Ⅰ. HIGHLIGHTS OF THE YEARRIKEN Accel. Prog. Rep. 50 (2017)

Optimization of mutation induction by heavy-ion beam irradiationin rice with seed water content change
Y. Hayashi,21 K. Ichinose,21 Y. Shirakawa,21 S. Usuda,21 R. Morita,21 H. Tokairin,21 T. Sato21,22 and T. Abe21
Radio sensitivity to X-rays and γ-rays are minimal atintermediate water contents in seeds of rice, 1), 2) wheat andbarley.3) It increases rapidly above and below this range. Incontrast, the seed water content has little or no effect on thesensitivity to fission neutron irradiation.2) Our previous
study revealed that the radio sensitivity to C-ion beam wasaffected by the water content of seeds as in the cases ofX-rays and γ-rays in rice.4) In this report, we investigatedthe effect of seed water content on mutation rate by C-ion
irradiation and estimated the efficient irradiation condition
for optimized mutagenesis in rice.
The dry seeds of rice (Oryza sativa L. cv. Nipponbare)
were equilibrated to 9% and 13% water contents throughstorage at constant humidity. Water-content-adjusted seedswere vacuum-packed in plastic bags for irradiation
treatment (Fig. 1). The dose ranges of the C-ion beam weredetermined according to our previous study4) as 12.5 to 50
Gy and 100 to 200 Gy for 9% and 13% water content,respectively. The liner energy transfer (LET) values were22.5, 30, and 50 keV/μm at each irradiation dose. M1 plants
were grown in a paddy field and M2 seeds were obtained
separately from each M1 plant. The number of surviving M1
plants after each treatment was counted to estimate survival
rates. The spikelet fertility was evaluated using the numberof fertile spikelets in the main panicle of the M1 plant. Table
1 lists the percentage of low-fertility M1 plants that haveless than fifty fertile spikelets in a panicle. The mean valueof the number of fertile spikelets per panicle in control
Nipponbare was 106.2 ± 12.2. Chlorophyll-deficientmutants (CDM) were observed in two-weeks-old M2
seedlings grown in a greenhouse. Mutation rates werecalculated based on the numbers of M1 lines, which showed
CDM in M2 generation.

water content without the extreme reduction of bothsurvival rate and spikelet fertility. There was no difference
between seeds with 9 % and 13% water content in theresponses to LET. Irradiations with 50 keV/μm were more
effective than the same doses of irradiation with 22.5 or 30
keV/μm on the survival rate and spikelet fertility. As aresult, we determined that the optimum condition of C-ion
irradiation for mutation induction was 175 Gy (30 keV/μm)
on seeds with 13% water content. In general, rice seeds arerecommended to be stored under cold and dry conditions tomaintain its activity. Therefore, the water content after
long-term storage tends to be less than 9%. It isindispensable to measure the water content of seed before
irradiation to set an appropriate condition in response to its
water content. To induce mutation with high probability, we
adjust the water content to 13% by keeping the seeds in athermo-hygrostat for a week under a temperature of 22℃
and humidity of 70%.
Table 1. Effect of C-ion beam irradiation on seeds with 9%
and 13% water content.
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Fig.1 Rice seeds set into a cassette of automatic sample
changer in our irradiation system.5) Each bag contains
three hundreds seeds of Nipponbare.
 The results of survival rate, spikelet fertility, andmutation rate are listed in Table 1. In the irradiation on
seeds with 9% water content, a higher frequency of CDM
was obtained by high dose irradiation, which resulted in alow survival rate. In contrast, the highest frequency of
CDM was obtained by the irradiation on seeds with 13%2122(+!(',!',

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References
1) C. Myttenaere et al., Radiat. Bot. 5, 443 (1965).
2) M. J. Conatantin et al., Radiat. Bot. 10, 539 (1970).
3) R. Biebi et al., Radiat. Bot. 5, 1 (1965).
4) Y. Hayashi et al., RIKEN Accel. Prog. Rep. 45, 214 (2012).
5) H. Ryutoet al., RIKEN Accel. Prog. Rep. 39, 245 (2006).

RIKEN Nishina Center
Graduate School of Agricultural Science, Tohoku University- 27 -