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

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

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

The impact of these changing conditions on agriculture are

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

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

The aims of this project are to

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

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

Plant height (cm)Height (cm)

No. of plants

121

20

-Dwarf40-54--Semidwarf

55-1002-Intermediate-tall

101-120

279

-Tall

121-155

218

RD31
M 2 of RD31

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

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

80

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

Panicle/hill
Number of panicles

Number of plants

RD31
M 2 of RD31

10
<5

0

5-9

176

10-15

323

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

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

Maturity (days)
Days to maturity

Number of plants

123

20

110-115333116-120

166

121

1

M 2 of RD31

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

81

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

Number of plants

1

-

3

-

5

95

7

222

9

2,680

Figure 3. Submergence tolerant mutants lines from field screening.

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

Site 1 (PCR)

Site 2 (KHS)

0

305

317

1

11

-

2

1

-

7

-

-

9

-

-

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

Classification

No. of plants

10-19

Low amylose

-

20-25

Intermediate amylose

16

26-34

High amylose

301

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

Description

No. of plants

0

None

-

1

Small (less than 10%)158

2Medium (11-20%)

147

3Medium (11-20%)

149

4

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

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

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

Mat.

Ht.

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

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

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

84

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

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

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

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

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

Bl'PCR
2018

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

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

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

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

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

Description

Scale

1

Minor visible symptom of injury

100

3

Some visible symptom of injury

95-99

5

Moderate injury

75-94

7

Sever injury

50-54

9

Partial to complete death

0-49

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

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

Disease severity

Host response

0

No lesion observed

Highly Resistant

1

Small brown specks of pin point size

Resistant2

Small roundish to slightly elongated, necrotic gray spots,

Moderately

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

Resistant

Lesion are mostly found on the lower leaves
3
4
56

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

Moderately

on the upper leaves

Resistant

Typical susceptible blast lesions; 3 mm or longer infecting

Moderately

less than 4% of leaf area.

Susceptible

Typical susceptible blast lesions of 3 mm or longer infecting

Moderately

4- 10% of the leaf area.

Susceptible

Typical susceptible blast lesions of 3 mm or longer infecting

Susceptible

11-25% of the leaf area.
7

Typical susceptible blast lesions of 3 mm or longer infecting

Susceptible

26-50% of the leaf area.
8

Typical susceptible blast lesions of 3 mm or longer infecting

Highly Susceptible

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

Typical susceptible blast lesion of 3 mm or longer infecting

Highly Susceptible

more than 75% leaf area affected
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88