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 andHitomebore.
(3) Evaluation of amylose mutants
Cultivate under various conditions to evaluate the phenotype of the amylose library created in theproject.
Table 1. Specific research methods and other actions taken throughout the research term

Year2006
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 HitomeboreHitomebore 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),
12C6+ (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 byHitomebore.

Amylosecontentsofmutants

andNILs

weredeterminedbyabsorption

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 otherhand, 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 -