VI. RNC ACTIVITIESRIKEN Accel. Prog. Rep. 54 (2021)

Accelerator Applications Research Division
Beam Mutagenesis GroupIon Beam Breeding Team
1. Abstract

Ion beam breeding team studies various biological eﬀects of fast heavy ions. It also develops new technique to breed plants andmicrobes by heavy-ion irradiations. Fast heavy ions can produce dense and localized ionizations in matters along their tracks, incontrast to photons (X rays and gamma rays) which produce randomly distributed isolated ionizations. These localized and dense ionization can cause double-strand breaks of DNA which are not easily repaired and result in mutation more eﬀectively than singlestrand
breaks. A unique feature of our experimental facility at the RIKEN Ring Cyclotron (RRC) is that we can irradiate living tissues inatmosphere since the delivered heavy-ion beams have energies high enough to penetrate deep in matter. This team utilizes a dedicatedbeam line (E5B) of the RRC to irradiate microbes, plants and animals with beams ranging from carbon to iron. Its research subjects
cover study of ion-beam radiation mutagenesis, genome-wide analyses of mutation, and development of new plants and microbial
varieties by heavy-ion irradiation. Thirty-five new varieties have already been brought to the market.

2. Major Research Subjects(1) Study on the biological eﬀects by heavy-ion irradiation(2) Study on the molecular nature of DNA alterations induced by heavy-ion irradiation(3) Innovative applications of heavy-ion beams

3. Summary of Research Activity

We study biological eﬀects of fast heavy ions from the RRC using 135 A MeV C, N, Ne ions, 95 A MeV Ar ions, 90 A MeV Feions and from the IRC using 160 A MeV Ar ions. We also develop breeding technology of microbes and plants. Main subjects are:(1) Study on the biological eﬀects by heavy-ion irradiation
Heavy-ion beam deposits a concentrated amount of dose at just before stop with severely changing the linear energy transfer(LET). The peak of LET is achieved at the stopping point and known at the Bragg peak (BP). Adjusting the BP to target malignant
cells is well known to be eﬀective for cancer therapy. On the other hand, a uniform dose distribution is a key to the systematic study
for heavy-ion mutagenesis, thus to the improvement of the mutation eﬃciency. Plants and microbes therefore, are irradiated using ionswith stable LET. We investigated the eﬀect of LET ranging from 23 to 640 keV/µm, on mutation induction using dry seeds of the model
plants Arabidopsis thaliana and rice (Oryza sativa L.). The most eﬀective LET (LETmax) was 30 keV/µm in Arabidopsis. LETmax
irradiations showed the same mutation rate as that by chemical mutagens, which typically cause high mutation rate. The LETmax
was 23–39 keV/µm in buckwheat, 23–50 keV/µm in rice and 50–70 keV/µm in wheat. By contrast, when LET was 290 keV/µm, themutation rate was low and the survival rate was greatly reduced in plants. In the case of microbe, filamentous fungus (Neurosporacrassa), the Ar ions at 290 keV/µm demonstrated higher mutagenic activity than the Fe-ions at 640 keV/µm. Thus, the LET is an
important factor to be considered in heavy-ion mutagenesis.
(2) Study on the molecular nature of DNA alterations induced by heavy-ion irradiation
A whole-genome analysis with high-throughput sequencing is a powerful tool used to characterize the nature of induced mutations.
We have been using whole genome sequencing to analyze DNA mutations in Arabidopsis and rice genomes. C ions with LETmax
mainly induced single nucleotide variants (SNVs) and small insertions and deletions (InDels), while the number of large deletions
and chromosomal rearrangements was low. However, 290-keV/µm Ar ions showed a diﬀerent mutation spectrum: SNVs and numberof small InDels was low, while the number of large deletions (≧100 bp) and chromosomal rearrangements was high. Number ofmutated gene induced by C-ion and Ar-ion irradiation is less than 10, relatively small, and often only 1 mutation is found near themapped location. Thus, irradiation with these ions can eﬃciently generate knockout mutants of a target gene and can be applied
to reverse genetics. Mutants of the causative gene of Arabidopsis induced by ion beam irradiation were compared at 30 keV/µm
and 290 keV/µm with two typical LETs. The most mutations irradiated with C ion at 30 keV/µm were small deletions (<100 bp).
Irradiation with 290-keV/µm C-ion and Ar-ion resulted in the most large-deletions and chromosomal rearrangements, and decreased
small deletions.
(3) Innovative application of heavy-ion beams
In 1999, we formed a consortium for ion-beam breeding consisting of 24 groups. In 2020, the consortium grew to 184 groups
from Japan and 20 from overseas. Previously, the ion-beam breeding procedures were carried out using mainly flowers and ornamental
plants. We have recently put a new non-pungent and tearless onion, ‘Smile ball,’ on the market along with ‘Kiku Meigetsu,’ anedible late flowering chrysanthemum. In addition, a new project was launched to expand the cultivation area of this variety ofchrysanthemum in Yamagata prefecture. Beneficial variants have been grown for various plant species, such as high yield sea weeds,
lipids hyperaccumulating unicellular alga, medicinal plant with high productivity of medicinal ingredient, peanuts without major
allergens, oranges with delayed coloring and one-month late harvest, and lettuce with a low browning property as a cut vegetable. As
a result of a collaborative study with the University of Hasanuddin, we have selected five useful aromatic rice mutants and increased
the percentage of fertile grains from 32% control to over 52%. By broadening the target of heavy-ion breeding extending from flowers- 288 -VI. RNC ACTIVITIESRIKEN Accel. Prog. Rep. 54 (2021)

to crops such as grains, the technology will contribute to solving the global problems of food shortage and environmental destruction.

Members
Team Leader
Tomoko ABE
Research/Technical Scientists
Tokihiro IKEDA (Senior Research Scientist)
Masako IZUMI (Senior Research Scientist)
Teruyo TSUKADA (Senior Research Scientist)

Kazuhide TSUNEIZUMI (Senior Research Scientist)
Ryouhei MORITA (Technical Scientist)

Research & Development Scientists
Hiroyuki ICHIDA
Technical Staﬀ I
Yoriko HAYASHI

Yuki SHIRAKAWA

Technical Staﬀ II
Sumie OHBU

Mieko YAMADASpecial Temporary Technical Scientists
Hiroshi ABE

Katsunori ICHINOSE

Research Consultant
Masahiro MII (Chiba Univ.)
Visiting Scientists
Ayumi DEGUCHI (Chiba Univ.)
Makoto FUJIWARA (Sophia Univ.)
Eitaro FUKATSU (Forestry and Forest Products Res. Inst.)
Tomonari HIRANO (Univ. of Miyazaki)
Akiko HOKURA (Tokyo Denki Univ.)
Yutaka MIYAZAWA (Yamagata Univ.)
Kazumitsu MIYOSHI (Chiba Univ.)
Toshikazu MORISHITA (Nat’l Agriculture and Food Res.
Org.)
Koji MURAI (Fukui Prefectural Univ.)

Kyosuke NIWA (Tokyo Univ. of Marine Sci. and Tech.)
Norihiro OHTSUBO (Kyoto Prefectural Univ.)
Tadashi SATO (Tohoku Univ.)
Yoichi SATO (Riken Food Co., Ltd.)
Kenichi SUZUKI (Suntory Flowers Co., Ltd.)
Hinako TAKEHISA (Nat’l Agriculture and Food Res. Org.)
Masanori TOMITA (Central Res. Inst. of Electric Power Industry)
Masao WATANABE (Tohoku Univ.)
Takeshi YAMAKI (Riken Vitamin Co., Ltd.)

Visiting Technicians
Yukiko KANAZAWA (Nippon Beet Sugar Manufacturing Co.,Ltd.)Norihide KISHINO (Sanwa Norin Co. Ltd.)

Miho MOGAMIYA (Riken Food Co., Ltd.)
Daisuke SAITO (Riken Food Co., Ltd.)
Takuji YOSHIDA (Takii & Co., Ltd)

Research Fellow
Hironari UCHIDA (Saitama Agricultural Tech. Res. Center)

List of Publications & Presentations
Publications[Original Papers]
T. Hirano, Y. Sato, K. Ichinose, M. Yamada, Y. Hayashi, N. Fukunishi, and T. Abe, “Mutant induction in gametophytes of Undariapinnatifida (Phaeophyceae) by heavy ion beam irradiation,” Phycol. Res. 68, 63–69 (2019).
N. Fukuda, N. Kitajim, Y. Terada, T. Abe, I. Nakai, and A. Hokura, “Visible cellular distribution of cadmium and zinc in the hyperaccumulator Arabidopsis halleri ssp. gemmifera determined by 2-D X-ray fluorescence imaging using high-energy synchrotron radiation,”
Metallomics 12, 193–203 (2019).
Y. Oono, H. Ichida, R. Morita, S. Nozawa, K. Satoh, A. Shimizu, T. Abe, H. Kato, and Y. Hase, “Genome sequencing of ion-beam-induced
mutants facilitates detection of candidate genes responsible for phenotypes of mutants in rice,” Mutat. Res. Fund. Mol. Mech. Mutagen.
(2020).
R. Tabassum, T. Dosaka, H. Ichida, R. Morita, Y. Ding, T. Abe, and T. Katsube-Tanaka, “FLOURY ENDOSPERM11-2 encodes plastid
HSP70-2 involved with the temperature-dependent chalkiness of rice (Oryza sativa L.) grains,” Plant J. 103, 604–616 (2020).
K. Kaya, Y. Kazama, T. Abe, and F. Shiraishi, “Influence of medium components and pH on the production of odd-carbon fatty acids by
Aurantiochytrium sp. SA-96,” J. Appl. Phycol. 32, 1597–1606 (2020).

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S. Muramatsu, K. Atsuji, K. Yamada, K. Ozasa, H. Suzuki, T. Takeuchi, Y. Hashimoto-Marukawa, Y. Kazama, T. Abe, K. Suzuki, andO. Iwata, “Isolation and characterization of a motility-defective mutant of Euglena gracilis,” Peer J 8, e10002 (2020).
H. Murata, S. Nakano, T. Yamanaka, T. Shimokawa, T. Abe, H. Ichida, Y. Hayashi, and K. Tahara, “Argon-ion beam induced mutantsof the ectomycorrhizal agaricomycete Tricholoma matsutake defective in β-1,4-endoglucanase activity promote the seedling growth ofPinus densiflora in vitro,” Botany, 99(3), DOI:10.1139/cjb-2020-0076 (2020).

[Proceedings]
A. M. Okasa, M. Riadi, K. Toriyama, K. Ishii, Y. Hayashi, T. Sato, T. Abe, Trisnawaty, N. J. Panga, and R. Sjahril, “Mutation breeding
for improvement of aromatic rice mutant by using ion beam irradiation,” IOP Conf. Ser. Earth Environ. Sci. 486, 012091 (2020).

Presentations[International Conferences/Workshops]
T. Kashiwabara, N. Kitajima, R. Onuma, N. Fukuda, S. Endo, Y. Terada, T. Abe, A. Hokura, and I. Nakai, “Synchrotron micro-X-ray
fluorescence imaging of arsenic in frozen-hydrated sections of a root of Pteris vittate,” Goldschmidt2020: Global Virtual Conference,
online, June 21–26, (2020).
T. Abe (invited), “Development for ion-beam breeding technology over the last two decades in Japan,” Virtual Meeting on FAO/IAEA
Mutation Breeding Network (MBN) for Asia Pacific Region, online, November 12–13, (2020).

[Domestic Conferences/Workshops]
常泉和秀, 山田美恵子, 一瀬勝紀, 市田裕之, 金禧珍, 萩原篤志, 川田実季, 片山貴士, 﨑山一孝, 手塚信弘, 小磯雅彦, 阿部知子, 「シオミズツボワムシの高増殖・大型変異系統の樹立」, 日本農芸化学会 2020 年度大会, 福岡県福岡市 (九州大学), 2020 年 3 月 26 日.
市田裕之, 阿部知子, 「オープンソースな変異タイピング手法の開発とイネをモデルとした概念実証」, 日本育種学会 第 137 回講演会, 東京都文京区 (東京大学), 2020 年 3 月 28 日.
渡邊遙, 大部澄江, 阿部知子, 風間裕介, 「ゲノム編集によるシロイヌナズナへの 758 kb の逆位の導入」, 同上.
A. M. Okasa, M. Riadi, 佐藤雅志, 鳥山欽哉, 石井公太郎, 林依子, 阿部知子, R. Sjahril, “An approach toward isolating early-heading
mutants from Tana Toraja local aromatic rice ‘Pare Bau’ irradiated with heavy ion-beam,” 同上.
橋本佳澄, 西浦愛子, 上田純平, 風間裕介, 阿部知子, 市田裕之, 村井耕二, 「2 倍体ヒトツブコムギにおける重イオンビーム照射による超極早生変異体 extra early-flowering 4 の原因遺伝子の同定」, 同上.
森田竜平, 市田裕之, 石井公太郎, 林依子, 安部弘, 白川侑希, 一瀬勝紀, 常泉和秀, 風間智彦, 鳥山欽哉, 佐藤雅志, 阿部知子, 「イネ長粒変異体 lin1 の単離および原因遺伝子同定」, 同上.
石井公太郎, 大部澄江, 白川侑希, 阿部知子, 「全ゲノム変異解析による重イオンビームの高頻度な変異誘発線量区の推定」, 同上.
村井耕二, 風間裕介, 阿部知子, 「2 倍体ヒトツブコムギにおける重イオンビーム突然変異体作出のための最適処理条件」, 同上.
Y. Ding(口頭発表), T. Katsube-Tanaka, H. Ichida, R. Morita, and T. Abe, “Studies on chalkiness of the rice cultivar ‘Kinmaze’under heatstress,” 日本作物学会第 250 回講演会, オンライン開催, 2020 年 9 月 3–4 日.
蝶野真喜子 (ポスター発表), 藤田雅也, 神山紀子, 松中仁, 氷見英子, 市田裕之, 阿部知子, 川上直人, 「粒の赤みが弱い新規コムギ変異体の農業特性」, 日本育種学会 第 138 回講演会, オンライン開催, 2020 年 10 月 10–11 日.
藤田悠生 (ポスター発表), 市田裕之, 風間智彦, 阿部知子, 鳥山欽哉, 「インディカイネ品種 Lebed に由来する雄性不稔遺伝子とその抑制遺伝子の解析」, 同上.
石井公太郎 (口頭発表), 風間裕介, 浅野円花, 阿部知子, 河野重行, 「Ar・Fe イオンビーム照射によって生じるクロレラ染色体の断片化と染色体再編成」, 同上.
南壮次郎 (口頭発表), 渡邊遥, 大部澄江, 阿部知子, 風間裕介, 「ゲノム編集を用いたシロイヌナズナへの染色体再編成の導入」, 同上.
野村文希 (ポスター発表), 風間裕介, 阿部知子, 村井耕二, 「イオンビーム照射により作出された Ppd-1 欠失『農林 61 号』変異系統の解析」, 第 15 回ムギ類研究会, オンライン開催, 2020 年 12 月 26 日.
橋本佳澄 (ポスター発表), 西浦愛子, 風間裕介, 市田裕之, 阿部知子, 村井耕二, 「重イオンビーム照射によって作出された超極早生
コムギ変異体 extra early-flowering 4（exe4）の花成関連遺伝子の発現解析」, 同上.

Awards
佐藤陽一, 斎藤大輔, 最上谷美穂, 令和 2 年度（第 21 回）民間部門農林水産研究開発功績者表彰（農林水産技術会議会長賞民間部門）, 「わかめ養殖種苗の優良系統開発と生産条件最適化による普及実用化」, 農林水産会議, 2020 年 11 月 12 日.

Patents
石井重久, 阿部知子, 林依子, サクラ「真和」, 品種登録出願番号 34356, 出願日 2019 年 11 月 27 日.
石井重久, 阿部知子, 林依子, サクラ「真理」, 品種登録出願番号 34356, 出願日 2019 年 11 月 27 日.
石井重久, 阿部知子, 林依子, サクラ「真星」, 品種登録出願番号 34356, 出願日 2020 年 4 月 14 日.

Outreach Activities
We established the “Asagao (morning glory) club” to deepen the understanding of our technology of mutation breeding. The club
distributes the morning glory seeds irradiated with C-ion on request, and collects and compiles the observation reports of their growth.

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