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 at200Gy 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 ofM3 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 ofPlant 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

aa

200 Gy300 Gya75.00b70.0065.00
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

abcc

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 -23.00

1000 seed-wt

aab

22.00

ab

21.00
20.00

b19.0018.00
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

toLengthofGrains/

Filled Grains/

flowerpanicle (cm)

Paniclepanicle

Check

93

23.20

124

78

Control

97

22.05

12190

200 Gy90

24.60

121

93

300 Gy

90

23.00

11691

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 toNo. with HighanalyzedIntermediate Amylose

AmyloseCheck

10010Control

10010200 Gy

15312141300 GY1271126

Treatment

3020300Gy
1200Gy1215Control

25Check

10501234567891011121314151617Cont…
Check% Amylose Content

35Samples

Figure 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

Check6.00

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 inTable 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 previousstudies, (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 perpanicle 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.atDays toNumberPanicle

Seed/Filled1000Maturity

flowerof tillers/

LengthpanicleGrains/seedPlant(cm)

paniclewt. (g)(cm)
Control

72.11 c

97

16 b

22.05

12190

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
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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.

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