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Ion-beam irradiation serves as a powerful tool of mutagenesis that engineers novel traits in a wide range of plants. A previous study indicated that coincidental changes of multiple traits are frequently induced by ion-beam irradiation in soybean, a paleopolyploid plant whose genome comprises a large number of duplicated genes. Here we analyzed the levels of isoflavones, the major secondary compounds accumulated in soybean seeds, in a population of mutants having chlorophyll deficiency of various degrees obtained by the mutagenesis. Significant reduction in the levels of four isoflavones, namely, daidzin, genistin, and their malonylglucosides, was detected in 4–9 plant lines among 28 lines analyzed. The average levels of chlorophyll in the leaf tissues and isoflavones in seeds are correlated with each other. In selected mutant lines, the levels of isoflavones were lower than the wild-type plants throughout seed development. Gene expression profiles indicated that the mRNA levels of multiple genes involved in the isoflavone synthesis pathway reduced during seed development, which suggests that the observed reduction in the levels of isoflavones may be attributable to gene expression changes. Along with the possibility that multiple and independent mutations that reduce the levels of chlorophyll and isoflavones occurred simultaneously, these observations suggest the presence of an unrecognized physiological link between the synthesis and/or accumulation of these compounds in soybean.

To investigate the involvement of the non-homologous end joining (NHEJ) pathway in plant mutagenesis by ionizing radiation, we conducted a genome-wide characterization of the mutations induced by gamma rays in NHEJ-deficient Arabidopsis mutants (AtKu70−/− and AtLig4−/−). Although both mutants were more sensitive to gamma rays than the wild-type control, the AtKu70−/− mutant was slightly more sensitive than the AtLig4−/− mutant. Single-base substitutions (SBSs) were the predominant mutations in the wild-type control, whereas deletions (≥ 2 bp) and complex-type mutations [i.e., more than two SBSs or short insertion and deletions (InDels) separated by fewer than 10 bp] were frequently induced in the mutants. Single-base deletions were the most frequent deletions in the wild-type control, whereas the most common deletions in the mutants were 11–30 bp. The apparent microhomology at the rejoined sites of deletions peaked at 2 bp in the wild-type control, but was 3–4 bp in the mutants. This suggests the involvement of alternative end joining and single-strand annealing pathways involving increased microhomology for rejoining DNA ends. Complex-type mutations comprising short InDels were frequently detected in the mutants, but not in the wild-type control. Accordingly, NHEJ is more precise than the backup pathways, and is the main pathway for rejoining the broken DNA ends induced by ionizing radiation in plants.

To investigate the involvement of the non-homologous end joining (NHEJ) pathway in plant mutagenesis by ionizing radiation, we conducted a genome-wide characterization of the mutations induced by gamma rays in NHEJ-deficient Arabidopsis mutants (AtKu70−/− and AtLig4−/−). Although both mutants were more sensitive to gamma rays than the wild-type control, the AtKu70−/− mutant was slightly more sensitive than the AtLig4−/− mutant. Single-base substitutions (SBSs) were the predominant mutations in the wild-type control, whereas deletions (≥2 bp) and complex-type mutations [i.e. more than two SBSs or short insertion and deletions (InDels) separated by fewer than 10 bp] were frequently induced in the mutants. Single-base deletions were the most frequent deletions in the wild-type control, whereas the most common deletions in the mutants were 11–30 bp. The apparent microhomology at the rejoined sites of deletions peaked at 2 bp in the wild-type control, but was 3–4 bp in the mutants. This suggests the involvement of alternative end joining and single-strand annealing pathways involving increased microhomology for rejoining DNA ends. Complex-type mutations comprising short InDels were frequently detected in the mutants, but not in the wild-type control. Accordingly, NHEJ is more precise than the backup pathways, and is the main pathway for rejoining the broken DNA ends induced by ionizing radiation in plants.

アジア原⼦⼒協⼒フォーラム(FNCA)放射線育種プロジェクトに関連する研究として、全ゲノム解析により明らかになったイオンビームとガンマ線で植物に生じる突然変異の分子レベルでの特徴について紹介する。, FNCAオンラインオープンセミナー

イオンビーム誘発変異の分子レベルでの特徴に関しては、欠失変異や逆位/転座などの染色体構造変化が電子線に比べて多く生じることが、2000年代半ばまでにわかっていたが、これ以降、変異の特徴に関する理解は大きく進んでいなかったが、近年、次世代シーケンサーの普及により、「どのような変異が、いくつ生じ、どれだけの遺伝子が影響を受けたのか」をゲノム全体で明確に調べられるようになった。本発表では、近年実施した全ゲノム解析で見えてきた量子ビーム誘発変異の特徴について紹介する。, QST高崎サイエンスフェスタ2021

クラスターイオンは複数の原子がnm距離に近接した状態で物質に入射することから、特徴的なエネルギー付与を示すが、生物試料に対する致死効果及び変異誘発効果は調べられていなかった。我々は、真空チャンバー内での照射が可能な枯草菌（Bacillus subtilis）の胞子をモデル試料として照射方法を確立し、生物効果に関する研究を行ってきた。ここでは、水素クラスターイオンの致死効果及び変異誘発効果に関して得られた結果を報告する。, QST高崎サイエンスフェスタ2021

微細藻類を利用したバイオ燃料は第3世代バイオ燃料と呼ばれ、生産性が高いことや食料生産と競合しないことから、次世代エネルギーの候補として注目されている。産業利用を図るには、既存のオイル産生藻類を改良することでオイルをより蓄積し、より速く増殖する株を創出する必要がある。我々はバイオ燃料候補として、これまで5種のハプト藻でしか合成することが知られていない超長鎖脂質アルケノンに着目した。本発表では、暗条件下でアルケノン分解率の低かった変異株を用い、アルケノンとそれ以外のオイル(トリグリセリド：TAG)や貯蔵多糖について、暗条件下での減少パターンを解析した結果について報告する。, QST高崎サイエンスフェスタ2021

微細藻類を利用したバイオ燃料は第3 世代バイオ燃料と呼ばれ、生産性が高いことや食料生産と競合しないことから、次世代エネルギーの候補として注目されている。産業利用を図るには、既存のオイル産生藻類を改良することでオイルをより蓄積し、より速く増殖する株を創出する必要がある。Tisochrysis lutea（T-Iso 株）は、現在知られている超長鎖脂質アルケノンを蓄積する5 種のハプト藻のうちの1 種である。アルケノンは、分子内の二重結合が一般的な脂肪酸が持つシス型とは異なり、トランス型であることから酸化耐性を持つ。また、炭素数37~39の長鎖直鎖構造を持つ中性脂質で、熱分解により炭化水素が生成されることからジェット燃料としての利用が提唱されている。現在まで、T-Iso 株の重イオンビームに対する放射線感受性を調べ、突然変異の誘導に最適な線量・照射方法を検討すると共に目的形質が安定した有用変異体の取得を行い、オイル高産生候補株を選抜してきた。これらオイル高産生候補株は、①急速な成長による高バイオマス化、②脂質への細胞内炭素分配の増加、③脂質分解の抑制などが起こっていると考えられる。脂質分解の抑制について、暗条件でのアルケノン分解を調べたところ、アルケノン分解抑制株が7 株得られた。本発表では、これらのアルケノン分解抑制株で分解が抑制される要因について検討した結果について報告する。, 2021 年度 関東学院大学 理工／建築・環境学会 研究発表講演会

Chronic gamma irradiation is one of the mutagenesis methods in practical plant mutation breeding, however, little is known about the characteristics of induced mutations at the molecular level. To evaluate the mutagenic effects of chronic gamma irradiation even at low dose rates, mutation accumulation lines were prepared by recurrent irradiation. Seven-day-old Arabidopsis plants were grown under a gamma-ray environment until the plants started bolting (approx. two weeks) at the dose rates of 500 mGy/h, 73–100 mGy/h, 0.6–1.9 mGy/h and 2–4 µGy/h. A significant reduction in seed fertility was only observed at 500 mGy/h. This irradiation process was repeated in five successive generations and the sixth-generation plants were subjected to whole genome resequencing. The mutation frequencies at 500 mGy/h and 73–100 mGy/h were 25 and 4 times higher than that of unirradiated control, respectively. The types of mutations were similar to those observed by the acute gamma irradiation of dry seeds, but the chronic gamma irradiation showed much higher mutation frequencies per dose. No significant increase in mutation frequency was observed for the other two low-dose-rate groups. The transition/transversion ratio decreased as the dose rate increased, and interestingly, the ratio at 0.6–1.9 mGy/h was significantly lower than that in the control. This suggests that the plants may responded to low dose rate of gamma rays around 1 mGy/h, even though the overall mutation frequency did not increase., 日本放射線影響学会第 64 回大会

連携重点研究として実施している課題「高速クラスタービームによる生命科学・表面界面工学への応用研究」の小テーマ１「生物影響に関するクラスター効果研究」について2020年度の成果を報告する。

18.1 植物・微生物へのイオンビ―ム照射 1.はじめに ２．突然変異育種とイオンビ―ム ３．イオンビームで誘発される変異の特徴 ４. 植物や微生物へのイオンビ―ム照射の方法 ５. イオンビーム照射による変異誘発と変異体の選抜 ６. イオンビ―ム育種の成果例 として 植物・微生物へのイオンビ―ム照射とその応用について主に実務的な面から解説

藻類を利用したバイオ燃料は、食料生産と競合しないなど次世代エネルギーの候補として注目されている。しかし、藻類を利用したバイオ燃料生産には、既存のオイル産生藻を改良し、よりオイル生産に適した有用変異体（オイル高蓄積株あるいは高い増殖速度を持つ株）を創出する必要がある。我々はその候補として、これまで5種のハプト藻でしか合成することが知られていない超長鎖脂質アルケノンに着目し、その1種であるTisochrysis lutea (T-Iso株)に重イオンビーム照射し、よりオイル生産に適した有用突然変異株の創出を行ってきた。T-Iso株は、オイルとしてアルケノンだけでなくトリグリセリド（TAG）も蓄積する。現在まで脂質の蛍光染色により、その蛍光強度からオイル高産生候補株を607株選抜した。これらオイル高産生候補株の要因としては、炭素固定量の増加や脂質への炭素分配の増加、脂質分解の抑制などが考えられる。これらのオイル高産生候補株から、脂質分解の抑制に注目し、アルケノンが分解される暗条件移行後の脂質蛍光強度の減少率が低い72株について今回は解析を行った。 アルケノン量は、変異体4株（#8, #13, #24, #39）で暗条件下での減少率が0~20%と野生株の30%より低かった。TAG量は、変異体3株（#14 #22, #24）で暗条件下での減少率が25%と野生株の70%より低かった。#24のみで、アルケノン量とTAG量共に減少率の低下が見られた。脂肪酸量は、TAG量の減少率が低かった3株（#14 #22, #24）で暗条件下での減少率が25%と野生株の50%より低かった。多糖量は、変異体と野生で共に暗条件下で減少したが、その減少率は70%前後で大きな差異は見られなかった。しかし、多糖減少率の増加が脂質減少率の増加より速かったため、多糖分解が脂質分解に先だって行われていることが示唆された。 今回調べた変異体6株全てでオイル分解抑制が見られたが、多糖量の減少率に変化は見られなかった。そのため、多糖分解と脂質分解が別々の経路や暗順応で行われていること、多糖分解が先だって行われているため脂質分解抑制の影響を多糖分解があまり受けないことが示唆された。オイル分解については、アルケノン量のみ、またはTAG量のみの分解抑制が見られた変異体株があったため、アルケノンとTAGの分解が異なる酵素や経路で行われている可能性が示唆された。また、今回アルケノンとTAG量で共に分解抑制を示した#24が得られたため、アルケノンとTAGの分解過程の関連性についても明らかになることが期待できる。, QST高崎サイエンスフェスタ2020

アジア原子力協力フォーラム(FNCA)放射線育種プロジェクトで2013年度から2017年度にかけて実施されたサブプロジェクト「持続可能な農業のためのイネの突然変異育種」に関連した報告であり、日本の在来イネ品種である「旭日」と「亀の尾」との交配、及びガンマ線照射による早生変異体選抜により、自然農法での栽培に適した優良系統を選抜した経過をとりまとめたものである。

Rice leaves and stems, which can be used as rice straw for livestock feed, accumulate soluble oxalate. The oxalate content often reaches 5% of the dry weight leaves. Excess uptake of oxalate-rich plants causes mineral deficiencies in vertebrates, so it is important to reduce the oxalate content in rice leaves to produce high-quality rice straw. However, the mechanism of oxalate accumulation in rice has remained unknown. To understand metabolic networks relating oxalate accumulation in rice. In this study, we performed metabolome analysis of rice M2 population generated by ion-beam irradiation using CE-MS. The result showed wide variation of oxalate contents in M2 plants compared with those of control plants. Multivariate analyses of metabolome dataset revealed that oxalate accumulation was strongly related with anionic compounds such as 2OG and succinate. For low-oxalate plants, four patterns of metabolic alterations affected oxalate contents in the M2 leaves were observed. In M3 plants, we found putative low-oxalate line obtained from low-oxalate M2 mutant. These findings would lead to produce the low-oxalate rice and to understand the oxalate synthesis in plants.These findings would lead to produce the low-oxalate rice and to understand the oxalate synthesis in plants.

Biofuel production by microalgae, is attracting attention as a candidate for next-generation energy, because it does not compete with food production. We have been trying heavy ion beam irradiation to create useful mutant strains that are more suitable for oil production. Here, we focused on cerulenin, which is known as inhibitor of fatty acid synthesis, to get fatty acid and alkenone synthesis enhanced mutants. We picked up mutants as following 3 groups, 1) top 10% mutants with high lipid fluorescence intensity after 4-week cultivation, 2) top 10% mutants with large increase in lipid fluorescence intensity between culture after 1 week and 4 weeks, 3) top 10% mutants with high chlorophyll fluorescence intensity and high optical density after 3-week cultivation.

Mutation breeding is advantageous and essential for carnation to extend the variation of genetic information and create novel traits. We have demonstrated that tissue culture combined with ion beam irradiation can create a wider spectrum in flower phenotype than that with gamma irradiation, which has been the most commonly used mutagen worldwide. We showed that chronic gamma irradiation also has the potential to generate a wide spectrum of flower color mutants comparable to ion beam irradiation. Furthermore, we found that non-acylated type anthocyanin is the key determinant in creating metallic coloration in carnation. We succeeded in diversifying the metallic coloration by ion beam irradiation. Through these studies, eight new varieties of carnation have been developed so far and sold worldwide for 8–15 years. Despite the relatively narrow genetic background of carnation, our research suggested that novel mutant varieties can be created by ion beam breeding system and also by finding key determinants that control flower phenotypes.

Gamma rays are the most frequently used ionizing radiation in plant mutagenesis; however, few studies are available on the characteristics of mutations at a genome-wide level. Here, we quantitatively and qualitatively characterized the mutations induced by acute/chronic gamma ray irradiation in Arabidopsis. The data were then compared with those previously obtained for carbon ion irradiation. In the acute irradiation of dry seeds at the same effective survival dose, gamma rays and carbon ions differed substantially, with the former inducing a significantly greater number of total mutation events, while the number of gene-affecting mutation events did not differ between the treatments. This may result from the gamma rays predominantly inducing single-base substitutions, while carbon ions frequently induced deletions ≥2 bp. Mutation accumulation lines prepared by chronic gamma irradiation with 100–500 mGy/h in five successive generations showed higher mutation frequencies per dose compared with acute irradiation of dry seeds. Chronic gamma ray irradiation may induce larger genetic changes compared with acute gamma ray irradiation. In addition, the transition/transversion ratio decreased as the dose rate increased, suggesting that plants responded to very low dose rates of gamma rays (∼1 mGy/h), even though the overall mutation frequency did not increase. These data will aid our understanding of the effects of radiation types and be useful in selecting suitable radiation treatments for mutagenesis.

Mutations caused by ion beams have been well-studied in plants, including ornamental flowers, rice, and algae. It has been shown that ion beams have several significantly interesting features, such as a high biological effect and unique mutation spectrum, which is in contrast to low linear energy transfer (LET) radiation such as gamma rays. In this study, the effects of double strand breaks and 8-oxo-2&prime, deoxyguanosine (8-oxodG) caused by ion-beam irradiation were examined. We irradiated repair-gene-inactive strains rad52, ogg1, and msh2 using carbon ion beams, analyzed the lethality and mutagenicity, and characterized the mutations. High-LET carbon ion-beam radiation was found to cause oxidative base damage, such as 8-oxodG, which can lead to mutations. The present observations suggested that nucleotide incorporation of oxidative damage gave only a limited effect on cell viability and genome fidelity. The ion-beam mutations occurred predominantly in 5&prime, ACA-3&prime, sequences, and we detected a hotspot at around +79 to +98 in URA3 in wild-type and mutant lines, suggesting the presence of a mutation-susceptible region.

Ion beams are physical mutagens used for plant and microbe breeding that cause mutations via a distinct mechanism from those of chemical mutagens or gamma rays. We utilized whole-exome sequencing of rice DNA in order to understand the properties of ion beam-induced mutations in a genome-wide manner. DNA libraries were constructed from selected carbon-ion-beam-induced rice mutants by capturing with a custom probes covering 66.3 M bases of nearly all exons and miRNAs predicted in the genome. A total of 56 mutations, including 24 single nucleotide variations, 23 deletions, and 5 insertions, were detected in five mutant rice lines (two dwarf and three early-heading-date mutants). The mutations were distributed among all 12 chromosomes, and the average mutation frequency in the M1 generation was estimated to be 2.7 × 10-7per base. Many single base insertions and deletions were associated with homopolymeric repeats, whereas larger deletions up to seven base pairs were observed at polynucleotide repeats in the DNA sequences of the mutation sites. Of the 56 mutations, six were classified as high-impact mutations that caused a frame shift or loss of exons. A gene that was functionally related to the phenotype of the mutant was disrupted by a high-impact mutation in four of the five lines tested, suggesting that whole-exome sequencing of ion-beam-irradiated mutants could facilitate the detection of candidate genes responsible for the mutant phenotypes.

藻類を利用したバイオ燃料は次世代エネルギーとして注目されており、我々は、その候補として、これまで5種のハプト藻でしか合成することが知られていない超長鎖脂質アルケノンに着目している。これまで、アルケノンを産生するハプト藻の1種であるTisochrysis lutea（T-Iso株）にイオンビーム照射を行い、高オイル産生変異株の選抜を行ってきた。アルケノンは暗条件ではエネルギー源として使われるため分解が進むことが知られている。これまでに単離したアルケノン高生産株を明条件で2週間培養後、暗条件で1週間培養を行い、ナイルレッド染色により脂肪酸量の推移を調査した。その結果、アルケノンの分解が遅いと思われる9系統を選抜した。さらに、実際のアルケノンの含量を測定する試験を行い、暗条件処理前の88%以上のアルケノンを含んでいた5系統を選抜した。

クラスターイオンは、複数の原子がnm距離に近接した状態で物質に入射することから、材料表面の改質や分析では単原子イオンに比べて特異なエネルギー付与を示すことが知られている。しかしながら、生物試料における研究は実施されていなかった。我々は、枯草菌の胞子を生物試料のモデルとし、クラスターイオンの生物効果の特徴について明らかにすることを目的とした研究を行っている。原子あたり2MeVの炭素イオンの致死効果を評価した結果、LETが非常に高いために1原子のヒットで致死するため、致死効果に関するクラスター化の効果は確認されないことがわかった。そこで、炭素よりも低いLETを持つプロトンのクラスターイオンの致死効果を評価した。2 MeV H2は1MeV Hに比べて原子あたりの致死効果が低く、負のクラスター効果を示す可能性が示唆された。これは、2つのプロトンが近接して通過するために、同じ遺伝子に影響する確率が高いことによると解釈できる。340 keV Hは2MeV H2と粒子あたりで同じLETを持つが、致死効果は340 keV Hの方が明らかに高かった。これは、イオントラック中心付近でのエネルギー密度が致死効果に大きく影響することによると考えられる。

Size of mutant sector and genetic damage were evaluated in Arabidopsis to further our understanding of effective ion beam use in plant mutation breeding. Arabidopsis seeds, heterozygous for the GLABRA1 (GL1) gene (GL1/gl1-1), were irradiated with 15.8 MeV/u neon ions (mean linear energy transfer (LET): 352 keV/μm), 17.3 MeV/u carbon ions (113 keV/μm), or 60Co gamma rays. The frequency and size of glabrous sectors generated because of inactivation of the GL1 allele were examined. The frequency and overall size of large deletions were evaluated based on the loss of heterozygosity of DNA markers using DNA isolated from glabrous tissue. Irrespective of the radiation properties, plants with mutant sectors were obtained at similar frequencies at the same effective dosage necessary for survival reduction. Ion beams tended to induce larger mutant sectors than gamma rays. The frequency of large deletions (>several kbp) increased as the LET value increased, with chromosome regions larger than 100 kbp lost in most large deletions. The distorted segregation ratio of glabrous plants in the progenies of irradiated GL1/gl1-1 plants suggested frequent occurrence of chromosome rearrangement, especially those subjected to neon ions. Exposure to ion beams with moderate LET values (30–110 keV/μm) is thought effective for inducing mutant sectors without causing extensive genetic damage.

Rice leaves and stems, which can be used as rice straw for livestock feed, accumulate soluble oxalate. The oxalate content often reaches 5% of the dry weight leaves. Excess uptake of oxalate-rich plants causes mineral deficiencies in vertebrates, so it is important to reduce the oxalate content in rice leaves to produce high-quality rice straw. However, the mechanism of oxalate accumulation in rice has remained unknown. In this study, we performed metabolome analysis of rice M 2 population generated by ion-beam irradiation using CE-MS to understand metabolic networks relating oxalate accumulation in rice. The result showed wide variation of oxalate contents in M 2 plants compared with those of control plants. Multivariate analyses of metabolome dataset revealed that oxalate accumulation was strongly related with anionic compounds such as 2OG and succinate. For low-oxalate plants, four patterns of metabolic alterations affected oxalate contents in the M 2 leaves were observed. In M 3 plants, we found putative low-oxalate line obtained from low-oxalate M 2 mutant. These findings would lead to produce the low-oxalate rice and to understand the oxalate synthesis in plants.

Rice (Oryza sativa L.) grain is a major dietary source of cadmium (Cd), which is toxic to humans, but no practical technique exists to substantially reduce Cd contamination. Carbon ion-beam irradiation produced three rice mutants with0.05 mg Cd⋅kg(-1) in the grain compared with a mean of 1.73 mg Cd⋅kg(-1) in the parent, Koshihikari. We identified the gene responsible for reduced Cd uptake and developed a strategy for marker-assisted selection of low-Cd cultivars. Sequence analysis revealed that these mutants have different mutations of the same gene (OsNRAMP5), which encodes a natural resistance-associated macrophage protein. Functional analysis revealed that the defective transporter protein encoded by the mutant osnramp5 greatly decreases Cd uptake by roots, resulting in decreased Cd in the straw and grain. In addition, we developed DNA markers to facilitate marker-assisted selection of cultivars carrying osnramp5. When grown in Cd-contaminated paddy fields, the mutants have nearly undetectable Cd in their grains and exhibit no agriculturally or economically adverse traits. Because mutants produced by ion-beam radiation are not transgenic plants, they are likely to be accepted by consumers and thus represent a practical choice for rice production worldwide.

With regard to fire safety for exterior walls of a building, fire-resistance performance is considered, according to the current building standard law of Japan. And it was revealed that the fire safety is not specifically regulated from the viewpoint of reaction-to-fire performance, such as fire propagation caused by combustible materials or products which are installed on the exterior side of fire-resistant load-bearing walls. Actual fire issues in the world have shown that massive facade fire could occur at the exterior side of building wall even when the wall itself is fire-resistant. In previous studies of the authors, a test method of facade fire was tentatively proposed for evaluating the vertical fire propagation over an external wall within the same building. Furthermore, especially at the high-density residential areas in Japan, fire spreading would occur mostly from window to window between adjacent buildings, while walls are not burning by facade fire. But in case of combustible facades, such as insulating materials, combustible coating, or even sandwich panel are installed on the wall surface, once they are ignited, facade fire would occur and accelerate both the fire propagation in the same building and the fire spreading to the adjacent building. In this paper, results of fire tests on combustible facades are discussed from the viewpoints of both vertical fire spread along facades and fire spread between adjacent buildings assuming the densely populated residential area in Japan. The tentative criteria were determined based on the time to reach 500°C and duration time exceeding 500°C on facade surface for vertical fire spread along facade. As for intensively burned cases, at 2500 mm height from upper edge of the opening, specimens of exterior thermal insulation system with 200-mm thick expanded polystyrene (EPS), aluminum composite panel with polyethylene core and wooden facade without fire-retardant treatment continued exceeding 500°C for 3.1 min, 5.7 min, and 3.6 min, respectively, that were longer duration compared with specimens of resin painting (approximately 1 min). On the other hand, the another tentative criteria were determined for horizontal fire spread to an adjacent building based on the duration time exceeding the calibration test’s peak incident heat flux on opposite surface 2000 mm from facade surface for horizontal fire spread to an adjacent building. At 500 mm height from upper edge of the opening, specimens of exterior thermal insulation system with high-density 100-mm thick EPS and wooden facade without fire-retardant treatment continued exceeding the peak incident heat flux of calibration test for 13.1 min and 13.6 min, respectively, while one specimen of resin painting and aluminum composite panel with polyethylene core continued for shorter duration of 4.2 min and 5.8 min, respectively, even though they showed relatively high peak facade surface temperatures.

We investigated the effect of tissue source selection in combination with ion beam irradiation on generating flower-color mutants in Chrysanthemum morifolium. Flower color mutation in the chrysanthemum cultivar YoMystery (purple flowers) was used as an index to estimate mutation frequency and the spectrum of mutations. Somaclones regenerated from petal and leaf tissues and ion-beam irradiated clones derived from the two tissues were analyzed for stem length and flower color mutation. Three Gy of argon ion beams was regarded as an appropriate dose for irradiation because the resulting color mutants maintained an adequate stem length suitable for commercial use even with high mutation frequencies. Statistical analysis of 7,258 individuals from three separate experiments revealed that (1) somaclones from petal tissue had a higher mutation frequency (2.91 %, r = 0.55) than leaf somaclones (2.01 %, r = 0.26); (2) the effect of tissue source on the frequency of flower color mutations was significantly enhanced when combined with ion-beam irradiation in that the mutation frequency of ion-beam irradiated petal clones was 6.47 % (r = 0.77), whereas that of ion-beam irradiated leaf clones was 3.89 % (r = 0.52); and (3) the spectrum of carotenoid color mutations was remarkably increased by ion-beam irradiation in that both leaf and petal ion-beam irradiated clones had brown, red and yellow flowers in contrast to the soma clones that only produced yellow flowers. These results suggest that intentional or directed induction of flower color mutants with practical value is possible by combining tissue source selection with ion beam irradiation.