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 recentyears, 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.

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

YearHybrids 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 tothe 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 3rdgrade 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 grade

Mean% 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

1758.6

EI (S)

48.9

44.4

14.1

8.3

20.6

45.8

00.0

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 stableand 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 withZhong 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 3Awith 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 inHangzhou. 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, JAEAlines (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. Inaddition 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 percentageof 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 (%)

Percentageof ripening(%)

2weeks after
germination

3weeks after
germination

87.25

63.09

50.34

30.33

100.0074.20

1083.39

63.76

47.65

29.67

97.85

73.30

20

76.16

51.6635.10

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

158.0625.7120 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 (%)

806040206040

20000204060

80

1000120

2040

100

10080

80

Percentage of ripening (%)

Growth rate (%)

60

8010012080

100120

Dose (Gy)Dose (Gy)

60

40

2060

40

200002040

60

801000120

2040

Dose (Gy)60
Dose (Gy)Figure 4. Z17 Dose-response curve for germination rate, survival rate,
growth rate and percentage of ripening .
100100Survival rate

8080Survival Rate(%)

Germination and survival rate (%)

Germination rate

604060402020003040050

20406080

100120

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’sexperience. 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 onHybrid 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 LineZhong 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.

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