Nobuhisa Fukunishi

Effects of heavy-ion beams on chromosomes of common wheat, Triticum aestivum

To investigate the nature of plant chromosomes irradiated by heavy-ion beams, the effects of nitrogen (N) and neon (Ne) ion beams on hexaploid wheat chromosomes were compared with those of X-ray. Chromosome aberrations, such as short, ring and dicentric chromosomes appeared in high frequency. The average numbers of chromosome breaks at LD-50 by irradiation with X-ray, N and Ne ion beams were 32, 20 and 20, respectively. These values may be underestimated because chromosome rearrangement without change in chromosome morphology was not counted. Thus, we subsequently used a wheat line with a pair of extra chromosomes from an alien species (Leymus racemosus) and observed the fate of the irradiated marker chromosomes by genomic in situ hybridization. This analysis revealed that 50Gy of neon beam induced about eight times more breaks than those induced by X-ray. This result suggests that heavy-ion beams induce chromosome rearrangement in high frequency rather than loss of gene function. This suggests further that most of the novel mutations produced by ion beam irradiation, which have been used in plant breeding, may not be caused by ordinary gene disruption but by chromosome rearrangements.

Molecular nature of mutations induced by high-LET irradiation with argon and carbon ions in Arabidopsis thaliana.

Linear energy transfer (LET) is an important parameter to be considered in heavy-ion mutagenesis. However, in plants, no quantitative data are available on the molecular nature of the mutations induced with high-LET radiation above 101-124keVμm(-1). In this study, we irradiated dry seeds of Arabidopsis thaliana with Ar and C ions with an LET of 290keVμm(-1). We analyzed the DNA alterations caused by the higher-LET radiation. Mutants were identified from the M(2) pools. In total, 14 and 13 mutated genes, including bin2, egy1, gl1, gl2, hy1, hy3-5, ttg1, and var2, were identified in the plants derived from Ar- and C-ions irradiation, respectively. In the mutants from both irradiations, deletion was the most frequent type of mutation; 13 of the 14 mutated genes from the Ar ion-irradiated plants and 11 of the 13 mutated genes from the C ion-irradiated plants harbored deletions. Analysis of junction regions generated by the 2 types of irradiation suggested that alternative non-homologous end-joining was the predominant pathway of repair of break points. Among the deletions, the proportion of large deletions (>100bp) was about 54% for Ar-ion irradiation and about 64% for C-ion irradiation. Both current results and previously reported data revealed that the proportions of the large deletions induced by 290-keVμm(-1) radiations were higher than those of the large deletions induced by lower-LET radiations (6% for 22.5-30.0keVμm(-1) and 27% for 101-124keVμm(-1)). Therefore, the 290keVμm(-1) heavy-ion beams can effectively induce large deletions and will prove useful as novel mutagens for plant breeding and analysis of gene functions, particularly tandemly arrayed genes.

Heavy-ion-induced mutants in sweet pepper isolated by M1 plant selection

We examined the effects of heavy-ion bombardment on mutagenesis in sweet pepper (Capsicum annuum L.). Dose–response studies indicated that 10 Gy irradiation of 12C or 20Ne ions on dry seeds is suitable for inducing mutations in plants. From 20Ne-irradiated M1 plants, putative mutants included two dwarf plants and one plant whose pericarp was yellow were isolated. Phenotypes of their M2 progeny were similar to those of the M1 plants and did not segregate. F1 plants resulting from reciprocal crosses between the mutants and wild-type plants showed the wild-type phenotype, but phenotypes of F2 and BC1F1 segregated at 1:3 (mutant:wild) and 1:1, respectively. These crossing experiments indicate that the three mutants have monogenic recessive mutations in nuclear genes. In light of these data, we discuss the effectiveness of using heavy-ion bombardment to mutate sweet peppers.

Characterization of a periclinal chimera variegated tobacco (Nicotiana tabacum L.)

Abstract A plant isolated from a population of tobacco (Nicotiana tabacum L. cv. BY-4) lines, which were generated with a heavy-ion beam, exhibited green and white variegation on its leaves, stems, and calyces. Fluorescence microscopic analysis of white and green sections of the leaves of the mutant showed an absence of developed chloroplasts in the epidermis and palisade layer. There were undeveloped chloroplasts in one or two layers of spongy parenchyma cells in the green sections of the mutant. In the F1 generation, the segregation ratio of green to white was 6:1163 and reciprocal crosses showed that the variegation was not maternally inherited. These results suggest that the mutant is a periclinal chimera with a White-White-Green histogenic composition. The mRNA levels of the photosynthetic genes rbcL and psbA in the green areas were normal, while they were significantly reduced in the white areas.

The Superconducting Ring Cyclotron in RIKEN

RIKEN Nishina Center for accelerator-based science in RIKEN is constructing the radioactive isotope beam factory (RIBF), which is capable of providing the worlds most intense RI beams over the whole range of atomic masses. The worlds first superconducting ring cyclotron (SRC) is the final booster in the RIBF accelerator complex which is able to accelerate all-element heavy ions to a speed of about 70% of the velocity of light. Assembling of six superconducting sector magnets in the SRC vault was completed in August 2005. This paper describes results of their cool-down and excitation tests, which were performed to check the magnet system from the various points of views: magnetic force, coil protection, magnetic field and operation of their cryogenic cooling system.

An effective method for detection and analysis of DNA damage induced by heavy-ion beams.

We have developed an efficient system to detect and analyze DNA mutations induced by heavy-ion beams in Arabiopsis thaliana. In this system, a stable transgenic Arabidopsis line that constitutively expresses a yellow fluorescent protein (YFP) by a single-copy gene at a genomic locus was constructed and irradiated with heavy-ion beams. The YFP gene is a target of mutagenesis, and its loss of function or expression can easily be detected by the disappearance of YFP signals in planta under microscopy. With this system, a 12C6+-induced mutant with single deletion and multiple base changes was isolated.

Sector magnets for the RIKEN superconducting ring cyclotron

The sector magnets of the K2500-MeV superconducting ring cyclotron (SRC), which is under construction for the RIKEN RI beam factory, are described. The SRC consists of six sector magnets, four rf resonators, etc. each sector magnet is 7.2 m in length and 6 m in height and 800 t (metric ton) in weight; the sector angle is 25 deg. The required maximum magnetic field is 3.8 T. the superconductor for the main and trim coil has a rectangular shape (8 mm by 15 mm) consisting of a Rutherford-type NbTi cable located at the center of an Al-alloy stabilizer housing. The main coil is bath-cooled and the trim coil is directly cooled. Each component of the sector magnet as well as the cryogenic cooling system was completed in the spring of 2003.

Phenotypic differentiation in the morphology and nutrient uptake kinetics among Undaria pinnatifida cultivated at six sites in Japan

Understanding the nutrient uptake kinetics of kelp populations will contribute to an improved understanding of environmental adaptation and the breeding of new cultivars. In this study, we examined the morphological characteristics, carbon (C) and nitrogen (N) contents, and NO3−–N and NH4+–N uptake kinetics of Undaria pinnatifida sporophytes cultivated at six industrial farms throughout Japan. We detected significant differences in morphology among sites. At Matsushima Bay (northern Pacific coast of Japan), where autumnal seawater temperatures fall in concert with increasing nutrient concentrations, sporophytes were significantly larger than at other sites from December to February. The C content of the sporophytes was seasonally stable at all of the locations, but the N content of sporophytes declined after February due to a decrease in seawater nutrients. We compared the uptake kinetics of NO3−–N and NH4+–N among cultivation sites. Vmax and Ks, which are Michaelis–Menten parameters that measure adaptation to nutrient concentrations, were highest in the Seto Inland Sea and lowest in the northern sector of the Sea of Japan. The Vmax/Ks ratio is a measure of adaptation to low nutrient concentrations; the highest values were measured in the northern sector of the Sea of Japan. The parameter ranges were broader than those previously reported for invasive populations of U. pinnatifida in other parts of the world. Thus, we detected population-level adaptations to the various nutrient conditions in Japanese waters, and these results suggest the existence of ecotypes according to nutrient uptake kinetics. The different populations may be used to provide sources of genetic material that could be of value in breeding programmes by improving productivity and quality.

Commissioning of the Superconducting Ring Cyclotron for the RIKEN RI Beam Factory

Since 1997, RIKEN Nishina Center has been constructing the Radioactive Isotope Beam Factory (RIBF) and succeeded in beam commissioning of its accelerator complex at the end of 2006. The worlds first superconducting ring cyclotron (SRC) is the final booster in the RIBF accelerator complex which is able to accelerate all-element heavy ions to a speed of about 70% of the velocity of light. Assembling of the superconducting sector magnets was completed in August 2005. The superconducting coils were successfully cooled down and excited for tests on many operational aspects: magnetic force, coil protection and quality of magnetic field, showing that they work as designed. After a series of tests the other components were installed and tested under stray fields from the sector magnets. Local magnetic shields were put to the components which could not work under the stray fields. After setups of beam vacuum and radio frequency, beam commissioning started. The first beam was extracted at the end of 2006 and the first uranium beam was extracted in March 2007.

Charge strippers for Radioisotope Beam Factory at RIKEN

The accelerator complex at the RIKEN Radioisotope Beam Factory accelerates heavy ions ranging from oxygen to uranium using triple stripping system to provide the beams at required charge. In many cases, the charge strippers cause problems during high-intensity beams accelerator operation. The charge stripper problem has been most significant during uranium beam acceleration because the lifetimes of the conventional carbon foils are extremely short. We conducted extensive R&D on the first stripper and found a solution using a low-Z gas stripper. We also plan to modify a second stripper operating recently with short-lifetime carbon foils. The stripper with better parameters will be needed when the beam intensity is increased by a new injector system for acceleration of uranium beam with higher charge.