Screening of Biocontrol Strain Bacillus subtilis by N^+ Ion Beam Implantation

[Objective] This study was to investigate the effect of N＋ ion beam implantation on the survival rate and mutation rate of biocontrol strain Bacillus subtilis. [Method] The factors influencing B. subtilis ion beam implantation, including culture time, dilution concentration, solvent, drying time of mycoderm were optimized. B. subtilis cells were implanted by using ion beam at dose of 2.0×10^14～4.0×10^14 ions/cm2 and the energy of 30 kev. Then the methods of culturing colonies confronting each other on plate and Oxford cup diffusion were used to screening strains. [Result] The optimal parameters were found as follows： culture in liquid for 20-24 h, dilution with sterile water to 106 cells/ml and drying time of 60 min for sample preparation; the optimal N＋ ion beam implantation dose of 2.0×10^14～4.0×10^14 ions/cm2 at the energy of 30 kev, the survival rate of 8.43%-26.71% and the mutation rate of 3.50%-5.43%. [Conclusion] This study provided reference for ion beam implantation mutation of B. subtilis.

A Mutant of Bacillus Subtilis with High-Producing Surfactin by Ion Beam Implantation

In order to generate a mutant of Bacillus subtilis with enhanced surface activity through low energy nitrogen ion beam implantation, the effects of energy and dose of ions implanted were studied. The morphological changes in the bacteria were observed by scanning electron microscope （SEM）. The optimum condition of ions implantation, 20 keV of energy and 2.6 × 10^15N^＋/cm^2 in dose, was determined. A mutant, B.s-E-8 was obtained, whose surface activity of 50-fold and 100-fold diluted cell-free Landy medium was as 5.6-fold and 17.4-fold as the wild strain. The microbial growth and biosurfactant production of both the mutant and the wild strain were compared. After purified by ultrafiltration and SOURCE 15PHE, the biosurfactant was determined to be a complex of surfactin family through analysis of electrospray ionization mass spectrum （ESI/MS） and there was an interesting finding that after the ion beam implantation the intensities of the components were different from the wild type strain.

Study on the Variation of the Distant Crossing Rice by Ion Beam Implantation

In this paper, the following contents including the original receptor EI213 and other two control materials, RAPD polymorphism, photosynthetic efficiency, and the number of vascular bundles of the first internodes below the peduncle have been studied for the eight F7 transgenic lines obtained from ion beam implantation. The results showed that there was a significant variation in genomes of maize-rice line, compared with the receptor EI213, after the total exogenous maize DNA was introduced into EI213. The number of the vascular bundles of maize-rice progeny’s lines was obviously much larger than those of the original receptor EI213 and other two controls GER-3 and MH63, and along with the photosynthetic efficiency of maize-rice progeny’s lines being gone up. Moreover, the parenchyma cells around the vascular bundles of the transgenic lines became much larger in number and in size than those around the controls. All these indicated that the maize-rice progeny’s lines are really different from and superio

Effect of Ar Ion Beam Implantation on Morphological and Physiological Characteristics of Liquorice(Glycyrrhiza uralensis Fisch)Under Short-Term Artificial Drought Conditions

Ar^＋ ion beam with low energy of 30 keV was implanted into liquorice （Glycyrrhiza uralensis Fisch） seeds at the doses of 0, 600, 900 and 1200 × （2.6 × 10^13） ions/cm^2, respectively. The seeds were sowed in pots and after one month the plants were subjected to different drought conditions for two months. Then the plants＇ morphological and physiological characteristics, antioxidation enzymes and levels of endogenous hormones were investigated. The results showed that ion implantation at a proper dose can greatly enhance the liquorice seedlings＇ resistance against drought stress.

Mutagenic Effects of BM302:GO112 Induced by Low-Energy Ion Beam Implantation

Mutant strains of GO112 and BM302 with a high 2-keto-L-gulonic acid （2KLG） transformation rate induced by ion beam implantation were separately and combinatorially compared with the original strains GO29 and BM80 to study the mutagenic effects of ion beam implantation. Both the sole GOl12 and mixed BM302：GOl12 demonstrated improved SNDH activity and 2KLG yield compared to the original strains. The mutant combinations of BM302：GOl12 showed a longer stationary phase and higher biomass than BM80：GO29. The mutant BM302 exhibited a stronger capacity to maintain a stable pH environment at mixed fermentation with Gluconobacter oxydans （G. oxydans） for 2KLG transformation and facilitated the growth of G. oxydans compared with the original strain BM80. The promotive capacity to L-sorbosone dehydrogenase （L-SNDH） from the supernate of BM302 was 1.6-fold higher than that of BM80. Genes encoded SNDH in GO29 and GOl12 were amplified and sequenced, and mutations including three transitions （CG →TA, CG →TA, GC → AT） and one transversion （AT→ TA） were confirmed from GO29 to GOl12. The corresponding amino acid was changed as Leu →Phe, Arg → Gln and Asn → Lys.

Enhancement of Gongronella sp.JG Chitosanase Production by Ion Beam Implantation

Gongronella sp. JG was a fungal strain which expressed extracellular chitosanase of about 800 U/L during its growth in production medium. To improve its enzyme production, low energy N＋ implantation was employed to mutate spores of JG. The implantation condition was optimized and the parameters of 15 keV and 60 × breeding experiments. A mutant designated as SG 2.6 × 10^13 ions/cm^2 were selected for further was obtained. It showed increased chitosanase production （1800 U/L） and shortened cultivation period （from 72 h to 60 h）. Five-generation cultivation of SG indicated that its chitosanase production was stable at about 1800 U/L.

Modification of the Properties of Cu_x S Thin Films by Low Energy Ion Beam Implantation

Cu x S thin films are implanted by low energy N + ion beam. The influences of the energy and dose of N + ion implantation on Cu x S films are investigated. The results show that the ratio of copper to sulfur is increased to some extent, the constituents of the film are turned to rich copper phase from rich sulfurous phase after ion beam irradiation. X-ray diffraction spectrum and optical transmission spectra of sample have confirmed the results.

Bioeffects of Low Energy Ion Beam Implantation:DNA Damage,Mutation and Gene Transter

Low-energy ion beam implantation （10 - 200 keV） has been proved to have a wide range of biological effects and is broadly used in the breeding of crops and micro-organisms.To understand its mechanisms better and facilitate its applications, the developments in the bioeffects of low energy ion beam implantation in the past twenty years are summarized in this paper.

Biological Effects on Fruit Fly by N^+ ion Beam Implantation

Mutation induced by low energy ion beam implantation has been applied widely both in plants and microbes. However, due to the vacuum limitation, such ion implantation into animals was never studied except for silkworm. In this study, Pupae of fruit fly were irradiated with different dosage N+ ions at energy 20 KeV to study the biological effect of ion beam on animal. The results showed a saddle like curve exists between incubate rate and dosage. Damage of pupae by ion beam implantation was observed using scanning electron microscope. Some individuals with incomplete wing were obtained after implantation but no similar character was observed in their offspring. Furthermore, about 5.47% mutants with wide variation appeared in M1 generation. Therefore, ion beam implantation could be widely used for mutation breeding.

Anomalous Phenomena in E<18 keV Hydrogen Ion Beam Implantation Experiments on Pd and Ti

Implantation experiments of low energy（1 keV【E【18 keV）hydrogen ion beamson hydrogen loaded metals are performed with high beam density(Jmax1.2 mA/cm2)and lowbeam density(Jmin0.02 mA/cm2).Palladium and titanium foils（plates）are bombarded withproton and deuteron beams in order to compare the atomic and nuclear interactions between dif-ferent ion beams.X-ray and charged particles are measured,and neutron and gamma doses arealso monitored during the implantation experiments.An anomalous peak in X-spectra,whoseenergy is about four times the beam energy,is observed during the high beam density experi-ment.The peak moves from about 40 keV to 62 keV and FWHM reduces rapidly,while thebeam energy and intensity increase.Another wide peak with over twice the beam energy is mea-sured in experiment with low beam density.It is located between 16 keV and 30 keV,and itspeak energy increases with the increase of implantation dose（implantation time）.Some anoma-lous intensities of neutrons correlated with a charged particle peak（2.93,3.85 MeV）are alsoobserved in the deuteron-palladium experiment.The highest neutron intensity reaches about8×104 n/s,while the beam energy and intensity are about 15 keV and 1.0 mA,respectively.

Purification and Properties of a New L-Sorbose Dehydrogenase Accelerative Protein from Bacillus megaterium Bred by Ion-Beam Implantation

Bacillus megaterium BM302 bred by ion-beam implantation produces L-sorbose dehydrogenase accelerative protein （SAP） to accelerate the activity of L-sorbose dehydrogenase （SDH） of Gluconobacter oxydans in the 2-keto-L-gulonic acid （2KLG） fermentation from L-sorbose by the mixed culture of B. megaterium BM302 and G. oxydans. The SAP purified by three chromatographic steps gave 35-fold purification with a yield of 13% and a specific activity of 5.21 units/mg protein. The molecular weight of the purified SAP was about 58 kDa. The SDH accelerative activity of SAP at pH 7 and 50℃ was the highest. Additionally, it retained 60% activity at a pH range of 6.5 ～ 10 and was stable at 20℃ ～ 60℃. After 0.32-unit SAP was added to the single cultured G. oxydans strains, the SDH activity was apparently accelerated and the 2KLG yield of GO29, GO112, GO and GI13 was enhanced 2.1, 3.3, 3.5 and 2.9 folds respectively over that of the strains without the addition of SAP.

Study of Genetics and Embryology of Polyembryonic Mutant of Autotetraploid Rice Induced by N^+ Beam Implantation

In the present study autotetraploid rice IR36-4X was treated by an ion implantation technique with nitrogen ion beams. A polyembryonic mutant （named IR36-Shuang） was identified in the M2 generation. The mutant line and its offspring were systematically investigated in regard to their major agronomic properties and the rate of polyembryonic seedling in the M3-M6 generation. The abnormal phenomena in the embryo sac development and the cytological mechanism of the initiation of additional embryo in IR36-Shuang were observed by Laser Scanning Confocal Microscopy. The results were as follows. 1） The plant height, the panicle length and 1000 grain weight of IRB6-Shuang were lower than that of its control by 35.41%, 5.08% and 15.72% respectively, Moreover, the setting percentage decreased 12.39% compared with that in normal IRB6-4X plants. 2） The polyembryonic trait of IRB6-Shuang was genetically stable and the frequency of the polyembryonic seedlings in the IR36-Shuang line was also relatively stable. 3） The rate of abnormal embryo sacs in IR36-Shuang was significantly higher than that in the control IR36-4X. 4） The additional embryo in IR36-Shuang might arise from the double Jet of embryo sacs in a single owry, antipodal cells or endosperm cells. These results suggest that IRB6-Shuang is a polyembryonic mutant and a new apomixis rice line induced by low energy ion implantation. The prospects for the application in production of the IR36-Shuang line are also discussed. The present study may provide a basis for future investigations of apomixis rice breeding via the ion implantation biotechnology.

Biological effects of implantation and penetration of nitrogenion beams on wheat seeds

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Enhancement of L(+)-Lactic Acid Production of Immobilized Rhizoous Orvzae Imolanted by Ion Beams

Immobilized Rhizopus oryzae culturing may be a solution to the inhibited production of L（＋）-lactic acid in submerged fermentation, which is caused by aggregated mycelia floc. In the present study, a R. oryzae mutant （RL6041） with a 90% conversion rate of glucose into L-lactic acid was obtained by N＋ implantation under the optimized conditions of a beam energy of 15 keV and a dose of 2.6 ×10^15 ions/cm^2. Using polyurethane foam as the immobilization matrix, the optimal L-lactic acid production conditions were determined as 4 mm polyurethane foam, 150 r/min, 50 g/L ～ 80 g/L of initial glucose, 38℃ and pH 6.0. 15-cycle repeated productions of L-lactic acid by immobilized RL6041 were performed under the optimized culturing conditions and over 80% of the glucose was converted into L-lactic acid in 30 hours on average. The results show that immobilized RL6041 is a promising candidate for continuous L-lactic acid production.

Mutation Breeding of β-carotene Producing Strain B. trispora by Low Energy Ion Implantation

Ion beam bioengineering technology as a new mutation approach has been widely used in the biological breeding field. In this paper the application of low energy nitrogen ion implantation in the β-carotene producing strain, Blakeslea trispora（-） was investigated. The effects of different fermentation conditions on β-carotene production by a high yield strain were examined. Results showed that two β-carotene high yielding strains B.trispora（-） BH3-701 and BH3-728 were screened out and the averaged production of β-carotene was raised by 178.7% and 164.6% respectively after five passages in the shaking flasks. Compared with the original strain, the highest yield strain BH3-701 was potent in accumulating β-carotene, especially in the later stage, and greatly increased production efficiency.

Impact of Ion Implantation on Licorice(Glycyrrhiza uralensis Fisch) Growth and Antioxidant Activity Under Drought Stress

Low energy ion beams are known to have stimulation effects on plant generation and to improve plants＇ intrinsic quality. In the present study, the growth and physiological index of licorice implanted with 0, 8, 10; 12 and 14× （2.6× 10^15） ions/cm^2 were investigated under well-watered and drought-stress conditions. The results showed that a proper dose of ion implan- tation was particularly efficient in stimulating the licorice growth and improving the plant biomass significantly in both the well-watered and drought-stress conditions. The physiological results of licorice measured by leaf water potential, lipid oxidation, soluble protein and antioxidant system showed a significant correlation between ion implantation and water regime except for leaf water potential. Therefore, the study indicated that ion implantation can enhance licorice＇s drought tolerance by increasing the activity of superoxide dismutase （SOD）, eatalase （CAT） and DPPH （1,1-diphenyl-2-picrylhydrazyl） radical scavenging ability to lower oxidative damage to lipids in plants. Ion beam implantation, therefore, provides an alternative method to enhance licorice drought tolerance.

Effects of Ion Implantation on in Vitro Pollen Germination and Cellular Organization of Pollen Tube in Pinus thunbergii Parl. (Japanese Black Pine)

Low-energy ion implantation, as a new technology to produce mutation in plant breeding, has been widely applied in agriculture in China. But so far there is a little understanding of the underlying mechanisms responsible for its biological effects at the cellular level. Here we report the biological effects of a nitrogen ion beams of 30 keV on the pollen grains of Pinus thunbergii Parl. In general, ion implantation inhibited pollen germination. The dose-response curve presented a particular saddle-like pattern. Ion implantation also changed the dimension of the elongated tubes and significantly induced tip swelling. Confocal microscopy indicated that the pollen tube tips in P. thunbergii contained an enriched network of microtubules. Ion implantation led to the disruption of microtubules especially in swollen tips. Treatment with colchicine demonstrated that tip swelling was caused by the disruption of microtubules in the tip, indicating a unique role for microtubules in maintaining the tip integrality of the pollen tube in conifer. Our results suggest that ion implantation induce the disruption of microtubule organization in pollen and pollen tubes and subsequently cause morphological abnormalities in the pollen tubes. This study may provide a clue for further investigation on the interaction between low-energy ion beams and pollen tube growth.

聚焦离子束(Focused Ion Beam)原理与其在半导体工业之应用

聚焦离子束被广泛应用于芯片电路修改、研磨、沉积和二次电子/离子成像。FIB对于新原型电路设计的修改以及离子间交互作用的基础研究具有独特之性能。使用极细的聚焦离子束,FIB技术可以进行比从前更加精确的产品失效分析。操作者可以快速地、选择性地去除绝缘层或金属层,以便进行集成电路下层信号的点针探测或材质分析。集成电路的断面切割还可以达到亚微米的精度。

Ion-Beam Surface Modification of Strut Dowel Used in ITER PF Support

In this paper, surface modification of the strut dowel used in ITER PF support is reported. Different ions （nitrogen/titanium） with different doses are implanted into the surface of strut dowel. The result of Auger Electron Spectroscopy （AES） indicates that nitrogen can be implanted more deeply than titanium under the implantation condition of 60 kV accelerating voltage and a dose of 8×10^17/cm2 nitrogen. Surface Micro Hardness （SMH） and wear resistance are improved remarkably. Further SEM observation shows that there are no obvious scratches and damages after wear test.

Aluminum ion beam surface modification of elastic metallic-plastic pads for improving tribological properties

Elastic metallic-plastic pads（EMP） were irradiated by low energy aluminum ion in a metal vapor vacuum arc（MEVVA） 80-10 implantation system. The samples were irradiated with 20keV Al ion with the influx from 1×1015 to 1×1016 Al/cm2. Then the as-irradiated samples were measured by ESCA, XRD, AFM/FFM and a nano-probe. It is found that the hardness of as-irradiated samples is 5-6 times as that of the pristine ones. The worn depth of sample implanted at ion influx of 1×1016 Al/cm2 is about one eighth of that of the pristine sample at the same load. The XRD results show that there are some Al2O3 and AlF3 intermingled with the phase of polytetrafluoroethylene（PTFE）. The experimental results reveal that the tribological properties of EMP can be significantly improved by the ion beam surface modification.