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3-14 Production of Soybean Mutants with Pale-Green-Leaf Phenotype by Ion Beam Irradiation S. Arase a) , J. Abe a) , S. Nozawa b) , Y. Hase b) , I. Narumi b) and A. Kanazawa a) a) Research Faculty of Agriculture, Hokkaido University, b) Radiation-Applied Biology Division, QuBS, JAEA Soybean (Glycine max) is an important crop in terms of production of food, oil, and forage. However, existing mutant lines of soybean is very limited, which is a constraint on performing a genetic study and breeding of this plant. Soybean is considered to have derived from ancestral plant(s) that have a tetraploid genome, and as a consequence, more than 90% of nucleotide sequence in the soybean 1) genome is duplicated . It is conceivable that such a duplicated nature of the genome brought about a low frequency of mutant production by conventional methods for mutagenesis such as γ-ray or X-ray irradiation as well as chemical treatments. In these circumstances, we have started to examine whether ion beam irradiation is effective in producing a mutant in soybean because ion beam irradiation is expected to cause genomic changes that are more drastic than those induced by conventional mutagenesis. We have previously analyzed the effects of ion beam irradiation on plant growth and morphology in soybean by exposing dried seeds to the 320 MeV carbon ions with the range of 0.2 – 25 Gy 2) . The irradiated seeds were sown on soil and plants were grown for three weeks in a greenhouse. We found that irradiation doses higher than approximately 5 Gy affected plant growth rate. In order to establish a plant population that can be available for screening mutants, we further examined the effects of irradiation by growing plants in a field. We found that both plant height and the ratio of the number of plants that survived until seed-setting per the number of seeds sown in the field depended on the doses of irradiation. We tentatively concluded that irradiation doses around 2.5 Gy are suitable for producing mutants 3) . Based on these results we grew plants from irradiated seeds in a large scale to obtain a population of M2 seeds: 3,200 seeds and 3,320 seeds irradiated at 5 Gy and 2.5 Gy, respectively, were sown in the field and seeds were harvested. The harvested M2 seeds were sown in the field next year and generation of individuals with visibly altered 4) phenotypes was examined . We detected plants with a visibly altered phenotype. The observed change was a chlorophyll deficiency, as evidenced by pale-green leaves (Fig. 1). Frequency of this type of change was 0.10% (n = 1,911) and 0.19% (n = 1,038) for M2 plants irradiated with 2.5 Gy and 5.0 Gy, respectively. We confirmed that this phenotypic change was heritable by growing the progeny of M2 plants with the altered phenotype. The M3 progeny of four out of six M2 plants maintained the altered phenotype (Fig. 1; the progeny of JAEA-Review 2010-065 - 70 - ‘D-2,’ ‘B-1,’ ‘B-2,’ and ‘C-2’ plants). Thus, at least these four M 2 plants had stable heritable change(s) that caused the altered phenotype, demonstrating that ion-beam irradiation at 2.5–5.0 Gy can induce mutation. The frequency of the chlorophyll-deficient phenotype was comparable to that observed in other plants. For example, the frequency of the mutation in petunia was 0.4% when irradiated at half the dose that caused a decrease in survival rate but which generated various mutants 5) . Thus, we consider that the irradiation conditions, including doses, to obtain mutants for use as breeding materials were effectively optimized in our study. References 1) R. C. Shoemaker et al., Genetics 144 (1996) 329. 2) A. Kanazawa et al., JAEA Takasaki Ann. Rep. 2006 (2008) 88. 3) A. Kanazawa et al., JAEA Takasaki Ann. Rep. 2007 (2008) 71. 4) A. Kanazawa et al., JAEA Takasaki Ann. Rep. 2008 (2009) 68. 5) Y. Hase et al., Plant Biotechnol. 27 (2010) 99. M3 generation Fig. 1 Inheritance of the pale-green-leaf phenotype. Four progeny plants from each M 2 plant are shown (except D-2, for which one progeny plant is shown): from left to right, non-irradiated control, progeny of M 2 plant with no visible change, progenies of D-2, B-1, B-2, and C-2 plants.
3-15 JAEA-Review 2010-065 Induction of Fusarium Wilt Resistant by Ion Beam Irradiation in Strawberry(Fragaria × ananassa) Leaf Explants M. Yotoriyama a) , J. Takano a) , K. Namai a) , R. Yoshihara b) , S. Nozawa b) , Y. Hase b) and I. Narumi b) a) Tochigi Prefectural Agricultural Experiment Station, b) Radiation-Applied Biology Division, QuBS, JAEA In order to obtain Fusarium wilt resistant mutant in strawberry cultivar “Tochiotome”, 1,516 leaf explants were irradiated with 320-MeV carbon ion beams. Regeneration rate was in the range of 11.7 - 15.3%. Three mutants that showed stronger resistance to Fusarium wilt were selected from 1.0 Gy and 2.5 Gy irradiated group. 栃木県が育成したイチゴ品種「とちおとめ」は、作付面積が全国一であり、優れた果実品質を有するが、 1) 最重要病害の萎黄病に罹病性であるため、生産者から耐病性品種の育成が強く望まれている。イオンビームによる変異誘発は、原品種の特性を維持しつつ、一部の特性を改良するのに適している。そこで本研究では、イオンビームを用いた「とちおとめ」の萎黄病耐病性個体を作出することを目的とした。イオンビームは日本原子力研究開発機構、イオン照射施設(TIARA)のAVFサイクロトロンを用いて、炭素イオン( 12 C 6+ , 加速エネルギー 320 MeV)を0.5, 1.0, 2.5 Gyの線量で照射した。とちおとめ無菌培養苗の葉片を約5 mm角に調製し、 TDZ 1.0 mg/L、2,4-D 0.1 mg/Lおよびブドウ糖18 g/Lを添加した1/3MS改変培地(前培養培地)で1日間液体培養した。その後、前培養培地に0.8%寒天を添加した培地(カルス誘導培地、60 mm径シャーレ)に置床し、イオンビーム照射まで4日間培養した。照射後、同様なカルス誘導培地(90 mm径シャーレ)で約1ヵ月間培養し、TDZ 1.0 mg/Lおよびブドウ糖18 g/Lを添加したMS 培地に移植して再分化を誘導した。その後、発根誘導培地(ショ糖30 g/L、寒天0.8%を含むMS培地)に移植し、約2ヵ月間培養した。発根した苗はバーミキュライトを含む50穴セルトレイで1~2ヵ月間順化した。培養条件は、カルス誘導時の光強度が7 mol m -2 s -1 、再分化誘導培養以降は40 mol m -2 s -1 とし14時間日長、25 ºCで管理した。再分化率は、再分化誘導1ヵ月後に調査した。萎黄病菌(FOF-10S菌株)はマングビーン培地で室温、110 rpm、10日間振とう培養後、滅菌二重ガーゼでろ過した。得られた胞子様菌体は、滅菌蒸留水で 2.2 × 10 6 による結果、対照の多芽体由来「とちおとめ」に比較して発病程度の軽かった1.0 Gy照射区の2個体、2.5 Gy 照射区の1個体の計3個体を選抜した(Table 1)。 Reference 1) Y. Ishihara et al., 栃木県農業試験場研究報告 44 (1996) 109. Fig. 1 Relationship between regeneration rate and ion beam irradiation. Table 1 Degree of disease resistance to Fusarium wilt in regenerated plants derived from irradiated Tochiotome leaves. bud cells/mLに調製し、1株当たり15 mLずつ灌注接種した。萎黄病菌接種苗は、人工気象器内で28 ºC、 14時間日長で管理した。接種77日後に発病程度(0:病徴無し、1:小葉のわずかな奇形、2:小葉に奇形・黄 No treatment 0 Gy 0.5 Gy 1 Gy 0 Degree of disease resistance to Fusarium wilt 1 2 3 4 5 Total 2 7 9 1 12 13 16 30 46 2 14 7 23 化、3:軽度の萎縮、4:萎縮・萎凋、5:枯死)を調査 2.5 Gy 1 14 26 41 した。なお、対照に多芽体由来の「とちおとめ」およ Control* び「アスカウェイブ(萎黄病抵抗性)」順化苗を用いた。 Tochiotome 4 13 17 16 本年度、萎黄病耐病性検定まで終了した供試葉片は Asuka wave 6 6 非照射(312葉片)まで含めると1,828葉片であった。 *: Growing points of runners were treated as a control 全照射区における再分化率は、11.7~15.3% (平均値は of disease resistance. 13.7%)であった(Fig. 1)。また、萎黄病耐病性検定 ☐: Selected plants. - 71 -
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JAEA-Review 2010-065 JAEA Takasaki
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JAEA-Review 2010-065 PREFACE This r
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JAEA-Review 2010-065 and pulsed hea
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JAEA-Review 2010-065 1-23 Studies o
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JAEA-Review 2010-065 3-24 Lethal Ef
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JAEA-Review 2010-065 4-07 Fabricati
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JAEA-Review 2010-065 5. Status of I
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JAEA-Review 2010-065 1-13 Alpha-rad
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1-01 Radiation Resistance of InGaP/
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1-03 Evaluation of Soft Error Rates
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1-05 Heavy-ion Induced Current in S
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Total Ionizing Dose Tolerance of Si
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1-09 Evaluation of Single Event Eff
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Hydrogen Diffusion in a-Si:H Thin F
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1-13 Alpha-radiolysis of Organic Ex
Page 35 and 36: Study on Stability of Cs·Sr Solven
Page 37 and 38: Irradiation Effect of Gamma-Rays on
Page 39 and 40: 1-19 Development of Radiation Resis
Page 41 and 42: 1-21 Alpha-Ray Irradiation Damage o
Page 43 and 44: 1-23 Studies on Microstructure and
Page 45 and 46: 1-25 Effect of Groundwater Radiolys
Page 47 and 48: 1-27 Simulation of Neutron Damage M
Page 49 and 50: 1-29 Irradiation Hardening in Ion-i
Page 51 and 52: 1-31 Preparation of Anion-Exchange
Page 53 and 54: 1-33 Radiation-Induced Graft Polyme
Page 55: JAEA-Review 2010-065 2. Environment
Page 58 and 59: 2-02 Development of Zwitterionic Mo
Page 60 and 61: Modification of Hydroxypropyl Cellu
Page 62 and 63: The Recovery of Precious Metals Usi
Page 64 and 65: 2-08 ESR Study on Carboxymethyl Chi
Page 67 and 68: JAEA-Review 2010-065 3. Biotechnolo
Page 69 and 70: JAEA-Review 2010-065 3-28 Electron
Page 71: JAEA-Review 2010-065 3-51 PET Studi
Page 74 and 75: 3-02 Mutagenic effects of He ion pa
Page 76 and 77: 3-04 Functional Analysis of Flavono
Page 78 and 79: 3-06 Generation New Ornamental Plan
Page 80 and 81: 3-08 Stability of Flower-colour Mut
Page 82 and 83: 3-10 JAEA-Review 2010-065 Effects o
Page 84 and 85: 3-12 Producing New Gene Resources i
Page 88 and 89: Assessment of Irradiation Treatment
Page 90 and 91: 3-18 Effect of Different LET Radiat
Page 92 and 93: 3-20 JAEA-Review 2010-065 Fungicide
Page 94 and 95: 3-22 JAEA-Review 2010-065 FACS-base
Page 96 and 97: 3-24 Lethal Effects of Different LE
Page 98 and 99: 3-26 JAEA-Review 2010-065 Ion Beam
Page 100 and 101: 3-28 Electron Spin Relaxation Behav
Page 102 and 103: 3-30 Target Irradiation of Individu
Page 104 and 105: 3-32 Carbon-ion Microbeam Induces B
Page 106 and 107: 3-34 Biological Effects of Carbon I
Page 108 and 109: 3-36 Difference in Bystander Lethal
Page 110 and 111: 3-38 Analysis of Lethal Effect Medi
Page 112 and 113: 3-40 Irradiation with Carbon Ion Be
Page 114 and 115: 3-42 Expression of Two Gelsolins in
Page 116 and 117: 3-44 Analysis of Bystander Cell Sig
Page 118 and 119: 3-46 Quantitative Evaluation of Ric
Page 120 and 121: 3-48 Visualization of 107 Cd Accumu
Page 122 and 123: 3-50 Uniformity Measurement of Newl
Page 124 and 125: 3-52 Imaging and Biodistribution of
Page 126 and 127: 3-54 Improvement of Spatial Resolut
Page 128 and 129: 3-56 The Optimum Conditions in the
Page 130 and 131: 3-58 Evaluation of Cisplatin Concen
Page 132 and 133: 3-60 JAEA-Review 2010-065 Analysis
Page 134 and 135: 3-62 JAEA-Review 2010-065 Sensitivi
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JAEA-Review 2010-065 4-14 The Effec
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JAEA-Review 2010-065 4-39 Relations
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4-01 Hydrogen Gasochromism of WO3 F
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4-03 Synthesis of Single-Crystallin
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4-05 Synergy Effects in Electron/Io
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Fabrication of Diluted Magnetic Sem
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4-09 Gas Permeation Characteristics
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4-11 Control of Radial Size of Poly
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4-13 Behavior of N Atoms in Nitridi
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4-15 JAEA-Review 2010-065 Annealing
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Low Temperature Ion Channeling of F
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4-19 Radiation-Induced Electrical D
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4-21 Study on Cu Precipitation in E
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4-23 Evaluation of Fluorescence Mat
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4-25 Evaluation of the ZrC Layer fo
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4-27 Structure Analysis of K/Si(111
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4-29 LET Effect on the Radiation In
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4-31 Stabilization of Measurement S
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Systematic Measurement of Neutron a
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4-35 Measurement of Neutron Fluence
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4-37 Evaluation of the Response Cha
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4-39 Relationship between Internucl
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4-41 Analysis of Radiation Damage a
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4-43 Positive Secondary Ion Emissio
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4-45 JAEA-Review 2010-065 Ion Induc
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4-47 Fabrication of Dielectrophoret
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4-49 Fast Single-Ion Hit System for
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4-51 Development of Beam Generation
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JAEA-Review 2010-065 5. Status of I
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Operation of the AVF Cyclotron T. N
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Operation of Electron Accelerator a
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5-06 FACILITY USE PROGRAM in Takasa
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5-08 Radioactive Waste Management i
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Appendix 2 List of Related Patents
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Appendix 4 Type of Research Collabo
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表1.SI 基本単位基本量 SI
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