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 -