Effects of Ion Irradiation on Seedlings Growth Monitored by Ultraweak Delayed Luminescence.

The optical technique based on the measurement of delayed luminescence emitted from the biological samples has demonstrated its ability to provide valid and predictive information on the functional status of various biological systems. We want to extend this technique to study the effect of ionizing radiation on biological systems. In particular we are interested in the action of ion beams, used for therapeutic purposes or to increase the biological diversity. In general, the assessment of the damage that radiation produces both in the target objects and in the surrounding tissues, requires considerable time because is based on biochemical analysis or on the examination of the evolution of the irradiated systems. The delayed luminescence technique could help to simplify this investigation. We have so started our studies performing irradiations of some relatively simple vegetable models. In this paper we report results obtained from mung bean (Vigna radiata) seeds submitted to a 12C ion beam at the energy of 62 MeV/nucleon. The dry seeds were irradiated at doses from 50 to 7000 Gy. The photoinduced delayed luminescence of each seed before and after ion irradiation was measured. The growth of seedlings after irradiation was compared with that of untreated seeds. A growth reduction on increasing the dose was registered. The results show strong correlations between the ion irradiation dose, seeds growth and delayed luminescence intensity. In particular, the delayed luminescence intensity is correlated by a logistic function to the seedlings elongation and, after performing a suitable measurement campaign based on blind tests, it could become a tool able to predict the growth of seeds after ion irradiation. Moreover these results demonstrate that measurements of delayed luminescence could be used as a fast and non-invasive technique to check the effects of ion beams on relatively simple biological systems.

Growth behavior of gold nanoparticles synthesized in unsaturated fatty acids by vacuum evaporation methods.

Physical vapor evaporation of metals on low vapor pressure liquids is a simple and clean method to synthesize nanoparticles and thin films, though only little work has been conducted so far. Here, gold nanoparticles were synthesized by vacuum evaporation (VE) methods in ricinoleic acid and oleic acid, two typical unsaturated fatty acids (UFAs). The two solvents formed black aggregates after deposition and then shrunk and finally disappeared with the progress of time. By transmission electron microscopy (TEM) images, nanoparticles in ricinoleic acids formed aggregates and then dispersed by time, while in oleic acid big aggregates were not observed in all timescales. From TEM images and small angle X-ray scattering (SAXS) measurements, the mean size of the nanoparticles was about 4 nm in both ricinoleic and oleic acids. UV-Vis spectra were also taken as a function of time and the results were consistent with the growth behavior presumed by TEM images. Air exposure had an influence on the behavior of the sample triggering the nanoparticle formation in both solvents. From control experiments, we discovered that oxygen gas triggered the phenomenon and nanoparticles function as a catalyst for the oxidation of the UFAs. It stimulates the phenomenon and in ricinoleic acid, specifically, electrons are transferred from riconleic acid to the gold nanoparticles, enhancing the surface potential of the nanoparticles and the repulsive force between their electronic double layers.

Low fragment polyatomic molecular ion source by using permanent magnets.

Electron-ionization-type polyatomic molecular ion source with low fragment was developed by using a pair of ring-shaped Sm-Co magnets. The magnets were placed forward and backward side of ionization part to confine electrons extracted from a thermionic cathode. Calculated electron trajectory of the developed ion source was 20 times longer than that of an ordinary outer filament configuration that has no magnetic confinement. Mass spectra of the molecular ions generated from n-tetradecane (C14H30) gas exhibited 4 times larger intensity than that of the ordinary configuration in a range of mass/charge from 93 to 210 u. This indicates that suppression of fragment ion was obtained by increase of low energy electrons resulted from the electron confinement.

Efficient modification of floral traits by heavy-ion beam irradiation on transgenic Torenia.

While heavy-ion beam irradiation is becoming popular technology for mutation breeding in Japan, the combination with genetic manipulation makes it more convenient to create greater variation in plant phenotypes. We have succeeded in producing over 200 varieties of transgenic torenia (Torenia fournieri Lind.) from over 2,400 regenerated plants by this procedure in only 2 years. Mutant phenotypes were observed mainly in flowers and showed wide variation in colour and shape. Higher mutation rates in the transgenics compared to those in wild type indicate the synergistic effect of genetic manipulation and heavy-ion beam irradiation, which might be advantageous to create greater variation in floral traits.

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 characterization of microbial mutations induced by ion beam irradiation.

A positive selection system for gene disruption using a sucrose-sensitive transgenic rhizobium was established and used for the molecular characterization of mutations induced by ion beam irradiations. Single nucleotide substitutions, insertions, and deletions were found to occur in the sucrose sensitivity gene, sacB, when the reporter line was irradiated with highly accelerated carbon and iron ion beams. In all of the insertion lines, fragments of essentially the same sequence and of approximately 1188bp in size were identified in the sacB regions. In the deletion lines, iron ions showed a tendency to induce larger deletions than carbon ions, suggesting that higher LET beams cause larger deletions. We found also that ion beams, particularly "heavier" ion beams, can produce single gene disruptions and may present an effective alternative to transgenic approaches.

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 (12)C(6+)-induced mutant with single deletion and multiple base changes was isolated.

The einkorn wheat (Triticum monococcum) mutant, maintained vegetative phase, is caused by a deletion in the VRN1 gene.

The einkorn wheat (Triticum monococcum) mutant, maintained vegetative phase (mvp), was induced by nitrogen ion-beam treatment and was identified by its inability to transit from the vegetative to reproductive phase. In our previous study, we showed that WAP1 (wheat APETALA1) is a key gene in the regulatory pathway that controls phase transition from vegetative to reproductive growth in common wheat. WAP1 is an ortholog of the VRN1 gene that is responsible for vernalization insensitivity in einkorn wheat. The mvp mutation resulted from deletion of the VRN1 coding and promoter regions, demonstrating that WAP1/VRN1 is an indispensable gene for phase transition in wheat. Expression analysis of flowering-related genes in mvp plants indicated that wheat GIGANTIA (GI), CONSTANS (CO) and SUPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) genes either act upstream of or in a different pathway to WAP1/VRN1.