. Several thousandsof hybrid rice varieties were released in the past 50 years. Until now total 96 super rice varieties wereconfirmed by Ministry of Agriculture (MOA) and were released to the farmers since MOA initiatedthe super rice breeding program in 1996. The planting area of super rice occupied 25% of total rice
planting area and reached 7.3 million hm 2 in 17 provinces in 2011 (Figure 1). These hybrid rice
varieties already made important contribution for food security in China.

- 12 -

Table 1. Yearly area of hybrid rice and percentage of the total rice area in China in 1976 -2010

Year

Hybrids planting area(m hm2)% of total rice area

1976

0.14

0.4

1978

4.34

12.6

1982

5.62

17.0

1986

9.00

27.9

1990

13.62

41.2

1997

17.73

55.8

1999

16.55

52.9

2002

15.48

54.9

2005

19.01

65.5

2008

18.45

63.1

2010

18.05

60.2

Figure 1. Increase of yield in rice through genetic improvement in China

Super Rice

With the great progress of hybrid rice research, the general quality of hybrid rice, especially in termsof head rice rate, chalk-ness and the amylose content etc., has dramatically improved. According to
the results in South China Rice Regional Trial in1998- 2004, comparing with the state standard forrice quality, the mean amylose content in every type of hybrids belongs to high quality rice (>3rdgrade). In regard to head rice rate (>3rd grade), over 60% single indica and late indica, over 90%
single-season late japonica hybrids reached the standard respectively (Table 2). However, among the560 hybrid rice combinations developed in 1998-2004, 106 of them reached state standard of the 3rd
grade of high quality rice. So the coordination of comprehensive quality is very important to hybrid
rice breeding. Hybrid rice breeding technology is different from that for inbred rice breeding.
Currently, the most popular male sterility system is the cytoplasmic male sterility (CMS, popularly
known in China as the three-line system). This utilizes three different lines, namely a cytoplasmic
male sterile line (A line), a maintainer (B line), and a restorer (R line). The quality of B and R line is
the key to determine the quality of hybrid rice regarding to three -line system.
- 13 -

Table 2. Performance of hybrids in some quality items in South China Rice Regional Trial in 1998 -2004

Head rice rate (%)
Type*

Chalkness (%)

Amylose content (%)

Over 3rd gradeMean% hybrids
reaching thestandard

Mean% hybrids
reaching thestandard

Mean% hybrids
reaching thestandard

No. ofhybrids%

EI (Y)

42.7

21.1

17.3

11.3

19.4

52.1

1

1.4

SI (Y)

57.9

81.0

9.9

30.0

20.7

76.2

44

21.0

LI (Y)

53.9

64.8

9.3

37.3

20.2

85.9

30

21.1

SJ (Y)

70.7

93.1

4.5

58.6

15.5

82.8

17

58.6

EI (S)

48.9

44.4

14.1

8.3

20.6

45.8

0

0.0

LI (S)

62.5

97.2

5.7

55.6

20.8

69.4

14

38.9

71.8

106

18.5

Total

66.3

29.6

*EI(Y or S)─ Early season indica rice (Yangtze River or south China)
LI(Y or S)─ Late season indica rice (Yangtze River or south China)
SI&J (Y) ─ Single indica or japonica rice (Yangtze River)

2.2 Objective of the Project
The main objective is to obtain ideal CMS lines and restorers w ith good quality including adaptive
amylose content and to get the optimum irradiation doses of heavy ion beam for early super rice Z7
and late-season inbred rice W2 to create efficient mutant.
2.3 Materials and Methods
Two experiments were carried out in this project during 2008-2010.
2.3.1 Experiment 1 Rice mutation breeding for hybrid rice with high yield and good quality(1) Mutation breeding of CMS lines
Zhong 3A is a good new indica CMS line with good-grain quality
and high disease resistance ability, developed from crossing
Zhong 9A to Zhenong996/Jin 23B and successively backcrossing
its filial male sterile plants to Zhenong/Jin 23B. It showed stable
and complete male sterility, vigorous stigma with high exsertion
rate, good disease resistance ability, high combining ability andhigh outcrossing rate. It had tall and thick stems with good lodging
resistance. It was technically identified in Zhejiang Province in
September 2005. However, most of the hybrids crossed with
Zhong 3A showed high amylose content (>22%). The amylose
content of Zhong 3B is 20.1%. Therefore we expect to decreaseamylose content of the CMS lines in order to obtain hybrid rice

Zhong 3A

with low amylose content (15-20%) by mutation breeding. In 2008,
seeds of Zhong 3B were irradiated with 350Gy and 400Gy from Co-60 gamma irradiator in
Hangzhou. Ten M4 lines of Zhong 3 B (maintainer) with low amylose content were selected forfurther backcross with CMS lines in 2010.
- 14 -

(2) Mutation breeding of restorers
Nineteen restorers including CR63, CR84, CR1577 had beencollected and irradiated with 250Gy from Co-60 gamma irradiator
in Hangzhou in 2006. Six crosses as CR1577(Mutant)/R501,
CR1577(Mutant)/R725,

Yangdao6/CR1577(Mutant),

Shuhui527/CR63(Mutant),

CR63(Mutant)/Minghui86,

R207/CR84(Mutant) were made and harvested for restorer
selection in winter of 2008. Two restorers (ZZ-2 and 07HY-1580)
were collected and irradiated with 350Gy and 400Gy from Co-60
gamma irradiator in Hangzhou in 2008. For each treatment 2500
seeds were used. The main objective is to obtain ideal restorers
with good quality including adaptive amylose content. Two mutant

AVF-cyclotron, TIARA, JAEA

lines (M 4) from a restorer as CR84 were crossed with CMS lines
as Nei 2A, Nei 5A, Nei 6A,, 618A and Zhong 3A respectively in winter of 2009. These hybrids
showed good appearance quality, weak potential ability of high yield in summer of 2010. 79 lines ofF4 generations were obtained from six combinations between mutant lines with the other restorers
through agronomic traits selection. These restorer mutants showed adaptive amylose content. Someof mutants were used as restorer parent for further utilization.
2.3.2 Experiment 2 Seed Irradiation Test by Ion Beam for Rice Mutation BreedingThrough the FNCA cooperation project we initiated heavy ion beam irradiation technology formutation breeding research in 2010. Two batch of hulled rice seeds were irradiated by the heavy ion
beam of the TIARA, JAEA. Two rice cultivars as Z17 and W2 were used. One is asuper-high-yielding early-season indica cultivar; another is a good-quality late-season indica
cultivar.
The dry seeds of Z17 and W2 were irradiated by carbon ion by the doses of 10, 20 , 40, 60, 80, 100,
120Gy. About 150 seeds were packed with plastic Petri dish for each experiment per test -dose. In
addition more dry seeds of Z17 were irradiated by carbon ion by the doses of 30, 40, 50Gy
respectively, 400 seeds for each irradiation. After irradiation, biological effects were be observed bymeasurements of the germination rate, survival rate, growth rate and ripening rate. Sowing date,transplanting date and harvesting date was June 10 th, July 9 th and Oct 12, 2010 respectively. Methodof measurement is same to J. Hidema’s experiment. After irradiation, the seeds were placed on wet
filter paper in petri dish and kept at 30 oC for 2 days and then the seeds were planted in plastic-tray
containing sterile field soil and placed in the control environmental greenhouse (12hr photoperiod,
day/night 27 oC/27oC). The germination rate was measured at 2days after planting. The survival rate
was measured by counting the viable plants at 2 and 3 weeks after germination. The growth rate (theratio of the value for plant length of each irradiated plant to the value for plant length of un -irradiatedplant) was measured, 30 days after sowing in the control environmental greenhouse. The percentage
of grain ripening was measured at 4 months after sowing (maturity stage).

- 15 -

2.4 Results and Discussion
Along with the irradiation dosage increasing, the germination rate, survival rate, growth rate,
ripening rate decreased in different extent. Ion beam made some damages to rice seed (Figure 2, 3) .
Regarding to Z17, when the irradiation dosage is up to 50Gy, the survival rate and ripening rate makea sharp decrease. When the dosage is down to 20Gy, there is no big difference in all investigated data.
Ripening rate significantly decreased at doses of up to about 60Gy, but panicle number significantly
increased at doses of up to about 100Gy (Table 3,4, Figure 4, 5). The sterile and low seed setting
plants probably kept tillering under water condition in the field. The survival rate of W2 after
irradiation decreased sharply (Figure 6).

(a)

(b)

10Gy

Control

Control

20Gy

(d)

(c)

Control

60Gy

Control

80Gy

Figure 2. Effect of different dose of ion-beam irradiation of Z17 at day 2.
(a)Germination rate at dose (0Gy and 10Gy), (b)Germination rate at dose (0Gy and 20Gy),(c)Germination rate at dose (0Gy and 60Gy), (d)Germination rate at dose (0Gy and 80Gy).

CK

10Gy

20Gy

40Gy

60Gy

80Gy

100Gy 120Gy

Figure 3. Emergency features of Z17 seedling in a phytotron.

- 16 -

Table 3. Z17 Performance after ion-beam irradiation

Dose
(Gy)

Germination rate(%)

0 (Control)

Survival rate (%)

Plant
Height*(cm)

Growthrate (%)

Percentage
of ripening(%)

2weeks after
germination

3weeks after
germination

87.25

63.09

50.34

30.33

100.00

74.20

10

83.39

63.76

47.65

29.67

97.85

73.30

20

76.16

51.66

35.10

30.19

99.55

61.70

40

73.83

51.01

33.56

30.04

99.05

51.20

60

70.34

46.21

32.41

26.32

86.79

32.40

80

73.51

43.05

26.49

27.09

89.32

13.20

100

72.85

33.11

17.88

28.15

92.82

25.70

120

67.11

20.81

10.74

26.59

87.69

12.80

*: Measured at 30 days after sowing in the control environmental greenhouse.

Table 3 showed the effects of carbon-ion beams on plant height, panicle number, seed number andripening rate in rice plant. Ripening rate significantly decreased at doses of up to about 60Gy, but
panicle number significantly increased at doses of up to about 100G y. The sterile and low seedsetting plants probably kept tillering under water condition in the field.

Table 4. Agricultural traits average performance of Z17 M 1 generation

Dose
(Gy)

Plant height(cm)

Panicle number(no./plant)

Seed number(no. /panicle)

Ripening rate(%)

Control

94.1

8.35

150.65

74.2

10Gy

93.6

9.30

150.86

73.3

20 Gy

93.0

8.40

152.24

61.7

40 Gy

93.5

9.85

150.90

51.2

60 Gy

95.0

9.70

170.62

32.4

80 Gy

89.9

9.15

168.0813.2

100 Gy

90.2

11.25

158.06

25.7

120 Gy

87.9

11.00

164.17

12.8- 17 -

100

100
2 weeks after germination

80

3 weeks after germination

Germination rate (%)

Survival rate (%)

80

60

40

20

60

40

20

0

0
0

20

40

60

80

100

0

120

20

40

100

100

80

80

Percentage of ripening (%)

Growth rate (%)

60

80

100

120

80

100

120

Dose (Gy)Dose (Gy)

60

40

20

60

40

20

0

0
0

20

40

60

80

100

0

120

20

40

Dose (Gy)60
Dose (Gy)Figure 4. Z17 Dose-response curve for germination rate, survival rate,
growth rate and percentage of ripening .
100

100
Survival rate

80

80

Survival Rate(%)

Germination and survival rate (%)

Germination rate

60

40

60

40

20

20

0

0
30

40

0

50

20

40

60

80

100

120

Dose (Gy)Dose (Gy)Figure 5. Z17 Dose-response curve (30, 40, 50Gy)

Figure 6. W2 Dose-response curve for survival rate.

for germination rate, survival rate.

2.5 Conclusion
Hulled seeds of Z17, local super rice cultivar, was irradiated with 7 different doses (10, 20, 40, 60, 80,
100, 120Gy) by the heavy ion beam of the TIARA, JAEA in 2010. These materials with 0 doses ascontrol were cultivated in summer of 2010. The germination rate, survival rate, growth rate andpercentage of ripening were investigated. These results suggested that the optimum irradiation doses- 18 -

of heavy ion beam for Z7 might be between 30 to 50Gy doses.

2.6 Acknowledgments
The authors would like to thank Japan Atomic Energy Agency (JAEA) for providing the ion -beamfacilities. This research was supported by China Atomic Energy Authority.
2.7 References
Cao L-Y，Zhan X-D，Chen S-G, Feng Y, Wu W-R, Shen X-H, Cheng S-H, Breeding Methodology andPractice of Super Rice in China. Rice Science, 2010, 17(2): 87-93
Cheng S-H, Cao L-Y, Yang S-H, Zhai H-Q. Forty years’ development of hybrid rice: China’s
experience. Rice Science. 2004, 11, 225-230.
Cheng S-H, Zhuang J-Y, Fan Y-Y, Du J-H, Cao L-Y. Progress in Research and Development on
Hybrid Rice: A super-domesticate in China. Annals of Botany, 2007, 100: 959–966.
Luo J, Tang S-Q, Jiao G-A, Hu P-S. Breeding of Good Quality and Resistance Indica CMS Line
Zhong 3A. Hybrid Rice, 2007, 22(1)：13-14
Hidema, J., Yamamoto, M., Kumagai, T., Hase., Sakamoto A., and Tanaka, A. Biological Effects ofCarbon Ion on Rice (Oryza sativa L.) JAERI-review.:85-87. (2003)
Yuan L-P. Strategy of hybrid rice breeding. Hybrid Rice, 1987, (1):1-3.

- 19 -

- 20 -

3. IndonesiaImprovement of Yield and Grain Quality of Ricethrough a Wide Cross and Mutation Breeding
Sobrizal
Center for the Application of Isotopes and Radiation
National Nuclear Energy Agency

3.1 Abstract
To meet the Indonesian domestic demand for both rice grain quality and quantity, high variability ofpure lines derived from a cross of Indica rice variety, IR36 and Japonica rice variety, Koshihi kari wasconstructed. Among these lines, KI237 line was treated by gamma ray irradiation to remove its
undesirable characters. KI237 shows good performance with high yield potency and red phericarp,
but susceptible to lodging because of tall plant stature. To reduce the plant height, 50g seeds of thisline were irradiated by 200Gy of gamma ray at Center for the Application of Isotopes and Radiation
Technology, Pasar Jumat, Jakarta, Indonesia. Selections and purifications were conducted in M 2 andM3 generation, finally, one dwarf and four semi-dwarf M3 mutant lines were selected. Based on the
segregation analysis in M 2 and M3 generation, it was concluded that the semi-dwarf of RKI237-1-17
mutant line was controlled by single recessive mutated gene. For breeding purpose, based on theiragronomical performances ten semi-dwarf lines were selected and subjected to yield and othercharacter evaluations. The results of multi-location yield trials showed that the highest average yieldof multi-locations yield trials in five locations was 7.39t/ha for RKI199 and followed by 7.24t/ha for
RKI241, while average yield of chiherang, a national leading variety is only 6.56 ton/ha. Out of tenlines, three lines are still with brown phericarp and the phericarp color of other lines change to white.
These ten lines have various amylose contents, ranging from 13.41% to 20.83%. Various amylose
contents and phericarp colors would be useful as genetic resources for improveme nt of rice grain
quality to serve the wide preference variability of Indonesian consumers. To fulfill the requirement ofvariety release in Indonesia, other multi-location yield trials as well as other examinations such as
pests, diseases and other grain quality examinations should be conducted.
Key words: yield, grain quality, intra-specific cross, mutation
3.2 Introduction
Rice is a steeple 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 is frequently attributed to loss of rice fields
caused by conversion to non-agricultural uses, and to the declining productivity. To fulfill thedomestic demand in which the annual population growth is still more than 1.3%, Indonesian riceproduction should be increased. This can be achieved by introducing the high yielding varieties to thefarmers.
Recently, the demand for superior grain quality increased for national markets in Indonesia. Riceconsumers in Indonesia exhibit wide preference variability and consumption patterns because ofarchipelagic nature of the country (Toquero 1991). Consumers in Medan, Padang and Makassar, for- 22 -

example, prefer non-sticky cooked rice, while consumers in Java prefer sticky and soft-cooked rice.
Nowadays, consumers have become more discriminating in terms of rice quality due to higher
incomes and better life quality. Indonesian rice consumers are now willing to pay higher price forbetter quality desired. These imply requirement of technology generation for better quality.
The primary components of rice grain quality include milling efficiency, appearance, cooking andedibility characteristics, and nutritional quality (Li et al. 2004). Generally, the milling efficiency isdetermined by head rice yield; appearance is determined by grain length, grain width, width -length
ratio, and translucency of endosperm; cooking and edibility characteristics are determine d byamylose content of endosperm, gelatinization temperature, and aroma; nutritional quality isdetermined by protein, oil, phytic acid, and micro nutrient contents.
IR36 is an Indica improved rice variety, tolerance to various pests and diseases, and ad apted well inalmost all Indonesian rice growing areas. Recently, IR36 rice variety is almost not grown inIndonesian because of its poor in cooking and edibility characteristics. Whereas, Koshihikari is avery popular Japonica variety and known having good cooking and edibility with low amylose
content, but it cannot grow well in Indonesia due to photoperiod sensitivity. By using these uniquegermplasms as well as mutations ten rice promising lines with high yield and grain q uality have been
constructed. The objective of this research is to develop high yielding and grain quality of rice
varieties.
3.3

Materials and Methods

To increase the genetic variability of breeding materials, IR36 and Koshihikari were cross, and the F 1
plants obtained from this cross were self-fertilized up to F 5 generation. F5 lines were developed from
100 F2 plants by pedigree method with 10 plants per line for each generation, and finally the size of
F5 population became 100,000 plants. Selection for agronomical and other major ch aracters was
performed in F5 populations. Selected plants were purified up to F 7 or F8 generations, then, selected
pure lines were designated as KI #. KI lines are used as basic materials in further breeding program.
Among these lines, the seeds of KI237 line were treated by gamma ray irradiation. This line shows
good performance with high yield potency and red pericarp, but susceptible to lodging because of tall
plant stature. To reduce the plant height, 50g seeds of this line were irradiated by 200Gy of gamma
ray at Center for the Application of Isotopes and radiation technology, Pasar Jumat, Jakarta.
Irradiated M 1 seeds were sown, and twenty day-old seedlings were transplanted to paddy field byplanting a single seedling per hole at experimental field, Sawangan, Depok with 20cm spacing
between plants.
Five hundred M 1 plants were harvested individually to obtain the M 2 seeds. Harvesting was
conducted by collecting only one main panicle in each M 1 plant. M2 seeds were sown, and 20 M 2plants derived from each M1 plant were transplanted to develop M 2 lines. Observations of semi-dwarf
plants were conducted in each M 2 lines before harvesting. The M 2 lines showed segregation between
normal and semi dwarf plants was harvested individually.

- 23 -

In the next growing season, 80 plants derived from each M 2 plant of selected M 2 lines were planted
to develop M 3 lines. Observations of segregation between semi-dwarf and normal plants wereconducted in each M 3 lines. The  2 test in segregated M 3 lines was performed to examine fitness ofthe frequencies of the semi-dwarf plants against expectation from Mendelian segregation.
Selected semi-dwarf plants were purified up to M 5 generations, then, selected pure lines were
designated as RKI #. RKI lines were subjected to yield trials, amylase content, and otherexaminations. Breeding scheme of plant materials used in this study can be shown in Fig. 1.
IndicaIR36

Selection &Purification

X

Japonica
Koshihikari

F1
F7

Breeding lines
KI #Irradiation of
KI 237Data
Growth duration
Panicle length
Seed length

Selection ofearly maturity

Plant highNo. of seeds/panicle
Seed width

Exam. of yield, pests,
diseases, quality

National leading
varieties

Figure 1. Breeding scheme of plant materials used in this study.

3.4 Results and Discussion
In this study, all the F 1 plants derived from cross of IR36/Koshihikari grew vigorously but theirpollens as well as seeds were semi-sterile. The fertilities were improved gradually from early to thenext generations. Selection was conducted in F 5 population with emphasis on agronomical characters,
seed fertilities, and pest and disease tolerances in the field. Of the 100,000 F 5 plants, 568 plants wereselected and they were purified up to F 7 or F8 generation to construct 568 fixed lines. Selected purelines were designated as KI #.

Fig. 2. Variability of selected pure lines derived from a cross of Koshihikari and IR36.
Figure 2 shows varying features of KI lines. Variations were shown in many characters such as plantheight, growth duration, number of tillers, flag life, panicle length, number of seeds per panicle, seedlength, seed width, and others. For rice breeding program, some of these lines showing good
performance were directly subjected to yield trials and several others were irradiated by gamma ray
to induce mutations for amelioration of one or two undesirable characters, or crossed each other to- 24 -

gather some desirable characters in one individual.
Among these KI lines, the KI237 was selected to improve by mutation breeding because this line
showed good yield potency but so tool that susceptible to lodging. To reduce the plant height of
KI237, its seeds were irradiated by 0.2kGy gamma ray. As much as 500 M 1 plants were harvested
individually to obtain M 2 seeds. Selection for dwarf and semi-dwarf were conducted in M 2 lines. Outof 342 M 2 lines, each M 2 line consisted of 20 plants, 5 lines segregated for plant height. In this study,
since the original of these lines is KI237, the plants having the same height as RKI237 were grouped
as normal plants. Among the segregated lines, the line of RKI237-1 segregated for 8 semi-dwarf and
12 normal plants. The semi-dwarf plant reached 60 – 62% of plant height of original plant KI237 at
the mature stage (Fig. 3A). It was also compared the length of internodes, panicle, and se ed between
these two plants (Fig. 3B; Fig. 4; Table 1). In rice, internodes elongation starts from the bottom at the
panicle initiation stage. All internodes of semi-dwarf plant were shortened in comparison with theoriginal pant. The elongation of the upper internodes was weakly inhibited. The retardation of the 1 st
(uppermost) internodes was 24%, moreover, the retardation of panicle and seed length was only 10%
and 2%, respectively. The elongation pattern of the internodes in this mutant was almost the sa me as
sd1 (Dee-geo-woo-gen), the original parent of the first release modern rice variety (Itoh et al. 2004).
To perform the genetic analysis of this semi-dwarf character, the segregated M 2 line of RKI 237-1
was harvested individually and 80 plants derived from each harvested M 2 plant were planted togenerate the M 3 lines. Four M 3 lines derived from normal M 2 plants (RKI 237-1-2, RKI 237-1-3, RKI
237-1-8 and RKI 237-1-12) and two M3 lines derived from semi-dwarf M 2 plants (RKI 237-1-1 and
RKI 237-1-17) were selected and use them for analysis of the mutated gene(s). It was observed thatall M3 plants derived from RKI 237-1-1 and of RKI 237-1-17 M2 plants showed semi-dwarf stature,
all M3 plants derived from RKI 237-1-2 and of RKI 237-1-8 M 2 plants showed normal stature, andthe other two M 3 lines derived from normal M 2 plants, RKI 237-1-3 and RKI 237-1-12 segregated for
57 normal and 22 semi-dwarf and 59 normal and 21 semi-dwarf plants, respectively.

The

segregation ratios between normal and semi-dwarf plants in both RKI 237-1-3 and RKI 237-1-12 M 3lines fitted well to Mendelian expected segregation 3 : 1 (Table 2). These results suggested that thesemi-dwarf character in these lines was controlled by a single recessive gene. This gene wastentatively designated as sd237-1.

RKI 237-1-1

KI 237

RKI 237-1-1

KI 237

Fig. 3. Morphological characterization of semi-dwarf mutant RKI237-1-1 and its original plant, KI237.

- 25 -RKI 237-1

KI 237

Fig. 4. Comparison of culm elongation between semi-dwarf mutant RKI237-1 and its original line KI237.
Table 1. The length of internodes, panicles, and seeds of KI237 and its semi-dwarf mutant.

In rice, At least 7 semi-dwarfing genes have been identified by classical genetic analysis (Nagato andYoshimura 1998), some of them, such as sd1 on chromosome 1 and d35 on chromosome 6 have been
isolated and have been extensively analyzed to elucidate the regulatory mechanisms of plant gr owthand development (Spielmeyer et al. 2002; Itoh et al. 2004). Even though the performance of ricemutants carrying sd 237-1 gene differed from that of rice mutant carrying sd1 or d35, the allelic test
between sd237-1 gene and other semi-dwarfism genes are necessary to clarify whether the sd 237-1 is a
new identified gene or not. Chromosome mapping concerning the sd237-1should also be conducted innear future to locate the sd 237-1 gene on rice chromosome linkage map.
Table 2. Segregation of normal and semi-dwarf plants in M 3 lines.

Semi-dwarfism is a valuable trait in crop breeding, because it increases lodging resistance anddecreases damages due to wind and rain. During the green revolution, in the second half of the 20 th- 26 -

century, a rice semi-dwarf variety, IR8, enabled dramatic yield increases and help to avert predicted
food shortages in Asia (Khush 2001). At the same time, a dominant wheat semi -dwarf cultivar, Rht1
as well as Rht2, facilitated a burst in productivity and lead to the wheat green revolution (Evans,1998). The original of sd237-1 is KI237, a pure selected line derived from Indica / Japonica cross. Thisline showed high yield potency, but susceptible to lodging. The sd237-1 mutant improved lodging
resistance without significantly changing its major characters. This mutant could be used as a genetic
resource for the improvement of KI237 line through back-cross breeding as well as be developed
further in breeding program directly to be new high yielding mutant varieties.
Recent developments of gene transfer technology have enormous promise for improvement of plant
productivity; however, there is a lack of available new genes which can be transferred to current
high-yielding varieties. In other words, there are no genes that have been identified which cancontribute to world crop production as much as sd1 (DGWG) in rice, and Rht1 as well as Rht2 inwheat (Maluszinki 1998). Since the elongation pattern of the internodes of sd 237-1 mutant was almost
the same as sd1, the sd237-1 mutant gene can be used with sd1 simultaneously to avoid genetic
vulnerability without reducing yield.
To evaluate the yield performances, ten selected semi-dwarf pure lines were subjected to yield trials
in five locations. The highest average yield in five locations is 7.39t/ha for RKI199 and followed by
7.24t/ha for RKI241, whereas the average yield of Ciherang, a national leading variety is only
6.56t/ha (Table 3). The lowest average yield is 6.54t/ha for KI237 line, an original line of mutant
lines. Multi-location yield trials will be continued in next growing season to reach at least 16
locations as a requirement of variety release in Indonesia.
Table 3. Multi-locations yield trials in Pusakanegara, Landak, Purbalingga, Pariaman and Banyuwangi.

Line

*)

Yield (ton/ha) *)Pskngr

Lndk

Prblg

Prmn

Bywgi

Average

RKI-124
RKI-130
RKI-131
RKI-198
RKI-199
RKI-2116.80 abc
6.14 c6.92 ab7.15 a7.12 a6.63 abc

4.95 a5.15 a4.64 a5.52 a5.60 a7.77 a6.80 d
7.40 c6.45 de6.10 ef
7.85 b
6.02 f7.30 ab7.25 a6.75 abc
7.20 ab7.15 a6.63 dc

8.85 a8.85 a8.68 a9.21 a9.21 a9.55 a

6.94
6.96
6.69
7.04
7.39
7.32

RKI-237
RKI-240
RKI-241
RKI-245
KI-237
Ciherang

6.18 c5.40 d
6.94 ab
6.39 bc
4.51 e6.52 abc

5.52 a6.35 a5.54 a5.44 a6.27 a4.54 a7.75 bc
7.95 b
8.75 a7.75 bc
6.50 de5.95 f6.80 abc
6.43 d6.97 abc
7.50 a7.00 abc
7.10 ab8.85 a9.03 a7.99 a8.51 a8.42 a8.69 a

7.02
7.03
7.24
7.11
6.54
6.56

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

The amylosa content of tested RKI lines are varied largely, ranging from 13.41% to 20.83%. The
amylose content of rice is recognized as one of the most important determinants of eating andcooking quality (Bao et al. 2002). The lines having various content of amylose should be useful as- 27 -

genetic resources for improvement of grain quality to meet the wide preference variability of
consumers. The amylosa contents of RKI199 and RKI241 are 17.16% and 15.27%, respectively(Table 4). The color of phericarp is white for RKI199 and brown for RKI241 (Fig 5). If the yield of
RKI199 and RKI241 is stable in next multi-location yield trials these lines can be recommended torelease as new leading varieties in Indonesia. To fulfill the requirement of variety release inIndonesia these data should also be completed with other examinations such as pests, diseases andother grain quality examinations.

Line
RKI-124
RKI-130
RKI-131
RKI-198
RKI-199
RKI-211
RKI-237
RKI-240
RKI-241
RKI-245
KI-237
Koshihikari
Ciherang

Amylose Content (%)

Endosperm color

20.83
19.89
20.69
17.35
17.16
14.17
14.25
13.41
15.27
16.71
24.36
16.90
18,00

White
White
White
White
White
Brown
White
White
Brown
Brown
Brown
White
White

Tabel 4. Amylose contents and phericarp color

RKI 124

RKI 130

RKI 131

RKI 198

RKI 199

RKI 211

RKI 237

RKI 240

RKI 241

RKI 245

KI 237

Ciherang

Fig. 5. Grain rice performances of selected lines

of selected RKI lines.

3.5 Conclusion
Based on the results it can be concluded as follow;
1) Large genetic variation was observed in fixed lines derived from a wide cross of IR36 andKoshihikari. These lines can be used as basic materials in breeding program.
2) Semi-dwarf character of RKI237-1 line was control by a single mutated gene, this gene wastentatively designated as sd237-1.
3) Out of ten selected lines, three lines are still with brown phericarp and the phericarp color of otherlines change to white.
4) These lines have various amylose contents, ranging from 13.41% to 20.83%.
5) The highest average yield was 7.39t/ha for RKI199 and followed by 7.24t/ha RKI241.
6) To fulfill the requirement of variety release in Indonesia, other multi -location yield trials and otherexaminations such as pests, diseases and other grain quality examinations should be conducted.
3.6 Acknowledgement
Thanks to my colleagues at the Division of Irradiation, Electro -mechanic 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 technical assistance.

- 28 -

3.7 References
Bao, J.S., Sun, M., Corke, H. 2002. Analysis of genetic behavior of some starch properties in indica
rice (Oryza sativa L.): thermal properties, gel texture, swelling volume. Theor. Appl. Genet.
104:404-413.
BAPENAS/DEPARTEMAN PERTANIAN/USAID/DAI FOOD POLICY ADVISORY TEAM. 2002.
Indonesian food policy program. Available at www.macrofoodpolicy.com
Evans, L. T. 1998. Fidding the ten billion. Plant and Population Growth. Cambridge University Press.
Cambridge.
Itoh, H., Tatsumi, T., Sakamoto, T., Otomo, K., Toyomasu, T., Kitano, H., Ashikari, M., Ichihara, S.
and M. Matsuoka. 2004. A rice semi-dwarf gene, Tan-Ginbozu (D35) encodes the gibberellinbiosyinthesis enzyme, ent-kaurene oxidase. Plant Moleculer Biology 54: 533-547.
Khush, G. S. 2001. Green revolution: the way forward. Nature Rev. Genet. 2: 815-822.
Li, J., Xiao, J., Grandilo, S., Jiang, L., Wan, Y., Deng, Q., Yuan, L., McCouch, S.R. 2004. QTL
detection for rice grain quality traits using an interspecific backcross population derived
from cultivated Asian (O. sativa) and African (O. glaberima S.) rice. Genome 47:697-704.
Maluszinski, M. 1998. Crop germplasm enhancement through mutation techniques. In Rutger, J. N .,Robinson, J. F. and R. H. Dilday (eds). Proceeding of the International Symposium on Rice
Germplasm Evaluation and Enhancement. Stuttgart, Arkansas, USA, p. 74 -82.
Nagato, Y. and A. Yoshimura, 1998. Report of the committee on gene symbolization, nomenclatureand linkage groups. Rice Genet. Nwsl. 15: 13Spielmeyer, W., Ellis, M. H. and P. M. Chandler. 2002. Semidwarf (sd-1) “green revolution” rice,contains a defective gibberellin 20-oxidase gene. Proc. Natl. Acad. Sci. USA

99:9043-9048.
Taquero, Z.F. 1991. Consumer demand for rice grain quality in rice grain marketing and quality
issues. Selected paper from the International Research Conference. 27 -31 August 1990,
Seoul, Korea. IRRI, Manila. p. 37-46.

- 29 -

- 30 -

4. Japan

Creation of Materials for Breeding Amylose Library of Primary Rice Varieties
M. NishimuraInstitute of Radiation Breeding
National Institute of Agrobiological Sciences

4.1 Introduction
Low-amylose rice holds promise for increased use in applications such as aseptically packed rice.
The Institute of Radiation Breeding has raised mutant strains of rice varieties such as Norin 8 andReimei with various amylose mutations, and has provided many of the breeding mother plants ofexisting low-amylose varieties. The institute is also working on raising isogenic lines of Koshihikarithat relate to the low-amylose genes of these varieties. The purpose of this research is to induce
mutations by means of gamma rays and other radiation to create near isogenic lines (NILs) withamylose-content gradients of about 2% from primary rice varieties, Koshihikari and Hitomebore.

Figure 1.

Figure 2.

Main rice varieties in Japan

Low-amylose varieties for pre-cooked rice

- 32 -

4.2 Materials and Methods(1) Creation of Koshihikari near isogenic lines (NILs) using existing amylose mutants(2) Selection of amylose-content mutants from primary varieties such as Koshihikari and
Hitomebore.
(3) Evaluation of amylose mutants
Cultivate under various conditions to evaluate the phenotype of the amylose library created in the
project.
Table 1. Specific research methods and other actions taken throughout the research term

Year
2006
2007-2010

Research objective
Raise

Koshihikari

NILs

relating to amylose

Research method
Use existing amylose variants

Expected achievements
Broadenedvariants

Screen amylose variants from Use mutagens such as γ rays , Creation
primary varieties

2011-2012 Evaluate amylose library

ion beams and EMS treatmentofamyloseofamyloselibrary

Evaluate amylose library under Completion
various cultivation conditions

amyloselibrary

4.3 Results and Discussion
4.3.1 Creation of Amylose Library of Koshihikari
We created the amylose library consisted of mutants backcrossed with original variety, Koshihikari.
Furthermore, we continued to screen amylose variants from Koshihikari. Consequently, we obtained
NILs consisted of F 3-5 lines backcrossed with Koshihikari and newly selected mutants to be added tothis library induced by EMS treatment and gamma-ray irradiation. We developed amylose libraryconsisted of 29 lines. As a result, we completed raising amylose library consisted of 29 mutant
strains with amylose-content gradients of about 2% (Fig.1). Amylose contents of mutants and NILs
were determined by absorption spectrophotometry method in 2011. In 2012, we will try t heevaluation of the response of amylose content of NILs to the temperature during grain -filling period.
Amylose content (%)
20.0

Koshihikari
15.0

10.0

5.0

0.0

-5.0

Figure 3. Koshihikari NILs relating to amylose content in 2011.

- 33 -

4.3.2 Selection of new mutants and raising NILs with low amylose content from Hitomebore
Hitomebore NILs were created by backcrossing with an original variety and ten mutants with waxy
or low amylose contents were newly selected. The generations of these mutants are M 7 to M 10 in2013.
Ion beam irradiation was conducted at the AVF cyclotron in the Takasaki Advanced Radiation
Research Institute of the Japan Atomic Energy Agency. The ion particles used were 12C 5+ (220 MeV),
12

C6+ (320 MeV), and 4He2+ (100 MeV). Gamma-ray irradiation was conducted at the Institute ofRadiation Breeding using a 10 Gy/h dose rate.
We obtained NILs consisted of 12 F5-6 lines backcrossed with Hitomebore, No.2 variety in Japan andnewly selected 10 mutants to be added to this library induced by ion beam and gamma -ray irradiation.
In culm length, some lines of NILs were shorter than original variety in spite of backcrossed by
Hitomebore.

Amylosecontentsofmutants

and

NILs

were

determinedbyabsorption

spectrophotometry method in 2011.
Amylose content (%)
25.0

20.0

Hitomebore
15.0

10.0

5.0

0.0

-5.0

Figure 4. Hitomebore NILs relating to amylose content in 2011.

4.3.3 Analysis of temperature response of low amylose mutant strains
The mutant strains developed from parental Koshihikari were cultivated a in a double cropping
system (planted on May 16 and transplanted on June 14 in 2012) with various grain -filling
temperatures. The fully matured grains were evaluated for endosperm amylose content.
In general, the temperature in August of 2012 was higher than that of 2011, therefore, amylose
content of mutant strains were relatively lower. As shown in Fig.5, the strains with relatively low
amylose content (around 5%) were stable throughout the range of tested temperatures. On the other
hand, the strains with an amylose content around 10% were classified into two types of stable andsharp response to the temperature. There are mutant strains that have an amylose content of about
12%, which is less than that of standard non-glutinous varieties. Some of them have a relatively- 34 -

stable temperature response and they could be promising candidates for the further breeding material.

Figure 5.

Response of amylose content to the temperature in Koshihikari NILs in 2012.

(vertical scale: amylose content %, horizontal scale: mean temperature during 20 days after heading )4.4 Conclusion
We completed amylose library consisted of Koshihikari and Hitomebore NILs with amylose -content
gradients of about 2%. They will be used for the genetic analysis in the new MAFF project from2013. We also analyzed the temperature response of low amylose mutant strains.

- 35 -

- 36 -

5. Korea

Molecular characterization of high level of VitE accumulating rice mutant induced
by in vitro mutagenesis
J.E. Hwang, D.S. Kim, S.-Y. Kang
Radiation Breeding Research Team, Advanced Radiation Research Institute,
Korea Atomic Energy Research Institute

5.1 Introduction
Vitamin E is an important constituent of the human diet (Evans and Bishop, 1922). Vitamin E is an
essential lipid-soluble nutrient that consists of eight tocochromanols in nature, which can beseparated into two groups, α-, β, -γ- and δ- tocopherols and four corresponding unsaturated
derivatives, α-, β-, γ- and δ-tocotrienols. Tocopherols help maintain membrane structure and integrity(Falk and Munne Bosch, 2010), act as antioxidants and free radical scavengers (Tappel, 1962; Behl
and Moosmann, 2002), and perform other nonantioxidant functions related to signaling andtranscriptional regulation (Ricciarelli et al., 2002). Both tocopherols and tocotrienols occur in plants,
as well as in photosynthetic microbes such as Synechocystis, with the highest concentration in seeds.
The seeds of most plants have significant amounts of γ- tocopherol, whereas leaves havepredominately α- tocopherol. Tocotrienols, however, are not as prevalent in plants as are tocopherols,
but they are the primary form of vitamin E in the seeds of most monocot species (Padley et al., 1994)
and fruits of some dicots (Aitzetmüller, 1997).
In the case of tocochromanol biosynthesis pathway, seven genes coding for tocochromanol
biosynthesis enzymes have been identified and characterized in Arabidopsis and rice (Mene -Saffraneand DellaPenna, 2010). This pathway diverges at the step where polyprenyl side chain is attached tohomogentisic acid (HGA) by homogentisic acid phytyl transferase (VTE2) or homogentisic acidsolanesyl transferase (HST) (Mene-Saffrane and DellaPenna, 2010). The first committed step in thebiosynthetic pathway is the prenylation of homogentisic acid with phytyldiphosphate to form
2-methyl-6-phytylbenzoquinol (MPBQ). The overall tocopherol composition is determined by the
combined activities and substrate specificities of the homogentisate prenyltransferase (VTE2),
tocopherol cyclase (VTE1), and two methyltransferase enzymes (VTE3 and VTE4) present in a given
tissue (Jo and Hyun, 2011). The action of the cyclase directly on MPBQ leads to the formation of
δ-tocopherol. By contrast, ring methylation at position R 2 by the first methyltransferase, MPBQ
methyltransferase (VTE3), leads to 2,3-dimethyl-5-phytylbenzoquinol and the subsequent action of
tocopherol cyclase leads to the formation of γ-tocopherol. The second methyltransferase, γ-tocopherol methyltransferase (VTE4), performs ring methylation at R 3, converting the γ – and δ
isoforms

to

the

α-and

β-isoforms,

respectively.

Tocotrienols

areformedwhen

geranylgeranyldiphosphate is used in place of phytyldiphosphate as the side chain precursor.
In this study, T1001-1 was isolated from in vitro mutagenized population by ionizing radiation andshown to have increased VitE contents. To study the molecular mechanism of VitE biosynthesis, we
identified rice genome encodes seven VitE biosynthetic enzyme and we analyzed its expression
patterns. The results of DNA sequencing analysis demonstrate that the OsVTE2 promoter and- 38 -

genomic sequences contain mutated region. In addition, we showed that the mutant confers retarded
seedling growth during the early seedling growth stage in rice. These observations su ggest that the
mutation of the OsVTE2 might affect a Vitamin E biosynthesis.

5.2 Materials and methods
5.2.1 Plant materials and growth conditions
The wild-type rice cv. Dongan embryo culture had been irradiated with gamma rays of 30–120 Gy
generated from a 60Co gamma irradiator (150 TBq of capacity; ACEL, Nordion, Ottawa, ON,
Canada) at the Korea Atomic Energy Research Institute. The mutant cell lines were regenerated on
medium containing 0.5 mM 5MT. Regenerated plants from 5MT-resistant calli were geneticallystabilized through consecutive generations of self-pollination and the homozygous M10 progenies
were obtained as reported elsewhere (Kim et al., 2004).
For seedling growth test, seeds were sown on 1/2 MS medium containing Murashige and Skoog salts(Duchefa, Haarlem, Netherlands), 3% Sucrose and 0.7% phyta-agar (Duchefa, Haarlem, Netherlands)
under 16 h light/8 h dark cycle at 24 oC.
5.2.2 HPLC analysis
Tocopherols and tocotrienols were determined using HPLC (Sykam system S1211). Resolution ofvitamin E species was achieved using an Agilent Eclipse LC -Si column (4.6 mm length, 5 μm) and asolvent system consisting of methanol:water (95:5, v/v) with a flow rate of 1.5 ml min -1 . Sample
components were detected and quantified by fluorescence with excitation at 292 nm and emission at
330 nm. A sample volume of 10 μl was injected for the chromatographic analysis. Peaks of
α-tocopherol,

β-tocopherol,

γ-tocopherol,

α-tocotrienol,

β-tocotrienolandδ

γ-tocotrienol,δ-tocotrienol were identified by comparing their retention times with commercially available
authentic standards. Pure tocopherols and tocotrienols were obtained from Sigma -Aldrich (St. Louis,
MO, USA) and Cayman Chemical (Ann Arbor, MI, USA), respectively. The content of each form wascalculated based on standard curves of external standards.
5.2.3 Semi quantitative RT-PCR analysis
Total RNA was isolated using Trizol reagents according to the manufacturer’s recommended
protocols (GibcoBRL, Cleveland, OH). 1 μg of total RNA was reverse transcribed in Power cDNA
Synthesis Kit (Intron Biotech Inc., Sungnam, Korea) for 60min at 42°C using 1 μg oligo(dT)15
primers. 1 μl of cDNA was used for PCR amplification with each gene speci fic primer (Table 1). The
resultant RT-PCR products were electrophoresed and analyzed on a 2.0% (w/v) agarose gel afterstaining with ethidium bromide.
5.2.4 Western-blot analysis
The OsVTE2 expression pattern was determined by western blot analysis using the anti-OsVTE2
polyclonal antibody (1:5,000 dilution), followed by the addition of peroxidase - conjugated goatanti-rabbit IgG (1:5,000 dilution) according to the manufacturer’s guidelines. Hybridization toprotein bands was detected using the SuperSignal West Pico Trial Kit (Thermo, USA)- 39 -

5.3 Results and Discussion
5.3.1 HPLC detection of tocopherols and tocotrienols in rice grains
Rice mutant lines in the M 10 generation and the control (Dongan byeo) were grown in the same filed
under normal cultural conditions, and mature seeds were harvested for determination of vitamin E
composition. Normally, the β- and δ-isomers usually appear in trace amounts, while α-tocopherol andγ-tocotrienol are the most abundant isomers. In our experiments, we also found t hat it was difficult to
detect δ-tocopherol. Therefore, this isomer was not included. In rice seeds, the major tocopherol
found is α-tocopherol, and amount of β-tocopherol and γ-tocopherol are relatively low (Fig. 1). As aresult, the contents of α-tocopherol increased to 42% in T1001-1 mutant seeds than that of thecontrols. HPLC analysis showed that α-tocotrienol and γ-tocotrienol were the major tocotrienols
present in seeds of mutants and control. However, we found that there was no significant change inthe content of each tocotrienol (Fig. 1). In the T1001-1 C mutant line, the γ-tocotrienol content wasincreased to 14% than in the control. The content of VitE (total of tocopherol and tocotrienol) was
increased to 26% in T1001-1 c mutant seeds. Tocotrienols, like tocopherols, are also potent
antioxidants (Serbinova et al., 1991), but they are less readily absorbed than α -tocopherol(Kamal-Eldin and Appelqvist, 1996). Thus, high accumulation of α-tocopherolin T1001-1 mutants
provides effective protection against oxidative damage.

Figure 1. Total and individual tocochromanol accumulation in seed of the Dongan and T1001 -1.

Tocopherol and tocotrienol composition of the seed of rice was determined by HPLC. The total VitE
content was 26% increased in the T1001-1 mutant seeds compared with Dongan.
5.3.2 Phenotypic analysis of T1001-1 mutants
As T1001-1 mutants seedling grew slowly, we compared seedling growth rates in mutant and control
plants (Fig. 2B). The primary root elongation pattern of T1001 -1 mutant was not different from thatof control (Fig. 2C). Sattler et al. (2004) reported that vte2 mutants exhibited seedling growth inArabidopsis. Thus, these T1001-1 mutant phenotypes have possibility by increasing of VTE2
expression.
- 40 -

Figure 2. Comparison of seedling growth in mutant and control plants .

(A)The early seedling growth differences between the control (Dongan) and mutant lines (T1001 -1)
for 7-days were determined by measuring the shoot and root lengths. (B) Quantitative analysis ofshoot lengths of 7-day-old seedlings from each mutant line. (C) Primary root length of 7-day-old
control and mutant line seedlings.
5.3.3 Expression of tocochromanol biosynthesis genes
To study the molecular mechanism of VitE biosynthesis, we identified rice genome encodes seven
VitE biosynthetic enzyme and we analyzed its expression patterns. Semi quantitative reverse
transcription analysis revealed that seven VitE biosynthesis genes were expressed at different levels
in four T1001-1 mutant lines. Interestingly, VTE2 transcripts were highly accumulated in the
T1001-1 C (Fig. 3), whereas the expression of VTE3 was decreased in T1001-1 lines (Fig. 3). This
result indicates that accumulation of VtiE in T1001-1 mutants is controlled by different expression of
VTE2 and VTE3 transcript.

Figure 3. Transcript levels of VitE biosynthesis genes in rice plants.

Expression profiles of 7 rice VitE biosynthesis genes in Dongan (Cont.) and mutant lines (T1001 -1).
Actin gene was used as loading control.
- 41 -

5.3.4 Sequence analysis of VTE2 and VTE3
Fig. 2 showed dramatic change of VTE2 and VTE3 expression in T1001-1 C mutant. Therefore, VTE2
and VTE3 genes were sequenced in both the control and T1001-1 C mutant. The A nucleotide at
position -245 is exchanged by a G nucleotide and the C nucleotide at position -106 is changed by Tof the VTE2 promoter in the T1001-1C mutant compared to the wild-type sequence (Fig. 4). The
position -245 mutation region was reported ZFP-TFs (zinc finger transcription factors) binding siteand -106 mutation region existence-box-cis element. ZFP-TFs were reported to upregulate theexpression of the endogenous Arabidopsis γ-tocopherol methyltansferage (GMT) gene (Van
Eenenaam et al., 2004). The presence of these motifs indicates that VTE2 may be regulated by
ZFP-TFs binding cis-acting elements. Genomic sequence of VTE2 was exchanged in only intron
region, therefore, there was no effect at amino acid sequence. In the T1001 -1 C mutant line, we
observed a G to A base change at position 662 and an added C of VTE3 promoter but not foundimportant cis-elements (Fig. 4). Sequencing of the VTE3 genomic DNA sequence was not different.
These results indicated that increasing of VTE2 transcript expression was controlled by VTE2
promoter sequence mutation in accumulation of tocochromanol.

Figure 4. Comparison of the promoter and genomic DNA sequences from Dongan (DA) and T1001 -1
mutant with the related region of VTE2 (A) and VTE3 (B).

Number of the DNA sequence is shown in the upper margin. The empty region at sequences indicatesthe different sequence in DA and T1001- 1C mutant.

- 42 -

5.3.5 Protein expression of VTE2
The VTE2 expression pattern was determined by western blot analysis using the prediction 4 ofanti-VTE2 polyclonal antibody. To precisely define the expression pattern of VTE2, we studied theexpression levels of VTE2 protein by western blot analysis (Fig. 5). These results are similar with theexpression patterns of VTE2 transcript that was accumulated more extensively in T1001 -1 mutant
than Dongan byoe in protein level.

Figure 5. Western blot analysis of VTE2 in T1001-1 mutants and Dongan byeo .

Total proteins from seedling of Dongan or T1001-1 mutants were isolated and subjected to western
blot analysis.

- 43 -

5.4 References
Aitzetmüller K (1997) Antioxidative effects of carum seeds. J Am Oil Chem Soc 74:185
Behl C and Moosmann B (2002) Oxidative nerve cell death in Alzheimer’s disease and stroke:
Antioxidants as neuroprotective compounds. Biol Chem 383: 521–536
Evans HM and Bishop KS (1922) On the existence of a hitherto unrecognized dietary factor essential
for reproduction. Science 56: 650–651
Falk J, Munne-Bosch S 2010 Tocochromanol functions in plants: antioxidation and beyond. J Exp
Bot 61: 1549–1566
Gunstone FD, HarwoodJL, Padley FB (eds) The lipid handbook, 2nd edn. Chapman & Hall, London,
pp 127–130
Kamal-Eldin A, Appelqvist LA (1996) The chemistry and antioxidant properties of tocopherols andtocotrienols. Lipids 31:671–701.
Padley FB, Gunstone FD, Harwood JL (1994) Occurrence and characteristics of oils and fats. In: Sen
CK, Khanna S, Roy S (2006) Tocotrienols: vitamin E beyond tocopherols. Life Sci 78:2088–
2098
Ricciarelli R, Zingg JM, and Azzi A (2002) The 80th anniversary of vitamin E: Beyond its
antioxidant properties. Biol Chem 383: 457–465
Tappel AL (1962) Vitamin E as the biological lipid antioxidant. Vitam Horm 20: 493–510- 44 -

6. Malaysia

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

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

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

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

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

Introduction

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

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

development

in

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

Materials and Methods

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

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

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

- 47 -

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

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

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

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

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

- 49 -

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

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

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

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

6.3

Results and Discussion

6.3.1 Radiosensitivity Test of MR219 irradiated with gamma irradiation

Figure 6. Radiosensitivity test of MR219

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

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

(a)

100

(b)

90
80
Survival rate (%)

Germination rate (%)

120

80
60
40

70
60
50
40
30
20

20

10
0

0
0 Gy

10 Gy

20Gy

40Gy

60 Gy

0 Gy

80 Gy 100 Gy 120 Gy 160 Gy 200 GY

10 Gy

20Gy

40Gy

120

Percentage of ripening (%)

Growth rate (%)

80 Gy 100 Gy 120 Gy 160 Gy 200 GY

120

(c)

100

60 Gy

Dose rate

Dose rate

80
60
40
20

(d)

100
80
60
40
20
0

0
0 Gy

10 Gy

20Gy

40Gy

60 Gy

80 Gy 100 Gy 120 Gy 160 Gy 200 GY

0 Gy

10 Gy

20Gy

40Gy

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

Dose rate

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

6.3.3. Germination rate of MRQ74 irradiated with ion beam

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

- 53 -

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

MR219-4 MR219-9

MR219

Difference Over parent

Flag leaf length(mm)

34.2

36.4

40.1

lower

Flag leaf width (mm)

13.7

16

14.3

Higher(MR219-9)

Leaf length(cm)

41.3

40.6

41.4

Lower
Higher(MR219-4)

Leaf width (mm)

15.3

12.3

13.7

Culm length

(cm)

74.3

75

72

HigherCulm number (No)

15

13

14

Higher(MR219-4)

3.61

3.62

3.62

same

Culm diameter (mm)Blast Reaction

Resistant Resistant Resistantsame

BPH Reaction

Resistant Resistant Resistantsame

Culm Strength

StrongStrongStrongPanicle length

28.2

28.4

26.4

same
Higher

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

Yield (kg/ha

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

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

107 a
104 a
107 a
99 a
109 a
ns

Percent
filledgrain

1000grain wt(g)

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

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

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

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

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

MR219-4 MR219-9 MR219 Difference Over parent

Panicles /plant

6.5

7.4

9

lower

Plant ht

100.1

100.4

103.4

lower

9
73.4

higher98Percent Filled Grain

78.1

87.7

1000 grain wt(gm)

30.3

30.3

28

higherGrain Length(mm)

9.69

9.69

9.86

lower
higherNo. of tiller/hilllower

Grain width (mm)

2.38

2.41

2.35

Panicle length

n.a

n.a

n.a

n.a

Yield (kg)

5259

6000

6444

lower

Maturity

110

112

116

lower

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

Field condition

MR219-4

MR219-9

Normal flooded conditions

4.8 t/ha

5.5 t/haSaturated soil

5.9 t/ha

6.8 t/haFlooded condition

7.2 t/ha

6.1 t/haAerobic soil condition

6.3 t/ha

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

Figure 11. Field Screening of mutants lines under

Figure 13. Advanced mutant of MR219-4

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

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

Standard rice samples% Amylose

Koshihikari (Japonica rice

17 (low)

MR219 (Local indica variety)20 (intermediate)

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

Figure 14. Standard curve of amylose

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

popular rice cultivars with Malaysian traditional rice

Figure 16. Comparison of amylose content between

Figure 17. Comparison of amylose content

ion beam and gamma rays

between MRQ74 control with mutant lines

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

6.4

Conclusion

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

Acknowledgement

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

6.6

References

Hidema, J., Yamamoto, M., Kumagai, T., Hase., Sakamoto A., and Tanaka, A. (2003). Biological
Effects of Carbon Ion on Rice (Oryza sativa L.) JAERI-review.:85-87.
Hase, Y., Tanaka., Baba, T. and H. Watanabe (2000). Plant Journal 24, 21-32;
Morita, R., Kusaba, M., Iida, S., Nishio, T. and Nishimura, M. (2007). Knockout of glutelin genes
which form a tandem array with a high level of homology in Rice by Gamma
Irradiation.Genes Genet.Syst.82, p.321-327.
Juliano, B.O. (1971). A simplified assay for milled rice amylose. Cereal Science Today.16:334-338.
Mohamad, O., MohdNazir, B., Alias, I., Azlan, S., Abdul Rahim, H.,Abdullah, M.Z.,Othman, O.,
Hadzim, K., Saad, A., Habibuddin, H. and Golam F. (2006). Development of Improved Rice- 58 -

Varieties through the Use of Induced Mutations inMalaysia.Plant Mutation Reports. 1:
27-34.
Yu, Z.L., Yu, L.D., Vailathong, T. and Brown, I.G. (2006). Introduction of Ion BeamBiotechnology(Transls.), Springer-Verlag, New Yo
Bhuiyan, S.I. (1992). Water management in relation to crop production: case study on rice: Outlook
Agric. 21(4): 293-299
Revandy.

I.D.,

Maziah.M.,Ismail.M.R.,Syahida.A.

Abdullah.M.Z(2010).

Responsesoftheantioxidative enzymes in Malaysian rice (Oryza sativa L.) cultivars under submergenceconditioin. Acta Physiology Plant.DOI 10.1007/s11738-009-0456-3.
Abdullah, M.Z., Mohamad,O., Azlan, S., Habibuddin,H., Saad, A., Kahzim,K. and Senawi,M.T.
(2003). Rice breeding and germplasm exchange: The Malaysian perspective. Proceedings ofan international rice conference 2003, 13-16 October 2003, AlorSetar, Kedah, Malaysia
Nishimura, M., Morita, R. and Kusaba, M. (2009).Utilization and Molecular Characterization of Seed
Protein Composition Mutants in Rice Plants. JARQ 43(1), 1-5- 59 -

- 60 -

7. The Philippines

Grain Quality Improvement in Rice (Oryza sativa L.) through Induced Mutation
Breeding
A. C. Barrida, F. G. Rivera and A. O. Dimaano
Agriculture Research Section, Atomic Research Division,
Philippine Nuclear Research Institute (PNRI)

7.1 Abstract
Gamma and ion beam irradiations were used for improving the grain quality and agronomic
characteristics of the Philippine rice variety IR72. Among the selections from gamma irradiatedplants, four mutant lines with intermediate amylose content were selected at 300Gy and one line at
200Gy using the quantitative method. Increased protein content was obtained on plants irradiatedwith 200Gy gamma rays.
Agronomic characteristics that were significantly affected by gamma and heavy ion beam irradiations
were plant height at maturity, number of tillers per plant, and 1000 seed -weight. Lines that are earlymaturing, short, high tillering, and with long panicle were selected on both irradiation methods used.
7.2 Introduction
Rice (Oryza sativa L.) is the main staple food crop in the Philippines. It is also the main source of
livelihood of more than 5 million farmers all over the country (Manila Bulletin, 2007). In 2011, theaverage rice yield per hectare for the first half improved to 3.8tons (or 76 cavans of 50kilos each).
This is roughly three cavans more than last year’s average yield of 3.64 tons per hectare (DA, 2011).
Rice production in the Philippines is important to the food supply in the country and economy. The
country is the 8th largest rice producer in the world, accounting for 2.8% of global rice production(FAO, 2011). However, the country was also the world's largest rice importer in 2010 (Reuters,2011).
When more rice become more available and countries achieved rice sufficiency, grain quality
becomes an important breeding objectives and consideration by consumers both in the domestic andinternational markets (Juliano and Duff, 1991). Physical properties such as length, width,translucency, degree of milling, color and age of milled rice are grain quality indicators. The amylose
content of rice starch is the major eating quality factor while protein content is an index of nutritional
value. In general, grain quality and quality preferences vary across rice growing countries andregions.

Filipinos prefer translucent, well milled, long grain rice with aroma and minimal broken

grains which is soft on cooling (Manila Bulletin, 2005).
The International Rice Research Institute (IRRI 1973) was able to breed a rice variety with 18 -23%
more protein than the check variety IR8 without any significant difference in grain yield. The grain
quality can be altered by gamma radiation. In Malaysia, rice mutant PS 1297 was developed withgood agronomic characteristics but with higher amylose content of 28.4%. Hag et al. (1971)
successfully used gamma radiation to obtain three rice mutants having higher prote in content thantheir parents. The quality of protein in rice is one of the best among the cereals but its content of 7%- 62 -

is one of the lowest (Manila Bulletin, 2005).
Given the importance of grain quality characteristic of rice for creating and stimulating demand, theForum for Nuclear Cooperation in Asia (FNCA), has initiated a cooperative project
entitled “Quality Crop Breeding in Rice (Oryza sativa L.)” among member countries (Bangladesh,
China, Indonesia, Japan, Korea, Malaysia, Philippines, Thailand and Vietnam). Such cooperative
project endeavors to use mutation breeding to widen the genetic variability for quality characteristics
and produce quality rice which is acceptable in each participating country.

7.3 Objectives
1) To develop mutants with desirable agronomic traits and physico - chemical attributes.
2) To develop mutants with good eating quality, high protein and low to intermediate amylose
contents.
7.4 Economic Impact
Development of high yielding mutants with low to intermediate amylose and high protein content
will open new market that will generally benefit the farmers. High income consumers pay higher
premiums for high quality rice, encouraging more hectarage to be planted to quality varieties. If
these are widely adopted, producers will benefit by retaining quality rice for home consumption andhaving a wider domestic market for their product. In addition, a count ry exporting rice will benefit
from quality improvement thus expanding the potential export market.
7.5 Methodology
7.5.1 Study I - Mutation induction by gamma and ion beam irradiations for improving theagronomic characteristics and grain quality of IR72
The study was conducted at the Philippine Nuclear Research Institute (PNRI) screen house andexperimental fields. The variety used was IR72, high yielding but with poor grain quality.
Radiosensitivity test was done to determine the optimum dose of gamma rays and heavy ion beam forirradiating IR72. For gamma irradiation, seeds were irradiated with 100, 200, 300, 400, 500, and
600Gy at the PNRI Gamma Irradiation Facility. Immediately after irradiation seeds were sown in theseedbed. Data on percentage emergence, percentage survival and seedling height were taken after
two weeks. After the M 1 generation and the results of analysis for amylose and protein contents of
M3 seeds, 200 and 300Gy were selected based on the rate of mutations exhibited. These dos es wereused for planting the succeeding generations for further selection of mutants. The control and thecheck variety IR 64 were grown for comparison.
Ion beam irradiation was conducted at the Takasaki Advanced Radiation Research Institute of theJapan Atomic Energy Agency Takasaki, Japan. Dehulled rice seeds were irradiated with 10, 20, 40,
60, 80, 100, 120, 160, and 200Gy using the AVF-cyclotron. Radiosensitivity test was also done inside
the screenhouse to determine the optimum dose. In planting the M4 generation, 20 and 40Gy wereselected based on the improved agronomic traits of the plants in the previous generations.
In field experiments the same methodology was employed both on gamma and ion beam-irradiated- 63 -

plants. The experiments were conducted with three replications using the Randomized Complete
Block Design (RCBD). Seedlings were transplanted in the field after three weeks after sowing at a
distance of 20 cm x 20cm. Only one seedling was planted per hill. Replanting of missing hill wasdone one week after transplanting. Proper cultural management was observed on the whole growing
season. Split application of fertilizer was done and based on the result of soil analysis. The first
application was made before transplanting and the second one was done before panicle initiation.
Spraying of pesticide and weeding were done whenever necessary. A desired water level wasmaintained for optimum growth of the plant. Sampling and harvesting were done at 85% ripening.
Plant sampling was done randomly and individual plants were selected based on agronomic
characteristics. At harvest, agronomic and yield data were recorded. Yield components such as
number of tillers, length of panicle, number of grains per panicle, number of filled and unfilled grains
were also determined.
7.5.2 Study II – Methods for Determination of Amylose and Protein
7.5.2.1 Determination of Amylose
Screening of amylose was done from M 3 to M6 generations using the iodine staining method. This
quick method of screening rice for amylose content was provided by the counterpart from Thailand.
The method was agreed upon by the working group, and the project leaders of each participating
member state. In the qualitative method, those seeds with pink to colorless in color after staining
were considered to have low amylose content and those with blue or dark purple in color was highamylose. On the other hand, the quantitative determination of amylose content of rice grain was
analyzed using the modified method being used by the Laboratory Services Division of the Bureau of
Plant Industry, Department of Agriculture (DA-BPI) and the simplified method of estimating
amylose by Juliano. This procedure was taken from rice grain quality evaluation procedures of IRRI.
Rice was pulverized into flour, weighed and an ethanol was added to wet the sample. A 1 N solutionof sodium hydroxide was then added to gelatinize to starch and was stand overnight for 18 -24 hours
or was placed in a water bath and boiled for 10 minutes.

The gelatinized sample was made tovolume and an aliquot of the solution was transferred to another volumetric flask. Acetic acid (1 N)
and iodine solution were added to form the deep blue colored starch - iodine complex. The coloreddeveloped in the sample was read in a UV-VIS spectrometer at 620nm wavelength against several
concentrations of known potato amylose standard following the same above mentioned
treatment/procedure made in the sample.
7.5.2.2 Determination of Protein
A modified semi-micro Kjeldahl method was used in the determination of protein in milled rice. The
organic nitrogen is converted to ammonia which reacts with the excess sulfuric acid forming
ammonium sulfate during digestion. A salt mixture of sodium or potassium sulfate, copper sulfate
and selenium or a readily available kjeltabs was used as catalyst to hasten the rate of decomposition
reaction. The solution is then made alkaline with 40-50% sodium hydroxide prior to steam
distillation. The ammonia being liberated during the process of distillation was trapped in a boric
acid containing receiver followed by titration using standardized 0.1 to 0.2 N sulfuric or hydrochloric
acid. Total nitrogen of the milled samples was computed based on the volume and normality of the- 64 -

acid used. The moisture of the rice samples was corrected and percentage protein was calculated bymultiplying the factor 6.25.

7.6 Results and Discussion
7.6.1 Selection and evaluation of mutant lines with improved agronomic characteristics
Planting of the selected putative mutants lines with improved agronomic traits and with low tointermediate amylose content was done continuously for further confirmation and evaluation. T here
was a significant increase in plant height at 200 and 300 Gy as shown in Fig. 1.

Plant Height (cm)

85.00
80.00

a

a

200 Gy300 Gy

a

75.00

b

70.00
65.00
Check VarControl

Treatment
Figure 1. Plant height at maturity as affected by gamma irradiation at M 7 generation(bars with the same letters are not significantly different at 5% level based on DMRT)The number of tillers per plant was significantly affected by gamma irradiation. (Fig. 2) There was a
33.34% increase at 300 Gy and 19.3% increase at 200 Gy compared to the control and check variety.
25.00

No. of tillers

20.00

a
b
c

c

Check VarControl

15.00
10.00
5.00
0.00

200 Gy300 GyTreatment
Figure 2. Number of tillers per plant as affected by gamma irradiation at M7 generation(bars with the same letters are not significantly different at 5% level based on DMRT)The weight of 1000 seed (g) increased at 200Gy. The control and 300Gy obtained similar weight aspresented in Fig.3.

- 65 -

23.001000 seed-wt

a

ab

22.00

ab

21.00
20.00

b

19.00
18.00
17.00
Check VarControl

200 Gy300 GyTreatment
Figure 3. 1000 seed - weight (g) as affected by gamma irradiation at M 7 generation(bars with the same letters are not significantly different at 5% level based on DMRT)The number of days to flower was not affected by the treatments. However, the control took lon ger toflower with 97 days as compared to irradiated plants with 90 days. Likewise, the length of panicle(cm), number of grains per panicle and filled grains were not significantly affected by the treatments(Table 1).
Table 1. Agronomic data at M 7 generation

Treatment

Days

to

LengthofGrains/Filled Grains/

flowerpanicle (cm)

Paniclepanicle

Check

93

23.20

124

78

Control

97

22.05

121

90

200 Gy

90

24.60

121

93

300 Gy

90

23.00

116

91

7.6.2 Selection of mutant with low to intermediate amylose content
The analysis of amylose content using the qualitative or the iodine staining method of M 6 seeds
irradiated with 200 and 300Gy gamma rays showed that at 200Gy, 12 out of 153 lines analyzed had
low to intermediate amylose, while at 300Gy only one out of 127 lines had low to intermediateamylose. Those with low amylose content were considered promising mutant lines. The control andthe check variety have high amylose content (Table 2).

- 66 -

Table 2. Lines at M 6 generation with varying amylose content

No. of Lines

No. with Low to

No. with HighanalyzedIntermediate Amylose

Amylose

Check

10

0

10

Control

100

10200 Gy

153

12

141

300 GY

127

1

126

Treatment

30

20

300Gy
1
200Gy
12

15

Control

25

Check

10
5
0

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
Cont…
Check

% Amylose Content

35

SamplesFigure 4. Percent amylose content using the quantitative method of analysis

For the M 7 generation, quantitative method of analysis for amylose content (Fig. 4) was applied forthe mutant lines selected in the previous generation and to confirm the result of qualit ative method.
Result showed that four lines at 300Gy and only one line at 200Gy had intermediate amylose content.
The control and check variety have high amylose contents.
7.6.3 Selection of lines with high protein content

Protein content (%)

12.00
10.00
8.00

Check

6.00

Control

4.00

200 Gy2.00

300 Gy0.00
M3

Generation

M4

Figure 5. Protein content at different treatments in two generations- 67 -

Fig. 5 shows higher protein content at 200 and 300Gy in the M 4 generation. The increase in protein
content was 31.11% higher at 200Gy and 8.58% higher at 300Gy compared to the control. The
control and check variety had similar protein content.
7.6.4 Heavy Ion Beam Irradiation for the Selection of Mutant with Improved Agronomic
Characteristics
Irradiation with heavy ion beam at 20 and 40Gy yielded plants that were early maturing (one weekearlier than the control), short, high tillering and with long panicle. The agronomic data are given in
Table 3.
The plant height at maturity was significantly affected by the treatment wherein the plants irradiatedwith 40Gy was the tallest followed by 20Gy with 83.13 and 78.36cm. The control was the shortest
with 72.11cm.
The number of tillers per plant significantly increased at 20 and 40Gy and was not significantly
different from each other at 23.00 and 24.00, respectively. At 40Gy, plants were 30.43% tiller thanthe control, while at 20Gy 33.33% tiller than the control.

Some authors reported that the mutation

frequencies per unit dose were higher with ion beams than with gamma rays, thus indicating that ion
beam had a higher mutation effectiveness than gamma rays, which confirms the result of previous
studies, (Fujii et al. 1966, Mei et al. 1994). Similar result was also obtained by Sobrizal et. al (2009)
that, based on the point of seed fertility of M 1 plants 20 Gy is the best dose. In addition, Kang (2009)
suggested that the proper irradiation doses of heavy ion beam for cultivar "Ilpum" might be between
20 to 30Gy doses. The weight of 1,000 seeds was significantly higher at 40Gy with 22.67gram.
However, there was no significant difference between 20Gy and the control. As in gamma irradiatedplants, the number of days to flower, panicle length, seed per panicle and number of filled grains per
panicle were not affected by heavy ion beam irradiation.
Table 3. Agronomic traits at M 4 generation as affected by ion beam irradiation
Treatment

Plt. Ht.at

Days toNumberPanicleSeed/

Filled1000

Maturityflowerof tillers/

LengthpanicleGrains/

seed

Plant

(cm)

paniclewt. (g)(cm)
Control

72.11 c

97

16 b

22.05

121

90

19.68 b20 Gy

78.36 b

90

24 a

22.99

118

79

21.94 b40 Gy

83.13 a

90

23 a

22.30

109

85

22.67 aIn a column, means with the same letters are not significantly different at 5% level based on DMRT.

7.7 Future Plans
The project will not just end by simply producing mutant lines with intermediate amylose content.
The mutant should be planted continuously to determine if the improved characteristics are alreadystable. Likewise, yield testing of the mutant is very important to find out if the yield is not affected- 68 -

by the alteration of amylose. In addition, determination of amylose on those plants irradiated with ion
beam and the use of molecular technique for the confirmation of the mutants will also be undertaken.

7.8 Conclusion
Gamma and ion beam irradiations showed similar effects for inducing mutations in plants as
exhibited by the results obtained in this study. Both methods of irradiations gave significant resultson some agronomic traits of the plants. Likewise, the grain quality specifically the amylose andprotein contents were also significantly affected by gamma irradiation.
7.9 Acknowledgement
The authors wish to thank the FNCA for this project on grain quality improvement in rice and thesponsors especially the government of Japan, NSRA, MEXT, Dr. Tanaka for irradiating the rice seeds
using ion beam and the PNRI Irradiation Facility Services Unit. The authors gratefully acknowledge
the support of Ms. Glenda B. Obra, Head of Agricultural Research Section of PNRI, Mr. Eduardo C.
Costimiano for assisting in the whole implementation of the project, and Mr. Fernando B. Aurigue
who did the editing of the manuscript.
7.10 References
Department of Agriculture - August 10, 2011 press release.
FAO Stat. FAO Statistics. Retrieved 30 March 2011.
Fujii, T., M. Ikenaga and J.T. Lyman, 1966.

Radiation effects on Arabidopsis thaliana, II. Killingand mutagenic efficiencies of heavy ionizing
Haq, M.S., N. Choudhury andparticles. Radiat. Bot. 6 : 297 - 306.

M.M.Rahman. 1970. Breeding for high protein content of ricethrough induced mutations. STI/PUB/258. IAEA, Vienna, Austria.
Hag, M. S., Chindhurg, N., and Rahman, M. M. Breeding for high protein content and quality of ricethrough induced mutation. Atomic Energy Center: Dacca, Pakistan (cited in Plant Breed.
Abst. 1971, 41(3):4436).
IRRI. 1973. Improvement of the protein content of rice. In: Annual Report for 1972. Int. Rice Res.
Inst.: Los Baios, Philippines.
Juliano, B.O. and Duff, 1991. Rice grain quality as an emerging research priority national rice
breeding programs in rice grain marketing and quality issue. IRRI, Philippines.
Kang Si-Yong. 2009. Seed irradiation test by heavy ion beam for rice mutation breeding. Paper
presented in the 2009 FNCA Workshop on Mutation Breeding. September 21 -24, 2009.
Hangzhou, China.
Kikuchi, H.1994. Improving the eating quality of rice using low amylose mutants. Gamma Field- 69 -

Symposia 33:18.
Manila Bulletin, 2005. The rice Filipinos like to eat: Physico-chemical basis. Sept. 4, 2005.
Manila Bulletin. 2007. NGOs Opposed Entry of GM rice into the country Manila Bulletin 410: 27
page B-12.
Mei, M., H. Deng, Y. Lu, C. Zhuang, Z. Liu, Q. Qiu, Y. Qiu and T.C. Yang. 1994. Mutagenic effectsof heavy ion radiation in plants. Adv. Space Rec.14: 363-372
Parpia, H..A.B.1970. Bridging the protein –calorie gap. STI/PUB/258. IAEA, Vienna, Austria
Reuters. 28 January 2011. Retrieved 30 March 2011.
Science Daily (Feb. 19, 2010)
Sobrizal, Carkum, Winda Puspitasari, Yulidar and Nana Supriatna.2009. Improvement of diah suci
variety by ion beam irradiation. Paper presented in the 2009 FNCA Workshop on Mutation
Breeding. September 21-24, 2009. Hangzhou, China
Tanaka, S. and Y. Takagi. 1970. Protein content of rice mutants Improving plant protein by nuclear
techniques. STI/PUB/258. IAEA, Vienna, Austria.

- 70 -

8. Thailand

Rice Mutation Breeding for Various Grain Qualities in Thailand
S. Taprab, W. Sukviwat, D. Chettanachit, S. Wongpiyachon and W. Rattanakarn
Bureau of Rice Research and Development, Rice Department,
Ministry of Agriculture and Cooperatives, Thailand

8.1 Introduction
Rice breeding through mutagenesis in Thailand had been continuously done for long period of time.
Recommended varieties of mutated glutinous rice (RD6) and RD 15 an earlier matured mutant of
KDML 105 were released. They are photoperiod sensitive varieties that can be grown once a year in
Thailand. RD10 was later a glutinous mutated variety released. It was a photoperiod insensitive
variety that can be grown more than one crop per year. Those released mutants were purposed forhigher yield per unit area. Rice breeders are consequently carrying on mutation breeding for better
grain quality. We reviewed previous mutation breeding activities in Thailand prior to FNCA Mutation
breeding project. The results showed some success in improving grain qualities of various purposes.
Seeds of recommended varieties were widely used for mutation induction through gamma ray andfast neutron irradiations. We have joined FNCA Sub-project on improvement of composition or
quality in rice since 2007. Using nuclear technique to induce mutation in rice for various grain
qualities was our breeding program proposal. We have just started by mutation induction by gamma
ray irradiation in 2008. We have not joined the Ion-beam mutation breeding project.
Collaborated with FNCA Mutation Breeding Project, five years plan of rice mutation breeding for
improvement of grain quality in Thailand started in 2007. Main objective is to develop mutants withvarious grain qualities such as amylose content, protein content and low phytic acid. Two groups ofvarieties used for mutation induction are low amylose content varieties (KDML105 and RD15) andhigh amylose content varieties (SPR1 and CNT1) including one international check variety, IR64.
Gamma ray with 200 and 300Gy, and fast neutron with 20 and 30Gy are used for irradiation.
KDML105 and RD15 are supreme grain quality aromatic rice in Thailand contain about 15%
amylose. They can be grown only one crop a year due to their photoperiod sensitivity. Mutants withvarious level of amylose content retained excellent physical grain quality with good cooking quality(aroma) and photoperiod insensitivity are intended to be obtained. KTH17, SPR1 and CNT1 are highyielding varieties and high amylose content of about 27%. They have very good physical grain
quality. Irradiated grains of those varieties were screened for low amylose content and low phyticacid. IR64 was used for international check variety of the FNCA mutation breeding project.

8.2 Materials and Methods
Rice seeds of varieties: KDML105, RD15, KTH17, SPR1, CNT1 and IR64
Mutation induction: -200 and 300Gy of Gamma ray irradiation,-20 and 30Gy of fast neutron irradiation
Grain analyses: -IRRI’s standard protocol for amylose content analyses,-SDS-PAGE technique (Laemmli, 1970) for protein and its component analyses,-Screening technique developed by Victor Raboy (2005) for low phytate analyses- 72 -

Mutant selection: -Pedigree selection method
Seeds had been irradiated and M 1 obtained in Wet season 2007. M2 plants of KDML105 and RD15
had been grown in Dry season 2008 where their wild types could not flower. During this period, M 2plants could be selected for photoperiod-insensitive mutants. Then, M3 seed of 200 mutant lines had
been separately harvested as single hill per line. Photoperiod sensitivity is an obvious trait and easy
to be selected in dry season whereas long day period presented. Those 200 photoperiod-insensitive
mutants were continuously screened for important agronomic traits and to be analysed for amylose
content. They were again tested for photoperiodism, tillering ability and maturity in Dry season 2009.
A half of single hill harvested M4 seeds had been grown in Wet season 2009 and another half of seed
had been analyzed for amylose content. In wet season, flowering time of the mutants were earlier
than their wild types but not much differ in plant height. Expected higher amylose content witharoma would be identified from those mutants. While, lower amylose mutants would be used forbreeding germplasm and for special purposes. Selected M5 mutants had been grown in Dry season 2010
and M6 and M7 seeds had been grown in Wet season 2010 and 2011. In 2008, we developed simple
technique for amylose content determination on breeding lines and distributed to FNCA members.
While amylose content was being analyzed, we developed a technique for protein analysis. Protein
extraction technique reported by Iida et al. (1993) and Tanaka et al. (2004) had been modified and tested
for indica rice protein analysis. Extracted proteins such as globulin, albumin, prolamine and glutelinfrom KDML105 and Koshihikari had been identified using SDS-PAGE technique (Laemmli, 1970).
In case of low phytic acid mutants, 2 mutant lines are going to be tested for bioavailability inartificial intestine digestion. New irradiated populations were screened for low phytic acid mutants.

8.3 Results
Wet Season 2010 and Dry Season 2011
8.3.1 Protein component analyses
Using SDS-PAGE technique adapted from Laemmli (1970) for protein and its component analyses todetermine total protein, albumin, globulin, glutelin and prolamine of KDML105 and Koshihikari.
The result showed clear bans of albumin, globulin and glutelin but not of prolamine (Figure 1). We
could distinguish KDML105 from Koshihikari by their pattern of protein components on SDS-PAGE
algarose gel (Figure 2).

Figure 1. SDS-PAGE analyses on total protein, globulin, albumin, prolamine and glutelin of rice variety, KDML105 .

- 73 -

Figure 2. Agarose gel electrophoresis of Koshihikari and KDML 105, lane M, DNA marker (GeneRuler™
1 kb DNA Ladder, Fermentas), lane 1, the PCR product amplified from genomic DNA with the primer set
F2 and F8, lane 2, the nested RT-PCR product amplified with the primer set F2 and F8, lane 3, the PCR
product amplified from genomic DNA with the primer set FC and F5, lane 4, the nested RT-PCR productamplified with the primer set FC and F5.

Molecular cloning of Glu-B from KDML105 was done by Nested reverse transcriptase-mediated
PCR. The cDNA was made from total RNA of 20 days seedling using RetroTools ® Two Step Kit(BIOTOOLS B&M LABS., S.A.) and random hexamers (pd(N)6 )as primers for 1st – Step Reverse
Transcription

(RT).

The

secondround

F2(5’-GTTTTGGAACGTTAATGCCCATAG-3’)-F8(5’-ATGAGCACCAAAAGATCCAC-3’) primers were used to amplified the read through LCC1
and the PCR product amplified from LCC1 genomic DNA with the primer set FC (5’CTCCTAGATATCAACAACAGAC -3’) and F5(5’- AGTTGTTGCTCTATATGTCTTCGACT-3’).
Then the PCR product was process to insert in pGEM®-T Easy(Promega, USA). Nucleotides
sequences analysis of Glu-B product were done by ABI 3130 automatic sequencer (Applied
Biosystems) using BigDye ® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). Results
show the nucleotide sequences with 600 bps are homologous to Oryza sativa glutelin precursor(GluB4) gene exon 1 through 4 and complete cds.
8.3.2 Mutant lines selection
Since 2008, mutant lines with photoperiod insensitiveness had been achieved. In the year 2010, twohundreds M 5 mutant lines of both KDML105 and RD15 derived from 2008 had been analyzed for
amylose content and protein components. Low amylose mutant lines had been achieved from KTH17
which is originally high amylose content local variety. Various amylose content mutant lines fromoriginal low amylose content varieties KDML105 and RD15 had been obtained. Some of thosemutants contained lower and higher amylose content than did of their wild types. 293 mutant lines of
RD15 showed wide variation of amylose contents from 10.58 – 28.08 % (Figure 3). 241 mutant lines
showed less variation of amylose contents than did of RD15, from 11.96-16.02 % (Figure 4). 191
mutant lines of KTH17 showed wide variation of amylose contents from 13.30 – 29.98 % (Figure 5).
Protein contents of those mutant lines are being analyzed.
- 74 -

Figure 3. Number of mutant lines of RD15 with various amylose contents.

Figure 4. Numbers of mutant lines of KDML105 with various amylose contents.

Numbers of mutant lines

Numbers of mutant lines of KTH17 with various amylose
contents

Amylose contents (%)
Figure 5. Numbers of mutant lines of KTH17 with various amylose contents.

- 75 -

When photoperiodism and dosages of gamma ray were considered, it was found that 20Kr (200Gy)
and 30Kr (300Gy) induced different frequency of mutation on amylose content. Photoperiod
sensitive mutants of RD15, KDML105 and KTH17 mostly contained lower amylose than wild types
did. From RD15, photoperiod sensitive mutants derived from 20Kr irradiation contained lower
amylose while 30Kr mutants contained slightly higher amylose (Figure 6). Highest frequency of
20Kr mutants of RD15 was 45 lines with 12% amylose content while 30Kr mutants was 43 lines with
13% amylose content. From KDML105, photoperiod sensitive mutants derived from 20Kr and 30Kr
were similar distribution to RD15 mutants. But 20Kr created wider range of amylose with 1 line
contained 18% amylose while 30 Kr had highest amylose of 15%. Both 20Kr and 30Kr had highest
frequency at 13% amylose content with 45 and 61 lines, respectively (Figure 7). Similarly, highamylose variety KTH17 had been also induced lower amylose mutants. KTH17 wild type contained
27-28% amylose while its mutants derived from both 20 Kr and 30 Kr contained lower amylose.
Their highest frequencies are 24 and 16 lines at 23% amylose content (Figure 8). In contrast,
photoperiod insensitive mutants showed different distribution from photoperiod sensitive mutants.
From RD15, lower dose of 20Kr induced higher amylose content than higher dose of 30Kr. It
produced very higher amylose content up to 28% mutants compared to its 15% amylose wild type. It
had highest frequency at 25% amylose with 32 mutant lines. The dose of 30Kr induced lower
amylose to the lowest of 10% amylose content. Its highest frequency was 40 mutant lines at 12%
amylose content (Figure 9).

Wild typeFigure 6. Amylose content (%) of RD15 photoperiod sensitive mutants irradiated with Gamma Ray 20Kr
and 30Kr- 76 -

Figure 7. Amylose content (%) of KDML105 photoperiod sensitive mutants irradiated with Gamma Ray 20Kr and 30Kr

Figure 8. Amylose content (%) of KTH17 photoperiod sensitive mutants irradiated with Gamma Ray 20Kr and 30Kr

Figure 9. Amylose content (%) of RD15 photoperiod insensitive mutants irradiated with Gamma Ray 20Kr and 30Kr

- 77 -

8.3.3 Grain analyses of mutants
Twenty five M7 -mutant lines of KDML105 and RD15 had been randomly selected to be analyzed on
grain physical properties and chemical quality. Grain size had been measured on brown rice in length,
width and thickness. Grain shape was determined by Length/Width ratio (L/W). The ratio of L/W thatwas greater than 3.0 indicated slender grain shape. Visually observation on chalkiness, pericarp colorand hull color had been also practiced. Means and standard deviations of mutants and wild types
were shown in Table 1.
Table1. Grain physical properties of 25 M 7 mutants of KDML105 and 25 M7 mutants of RD15 compare to
its wild type.
Designation

Size of brown rice (mm)Length

Width

Thick

L/W
ratio

Chalkiness Pericarp
color

Hull
color

M7-KDML105 mutants

7.16 ± 0.08 2.05 ± 0.02

1.68 ± 0.03

3.34 ± 0.05

dull

White

Straw

M7-RD15 mutants

7.46 ± 0.09 2.14 ± 0.02

1.75 ± 0.03

3.48 ± 0.04

dull

White

Straw

KDML105 (wild type)

7.40

2.12

1.76

3.49

dull

White

Straw

RD15 (wild type)

7.49

2.11

1.74

3.55

dull

White

Straw

It had been found that all mutants shown dull endosperm as well as their wild type. Chalkiness could
not be identified in these samples. We have an experience that aromatic rice especially KDML105
and RD15 shown dull endosperm when grown in acid soil. Since our experimental field was acidsulfate soil, dull endosperm occurred. All mutants had white color pericarp and straw color hull aswell as their wild types. Mutants of KDML105 had shorter grain length (7.16mm) than their wild
type (7.40mm) and shown slightly bold shape (3.34 L/W) compared to their wild type (3.49 L/W).
Mutants of RD15 shown almost same grain length but they were wider and thicker that gave smaller
L/W ratio. This indicated that mutants of RD15 had bigger grain size than their wild type (Table 1).
Grains from 25 mutants of KDML105 and 25 mutants of RD15 that had been divided to be analyzed
on physical properties were synchronously analyzed on chemical quality. Amylose content, Gelconsistency, Alkali spreading value, Elongation ratio were important chemical quality that had been
analyzed. Total protein (%) was analyzed by Kjaldal Method. Aroma or odor of milled rice had beentested by human sensory test. Aroma expressed in 4 levels of sensory test are 0 = odorless (notaroma), 1 = mild aroma, 2 = aroma and 3 = strong aroma. 2-Acetyl-1-pyrroline (2 AP) (Fig 10) was
also analyzed through gas chromatography. 2-Acetyl-1 pyrroline is a pyrroline that is 1-pyrroline inwhich the hydrogen at position 2 is replaced by an acetyl group. It is an aroma and flavor compound
present in jasmine rice and basmati rice. It is responsible for the 'popcorn' aroma in a large variety of
cereal and food products. It is one of the key odourants of the crust of bread and considered to be
responsible for the cracker-like odour properties. In bread, it is primarily generated during baking but
amounts are influenced by ingredient composition and fermentation conditions.

- 78 -

Figure 10. Structural formula of 2-Acetyl-1-Pyrroline

It found that M7 mutants of KDML105 had low amylase content similar to their wild type. They
contained amylose within range of 15.03 to 16.06% of amylose or 15.50≠0.2% in average, while theirwild type contained 15.42% amylose. In contrast, M 7 mutants of RD15 contained higher amount ofamylase than did of their wild type. The mutants had wider range of amylose content. Their amylase
content varied from 15.40 to 27.79% while their wild type contained 15.55% of amylase. Out ofthese 25 mutants, there were three mutants that contained 15.40, 15.87 and 15.93% of amylose.
Mutants of RD15 had harder gel than mutants of KDML105 and their wild type. Mutants of
KDML105 had softer gel than their wild type. Alkli spreading value of all mutants was almost notdiffered from their wild type as well as elongation ratio. Total protein content of mutants averagely
higher than their wild type in both KDML105 and RD15. But it was still in normal level of thosevarieties. Unfortunately, we could not find low protein content mutants in these samples. Mutants of
KDML105 contained 9.1% while those of RD15 contained 9.8% protein. Aroma tested by smellingshown different levels of aroma scented between KDML105 and RD15 mutants. Mutants of
KDML105 had strong aroma as well as their wild type with score of 3. Mutants of RD15 showedaroma in score 2 of sensory test level as well as their wild type. When 2-acetyl-1-pyrroline had been
detected it was found that all mutants of KDML105 contained 2 AP in higher amount than their wild
type in both milled rice and brown rice. Brown rice and milled rice of KDML105 mutants contained
2.00 and 2.32ppm of 2 AP, respectively. Their wild type KDML105 contained 2.06 and 2.24ppm of 2
AP in its brown rice and milled rice. We could detect 2 AP in only 3 mutants of RD15 that contained
low amylase content. The mutants of RD15 those contained high amylose were not aroma with 2 AP
detection, but sensory test detected aroma from those mutants. It would be an expression of othervolatile compound. One mutant with 15.87% amylase content showed 2.18 and 2.16ppm 2 AP in its
brown rice and milled rice which was almost similar to its wild type (2.99 and 2.06ppm 2 AP) (Table2). It was very rare opportunity to find aromatic rice with high amylose content in our breeding
material. This will be new chance for us to produce various products from our aromatic mutants forworld market.
Table 2. Chemical quality of M7 mutants of KDML105 and RD15 compare to their wild type
Designation

amylose

Gel1

(%)

(mm)

Alkali

E.R.2

ProteinAroma3 2 AP4 (ppm) 2 AP4 (ppm)(%)

Milled Rice

Brown Rice

M7-KDML105 mutants

15.5±0.2 72.9±3.9 6.9±0.28 1.6±0.03 9.1±0.39 2.7±0.48

2.32±0.35

2.00±0.28

M7-RD15 mutants

25.6±3.8 54.0±9.9 7.0±0.0 1.7±0.03 9.8±0.62 2.0±.00

0.1±0.43

0.1±0.44

KDML105 (wild type)

15.42

69

7.0

1.66

8.77

3

2.24

2.06

RD15 (wild type)

15.55

71

7.0

1.66

9.24

2

2.06

2.99

1

Gel

E.R.

= Gel consistency
2= Elongation Ratio- 79 -

Aroma
4

2 AP

3

= aroma by sensory test
= 2 Acetyl-1-pyrroline detectionIn other purpose, low phytate mutant lines achieved from SPR1 are being evaluated on their grain
yield and other agronomic characters. Forty eight mutant lines of low phytic SPR1 are going to be
examined their protein content.

8.4 Conclusion
Gamma ray irradiation can be applied for mutation induction in rice. Dosages of 20Kr (200Gy) and
30Kr (300Gy) showed advantage on mutation induction for grain quality improvement. Thai rice
varieties namely, SPR1, KTH17, KDML105 and RD15 had been irradiated by 20Kr and 30Kr of
gamma ray to induce mutation for various grain qualities such as phytic acid, amylose content andprotein and its compositions. Amylose content had been analyzed using standard method for ricedeveloped by IRRI. Globulin, albumin, prolamine, and glutelin were detected using SDS-pageanalyses. Low phytate mutants had been screened using technique developed by Raboy (2004). Low
amylose mutant lines had been achieved from KTH17 which is originally high amylose content local
variety. Various amylose content mutant lines from original low amylose content varieties KDML105
and RD15 had been obtained. Some of those mutants contained lower and higher amylose content
than did of their wild types. Photoperiod sensitive mutants from RD15, KDML105 and KTH17 hadlower amylose content than did of their wild types. Both 20Kr and 30Kr induced similar distributionof mutants in case of photoperiod sensitive. Among photoperiod insensitive mutants derived from
RD15, 20Kr induced higher amylose while 30Kr induced lower amylose than its wild type. M7mutants shown bigger grain size but shorter length than did of their wild types. Aromatic rice withlow amylose content obtained from KDML105 mutants. Newly found aromatic rice with highamylose content derived from RD15 mutants could not be detected on its 2-acetyl-1 pyrroline
compound. Those mutants might contain other volatile aromatic compound.

- 80 -

9. Vietnam

Breeding New Rice Variety DT39 Quelamby Gamma Ray
Bang. D. T. T. Nhung N. T. H., Duc N. M. and Hung N. P.
Institute of Agriculture Genetics

9.1 Introduction
In 2012, Vietnam rice export reached 7.7 millions of tons and overcomes Thailand and becomes thebiggest country for rice export in the world. However, price of Vietnamese rice in international
market is often lower than of Thailand’s because the quality is lower. Vietnam government conducts
many breeding projects for rice quality improvement. One of the most effective methods for rice
quality improvement is mutation breeding by irradiation of Gamma rays 60Co source combined withselection process. Generally, mutation breeding by irradiation of Gamma rays has many advantages
compared to other breeding methods such as improvement of one or some characteristics while
maintaining the rest characteristics of variety. In Vietnam, rice variety Bacthom No.7 has very goodcooking quality, easily cultivated, relatively short growth duration, stable grain yield potential but
poor resistance to bacterial leaf blight. That’s why, Bacthom No.7 is not widely cultivated in many
locations, especially in summer season.
For the above reasons and also as a follow up on the sub-project on composition or quality in rice
under the FNCA, we carried out the project “Improvement of Bacthom No.7 by irradiation of dried
seeds with gamma rays of 60Co source”.
Aims of research: Creation of new rice variety with good cooking, high quality, easily cultivated,short growth duration, high and stable grain yield, and good resistance to bacterial leaf blight.

9.2 Materials and Methods
3,000 dry seed at 13% moisture content was used for irradiation by gamma ray 60Co source at therange doses of 150, 200 and 250Gy in the K hospital, Hanoi. The experiment was started in summer
season 2008.
9.3 Results and Discussion
9.3.1 Selection Process
9.3.1.1 M 1 generation: All main panicles in M1 generation were harvested to develop M2 population.
9.3.1.2 M2 generation: At the dose of 150Gy, there were no differences observed. However, from1,753 individual plants harvested from 200Gy, the frequency of variation observed was around 3%.
Mutants with changed characteristics consist of morphological traits, growth duration, tillering
ability, hard stem, seed size and so on. From those variations observed, 112 mutant lines were
harvested individually for M3. At 250Gy, obtained variations included high sterility ratio and shortplant height. However, most of these variations were not useful for breeding, therefore all plantsfrom this dose were not included in the next experiment.
- 82 -

9.3.1.3 M3 generation: Single plants were cultivated and evaluated separately, each plant is
considered as one family. Selection was continued to discard unfavorable lines for example less seed,less panicle, or sterile plants and so on. Elite individuals in M3 were selected to develop promising
rice lines in M4 generation. There were some elite individuals with better economic characteristics
compared to control variety namely BT15-1, BT15-2, BT15-3 BT20-1, BT20-2, and BT20-4 withlong seed, good cooking quality, high grain quality, and resistant to bacterial leaf blight. BT20-2 andBT20-2 have higher 1,000 seeds weight, 25.89g and 31.73g, respectively.
9.3.1.4 M4 - M6 generation: From 4 promising lines in M5, one outstanding lines M 6 -20.3 wasselected and showed no segregation, hard stem, long seed, stable grain yield potential, and good
resistant to bacterial leaf blight. Line M6 -20.3 was named DT39 Quelam and further evaluated in
narrow scale for agronomic and biological trait by author. Beside of that DT39 Quelam was officiallysent to the National Testing Center for Crop to test for Value of Cultivation and Use of crop (VCU)
from M6 - M 8 generation (during 2011- 2012).
9.3.2 Evaluation Process
9.3.2.1 Evaluation results of author
Evaluation experiment was carried out in order to compare yield performance and agronomic trait
with the control Bacthom 7 in both seasons. The result showed that the yield in spring season was
higher than summer season in both varieties, and DT39 Quelam had higher yield than Bacthom 7 inboth season.
Table 1: Evaluation agro-biological characters and yield of DT39 Quelam by author(Spring and summer 2010 in Tuliem, Hanoi)*

Variety

DT39
QuelamBacthom
Control

Growthduration(days)

Plant
height(cm)

No panicle/
plant

Paniclelength(cm)

Numberseed/
panicleSeed set
ratio
(%)

Yield
(tons/ha)Spring:
132

102,2
±
0,30

5,5
±
0,28

30,2
±
0,24

162,3
±
6,13

89,5
±
0,32

6,3
±
0,31

Summer:
105

112,2
±
0,22

5,0
±
0,30

28,5
±
0,27

147,3
±
6,05

88,2
±
0,25

5,9
±
0,30

Spring:
134

98,6
±
0,27

5,2
±
0,22

22,3
±
0,30

152,5
±
5,76

87,5
±
0,31

5,6
±
0,26

Summer:
106

107
±
0,32

5,2
±
0,30

20,6
±
0,23

140,2
±
6,20

82,3
±
0,28

5,0
±
0,31

*Transplanting 1 seedling per hill, 45 hills per square meter.

The results in the above table showed that mutant line DT39 Quelam and Bacthom 7 have the same
growth duration. Panicle length of DT39 Quelam is longer than Bacthom 7 and number of seeds was
also higher. The resulting grain yield of DT39 Quelam was higher than that of Bacthom 7.
- 83 -

Table 2 shows the main characteristics of DT39 Quelam and Bacthom 7.
Table 2: Some main characteristics of mutant DT39 Quelamand control Bacthom 7

Bacthom 7(control variety)DT39 Quelam(mutant variety)- Spring

130-135

130-135- Summer

105-108

104-1062

Plant height (cm)

106

110

3

Tilerring ability

MediumMedium4

Leaf colour in harvest

Ligh yellow

yellow

5

Flag Leaf type

short, narrow, incline

short, narrow, arrect

7

Stem hardness

Hard

Harder8

Flowering duration (days)

5

5

9

No. of panicle/hills

5.4

5.5

10

Panicle length (cm)

25.2

28.2

12

No. of seeds/panicle

140

155

13

Abort grain ratio (%)

12-15

12-17

14

Awn

AbsentShort and partly

15

Weight of 1000 seeds (gram)

19.2

23.8

16

Seed type

Small, short (2.0-7.2 mm)

Small, length (2.2-9.3 mm)

17

Husk color

Light brown

Dark brown

Characteristics
1

Growth duration (days)

The data in table 2 showed that mutant variety DT39 Quelam7 was not so much different from
Bachom 7 in 17 phenotype characters. In some main characters such as length of panicle, grain
weight of 1,000 seeds, number of seeds per panicle DT39 Quelam was higher than Bacthom 7. DT39
Quelam had harder stem, darker husk color than Bacthom 7.
9.3.2.2 Evaluation results of The National Testing Center for Crop (NTCC)The National Testing Center for Crops (NTCC) is the unique office that is responsible forindependently evaluating and testing new varieties in Vietnam before release.
DT39 Quelam had been sent to NTCC for testing and evaluation in three continuous seasons(summer season 2011, spring and summer season 2012).

- 84 -

Table3: Assessment for agronomic traits of DT39 Quelam variety by NTCC (Source: NTCC)*

Seasons

Variety
BT7

Summer(control)

2011

DT39
QuelamBT7

Spring(control)

2012

DT39
QuelamBT7

Summer(control)

2012

DT39
QuelamSeedling
vigor(points)

Flowering
duration(points)

Plant
hardness(points)

Plantheight (cm)Growthduration(days)

5

5

3

112.8

106

5

5

1

120

105

5

5

1

101

139

1

5

1

106

140

5

5

1

108.5

107

5

5

1

112.8

104

*points: from 1 to 9 means best to worst

The result of NTCC for Crop for three continuous seasons (Table 3) showed that generally, DT39
Quelam and Bacthom 7 (BT7) have the approriate growth duration for rice production in many
locations in spring season and shorter in summer season, 104-105 days (DT39 Quelam), 106-107
days (Bacthom 7). In spring season 2012, seedling vigor of DT39 Quelam was point 1 (cold tolerant)better than Bacthom 7, point 5. Plant height of DT39 Quelam was higher than Bacthom 7, flowering
duration was similar in both varieties.
Table 4 induced that grain weight of DT39 Quelam was higher than Bacthom 7 in three tests. But thedata also showed the disadvantage of DT39 Quelam. The abort ratio in three test was higher than
Bacthom 7 and the purity (point 1, 5 and 3 for each season) was lower than Bacthom 7 (point 1) inthree tests. However, field puritication index of variety could be improved by selection proccess.
Evaluation of 1,000 grain weight of DT39 Quelam and Bacthom 7 (table 5) showed clearly different
between two varieties. 1,000 grain weight of origin variety, Bacthom 7 was from 18.5g to 19.2g
meanwhile that of DT39 Quelam was from 23.2g to 25.2g in three seasons. It may result that theyielding performance of DT39 Quelam is often higher than Bacthom 7 (table5).

- 85 -

Table4: Variety purity and other traits

Seasons

Variety(Source: NTCC)*

Purity(points)

No.panicle
per hill

No. Seeds
per panicle

Abort ratio(%)

Grain Weight of
1,000 seeds (g)Summer

BT7

1

5.2

141

17.6

18.5

2011

DT39 Quelam

1

5.0

147

22.0

23.2

Spring

BT7

1

5.2

152

13.0

18.6

2012

DT39 Quelam

5

4.9

148

16.6

24.8

Summer

BT7

1

5.6

139

11.9

19.2

2012

DT39 Quelam

3

5.6

145

22.5

25.2

*points: higher means better.

Table5: Yield performance evaluation in rice production trials (Source: NTCC)Unit: tons/hectare
Location
Season Variety

Thái

Thanh

Vĩnh

Hòa

Hà

Hải

Yên Dương

An

Bình

Hoá

Phúc

Bình

Tĩnh

Phòng Quang Giang Biên

5.47

5.18

-

3.79

4.28

5.07

-

4.81

6.24

4.99

-

-

4.98

5.67

5.39

-

3.83

4.13

5.59

-

5.15

6.60

5.59

-

-

5.23

Spring
2012

BT7

5.23

3.87

6.00

3.56

4.81

5.10

5.73

4.76

-

-

-

-

4.88

DT39
Quelam

5.47

4.24

5.57

3.41

5.67

6.13

5.63

5.23

-

-

-

-

5.17

5.79

5.74

-

3.90

4.83

3.47

4.97

-

-

-

5.77

5.37

4.98

6.06

6.00

-

5.03

5.44

4.60

4.67

-

-

-

6.73

6.20

5.59

BT7
Summer2012
DT39
Quelam

Hải

Tuyên

Bắc

Điện

Aver.

Nghệ

BT7
Summer2011
DT39
QuelamHưng

In three continuous seasons of the test for value of cultivation and use, the mutant variety gavehigher yield compare to Bacthom 7. In summer 2011, DT39 Quelam gained 5% compare to Bacthom
7. Especially at 6/8 location trail test in summer 2012, the yield of DT39 Quelam reached average
5.59 t/ha higher by 12.24% while that of Bacthom 7 was 4.98 t/ha.
For the rice production in northern part of Vietnam, Bacthom 7 variety is mainly grown in spring
season, because this variety often gives high yield, good quality and moreover but it is very sensitive
to bacterial leaf blight in summer season. Therefore, the cultivation area is significant decreased eventhe quality is accepted for consumption. The result from the NTCC indicated that new mutant variety
DT39 Quelam could be used for summer season.

- 86 -

Table6: Resistant ability for pests and diseases (Source: NTCC)

Seasons

Summer
2011

BT7
DT39
QuelamSpring
2012

BT7
DT39
QuelamSummer2012

Blast

SheathBacterial

Brown

Stem

Leaf

Brown

In leaf In panicle

blightLeaf blightblightborer

rollerhopperVariety

BT7
DT39
Quelam

-

-

0-1

3-5

-

1-3

0-1

0-1

-

-

0-1

1-3

-

0-1

1-3

1-3

0-1

-

1-3

0-1

0-1

0-1

0-1

0-1

0-2

-

1-3

0-1

0-1

0-1

0-1

1-3

1-3

0-1

1-3

1-3

1-3

1-3

1-3

1-3

1-2

0-1

1-3

1-3

0-1

0-1

1-3

1-3

*points: smaller means better.

Vietnam is tropical country, where pests and diseases appear every year. So, resistance to pests anddiseases in rice is a very important criterial standard for commercial production. The data from table
6 indicated that the resistance to pests and diseases in the field condition for blast, bacterial leaf
blight, brown blight, and stem borer of DT39 Quelam was similar to Bacthom 7. Especially, insummer season 2011, DT39 Quelam showed resistance to bacteria leaf blight at 1-3 point while
Bacthom 7 showed it at 3-5 point.
Table7: Grain quality evaluation of DT39 Quelam (Source: NTCC, 2012)

Variety

DT39
QuelamBT7

Milled rice
HuskedAmyloseRice ratio ofratio
rice length
Content (%)
length/width
(%)
(mm)

Protein
CollagenicGelcontent
temp.
Durability
(%)

71.25

17.82

6.47

3.09

(70 – 74 oC)

medium

9.10

69.40

15.65

5.59

2.83

(70 – 74 oC)

soft

8.21

Evaluation for the quality according to phenotype of milled and cooked rice indicated that DT39
Quelam had some advantage characters (table 7), and amylose content (17.82%) was higher than
Bacthom 7 (15.8%). In this case, the higher amylose content leads to the better quality because low
amylose content results in sticky cooked quality. In this experiment, after mutation induction protein
content was increased from 8.21% of Bacthom 7 to 9.10% in DT39 Quelam. The most importantcharacter of new mutant variety DT39 Quelam is that the cooked rice is soft, not so sticky, highprotein and with light aroma.
- 87 -

Table8: Testing for cooking quality of DT39 Quelam (Source: NTCC, 2012)

Variety

Aroma

Softness

Stickness

Whiteness

PolishAttractive

DT39 Quelam

1

4

4

5

3

3

Huongthom 1

2

3

4

5

3

2

Bacthom 7

2

4

4

5

3

3

*points:- Aroma: bigger means better; - Softness: smaller means harder- Stickiness: bigger means sticker ; - Attractive: bigger means better cooking

Test for cooking quality of DT39 Quelam was carried out by committee consisted of 7 persons who
worked independent evaluation. The result showed that in most indexes, DT39 Quelam was similar to
Bacthom 7 except for aroma which was lower in DT39 Quelam. Due to irradiation treatment, aroma
character of Bacthom 7 was decreased in DT39 Quelam.

Bacthom 7

DT 39 Quelam

Photos of DT39 Quelam in experiment and exhibition field

9.4 Conclusion
1) New rice variety DT39 Quelam was created by gamma ray irradiation of cobalt source at the doseof 200Gy from Bacthom 7.
2) Main characteristics of DT39 Quelam: Grow duration in spring season: 130-135 days, summer
season: 104-105 days; good tolerance to main pest and disease. Good cooking quality, high yield,
hard stem, high protein, better resistance to bacteria leaf blight and better cold tolerance comparedto the original variety.
- 88 -

3) DT39 Quelam has been planting in some provinces in northern part of Vietnam in more than 87 ha.
In most location of trial field, the mutant variety gave higher yield around 5-14% from the original
variety.

9.5 Acknowledgement
The authors wish to express sincerely thank to the IAEA and FNCA for its long time supports on ourrice mutation breeding programs and my attendance at the annually FNCA work shop 2009-2013. I
also wish to thank Dr. Atsushi Tanaka, Dr. Hirokazu Nakai and Ms. Aki Koike for their kind
hospitality and help me during the meeting.
9.6

References

Bang Dao ThiThanh et al., 2008, new rice variety, DT38 selected sucessfully by gamma ray
irradiation, poster at International Symposium on Induced Mutation in Plant organized by
FAO/IAEA 12-15 August, 2008-Vienna, Austria.
Bang Dao ThiThanh et al. 18-19 August 2011, Primary results of application of carbon ion beam andgamma ray for rice beeding improvement. The IX National symposium on Nuclear Scienceand Technology, Phanrang-Vietnam
ProccedingInternational Symposium on Induced Mutation in Plant, FAO/IAEA 12-15 August,
2008-Vienna, Austria
Recently progress of rice mutation breeding and germplasm in China. Plant breeding report, Vol.1
No.1; 2006 pag 4
THINH DO KHAC et al., 2008. Socio-Economic impacts of mutant rice varieties in Southern
Vietnam, reported at International Symposium on Induced Mutation in Plant organized by
FAO/IAEA 12-15 August, 2008-Vienna, Austria.
Yu Zengliang, Introduction to ion beam Biotechnology, 2006, P 183-199- 89 -

- 90 -