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.     

The Effects of Low-Energy Nitrogen Ion Implantation on Pollen Exine Substructure and Pollen Germination of Cedrus deodara

The aim of this study is to investigate the biological effects of ion beams on pollen. Pollen grains of Cedrus deodara were implanted with 30 keV nitrogen ion beams at doses ranging from 1 × 10^15 ions/cm^2 to 15 × 10^15 ions/cm^2. The effects of N^＋ implantation on the pollen exine substructure were examined using an atomic force microscope （AFM）, and the structure and morphology of pollen and pollen tubes were observed using a laser scanning confocal microscope （LSCM）. AFM observations distinctly revealed the erosion of the pollen exine caused by N^＋ implantation in the micrometer to nanometer range. Typical results showed that the erosion degree was linearly proportional to the ion dose. Pollen germination experiments in vitro indicated that N^＋ implantation within a certain dose range increased the rate of pollen germination. The main abnormal phenomena in pollen tubes were also analyzed. Our results suggest that low energy ion implantation with suitable energy and dosage can be used to break the pollen wall to induce a transfer of exogenous DNA into the pollen without any damage to the cytoplasm and nuclei of the pollen. The present study suggests that a combination of the method of ion-beam-induced gene transfer and the pollen-tube pathway method （PTPW） would be a new plant transformation method.     

Effect of Ar^＋ Implantation and Maize Genome DNA on Autotetraploid Rice

The effect of Ar^＋ beam implantation and maize genome DNA on autotetraploid rice is studied. Better mutation types and higher mutation rates were discovered in M2 of T3 with ion implantation and immersion in maize genome DNA. In the five agronomic categories investigated, the mutation rate of the seed setting rate was 9.1%, and the total mutation rate was 14.8% in the T3. However, the total mutation rate was 2.1% with the treatment of only ion implantation and 1.3% with the treatment of only immersion in maize genome DNA. Mutant FA36（4） was discovered in M1 with ion beam implantation and immersion in maize genome DNA. Its RuBPCase activity, PEPCase activity and seed setting rate were 32%, 153%, and 36.79%, respectively, higher than its parent IR36（4）. Rapid analysis of polymorphicDNA （RAPD） analysis of three M2 plants of FA36（4） （FMI, FM2, FM3） and two controls （purple maize and IR36（4）） were also conducted with 40 random primers. S5-3 was RAPD fragment amplified with a template of purple maize, FM2 and FM3 genome DNA using primer S5. There was no S5-3 in the RAPD pattern of IR36（4） or FMI.     

Pigment analysis of a color-leaf mutant in Wandering Jew （Tradescantiafluminensis） irradiated by carbon ions

Many mutants of plant induced by heavy ion beam irradiation have been reported in recent years,but leaf anthocyan mutants induced by ion irradiation in evergreen were rarely found.In this study,a color-leaf mutant with purple leaves,stems and petals was isolated from clones of Wandering Jew irradiated by 95.8 MeV/u carbon ion beam.The concentration and histological distribution of leaf pigment were surveyed in wild type and mutant.In mutant,contents of total chlorophylls (Chl),chlorophyll a (Chl a),chlorophyll b (Chl b) and carotenoids (Car) decreased significantly,while concentration of the anthocyanins was 6.2-fold higher than that of wild type.Further composition analysis of anthocyanins by electrospray ionization mass spectrometry (ESI-MS) indicated that the purple pigmentation of leaves in mutant was caused by accumulation of petunidin anthocyanin.Microscopic examination showed that most petunidin anthocyanin accumulated in the lower epidermis,and little in vascular parenchyma of mutant,while there was no pigment in wild type.Meanwhile,in spongy parenchyma of mutant we observed little Chl,which the wild type abounds in.In conclusion,the color-leaf mutant of Wandering Jew induced by irradiation of carbon ions was improved in ornamental value,and it could be contribute to variation in level,component and distribution of foliar pigment.The possible mutation mechanisms were discussed.     

Effects of Annealing on the Structural and Photoluminescent Properties of Ag-Doped ZnO Nanowires Prepared by Ion Implantation

ZnO nanowires deposited on Si substrates were prepared by thermal evaporation of a mixture of ZnO and carbon powder. Ag ions with an energy of 63 keV and a dose of 5×1015 ions/cm-2 were implanted into the as-prepared ZnO nanowires. After ion implantation, the Ag-implanted ZnO nanowires were annealed in air at different temperatures from 600℃ to 1000℃. Effects of ion implantation and thermal annealing on the structural and photoluminescent (PL) properties of the ZnO nanowires were investigated by transmission electron microscopy (TEM), selected area energy dispersive X-ray spectroscopy (SAEDX), X-ray diffraction (XRD), and fluorescence spectrophotometry. TEM, HR-TEM, and SAEDX analyses demonstrated that efficient doping of Ag was achieved by ion implantation and the subsequent annealing process. XRD patterns revealed that the hexagonal wurtzite structure of ZnO nanowires was maintained after ion implantation. Photoluminescent emissions of ZnO nanowires were decreased significantly by Ag implantation but could be recovered by thermal annealing. The mechanism of the influence of ion implantation and annealing on the PL intensity was assessed.     

Mutation induction of Pleurotus ferulae by low-energy N^＋ ion implantation and characters of the selected mutant

In order to obtain Pleurotus ferulae with high temperature tolerance,mycelium mono-cells of wild type strain ACK was treated by nitrogen ion(5～30 keV,1.5×1015～1.5×1016cm-12)implantation,and mutant CGMCC1762 was selected through auxotrophy screening method,which was Lys.VB6 auxotrophy stress with high temperature.We found that during riper period the surface layer mycelium of the mutant was not aging neither grew tegument even above 30℃.The mycelium endurable temperature of the mutant was increased 70℃ compared with that of the wild type strain.The fruiting bodies growth temperature of the mutant was 16-20℃ in daytime and was 6～12℃ at night.The highest growth temperature of fruiting bodies of the mutant Was increased by 5℃ than that of original strain.Through three generation investigation,we found that the mutant CGMCC1762 was stable with high temperature tolerance.     

Optimization of L（＋）-Lactic Acid Fermentation Without Neutralisation of Rhizopus Oryzae Mutant RK02 by Low-Energy Ion Implantation

In order to get an industrial strain which can yield a high concentration of lactic acid for ISPR （in situ product removal）, the original strain Rhizopus oryzae RE3303 was mutated by low-energy ion beam implantation. A mutant RK02 was screened, and the factors such as the substrate concentration, nitrogen source concentration, inoculum size, seed age, aeration and temperature that affect the production of lactic acid were studied in detail. Under optimal con- ditions, the maximum concentration of L（＋）-lactic acid reached 34.85 g/L after 30 h shake-flask cultivation without adding any neutralisation （5% Glucose added）, which was a 146% increase in lactic acid production after ion implantation compared with the original strain. It was also shown that RK02 can be used in ISPR to reduce the number of times of separation.     

Optimization of L（＋）-Lactic Acid Production from Xylose with Rhizopus Oryzae Mutant RLC41-6 Breeding by Low-Energy Ion Implantation

In order to obtain an industrial strain with a higher L（＋）-lactic acid yield, the strain Rhizopus oryzae PW352 was mutated by means of nitrogen ion beam implantation and the mutant strain Rhizopus oryzae RLC41-6 was obtained. An experimental finding was made in surprise that Rhizopus oryzae mutant RLC41-6 is not only an L（＋）-lactic acid producer from corn starch but also an efficient producer of L（＋）-lactic acid from xylose. Under optimal conditions, the production of L（＋）-lactic acid from 100 g/L xylose reached 77.39 g/L after 144 h fed-batch fermentation, A high mutation rate and a wide mutation spectrum of low-energy ion implantation were observed in the experiment.     

Mutation induction of Pleurotus ferulae by low-energy N~ ion implantation and characters of the selected mutant

In order to obtain Pleurotus ferulae with high temperature tolerance, mycelium mono-cells of wild type strain ACK was treated by nitrogen ion (5~30 keV, 1.5×1015~1.5×1016 cm-2) implantation, and mutant CGMCC1762 was selected through auxotrophy screening method, which was Lys, VB6 auxotrophy stress with high temperature. We found that during riper period the surface layer mycelium of the mutant was not aging neither grew tegument even above 30°C. The mycelium endurable temperature of the mutant was increased 7°C compared with that of the wild type strain. The fruiting bodies growth temperature of the mutant was 16~20°C in daytime and was 6~12°C at night. The highest growth temperature of fruiting bodies of the mutant was increased by 5°C than that of original strain. Through three generation investigation, we found that the mutant CGMCC1762 was stable with high temperature tolerance.     

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 superior to the receptor and the controls. It is a novel and useful way to apply ion beam implantation in transferring DNA from C4 plant maize into C3 plant rice.     

Optical Emission Spectroscopic Studies of ICP Ar Plasma

The ion line of 434.8 nm and atom line of 419.8 nm of Ar plasma produced by an inductively coupled plasma （ICP） were measured by optical emission spectroscopy and the influences from the working gas pressure, radio-frequency （RF） power and different positions in the discharge chamber on the line intensities were investigated in this study. It was found that the intensity of Ar atom line increased firstly and then saturated with the increase of the pressure. The line intensity of Ar^＋, on the other hand, reached a maximum value and then decreased along with the pressure. The intensity of the line in an RF discharge also demonstrated a jumping mode and a hysteresis phenomenon with the RF power. When the RF power increased to 400 W, the discharge jumped from the E-mode to the H-mode where the line intensity of Ar atom demonstrated a sudden increase, while the intensity of Ar^＋ ion only changed slightly. If the RF power decreased from a high value, e.g., 1000 W, the discharge would jump from the H-mode back to the E-mode at a power of 300 W. At this time the intensities of Ar and Ar^＋ lines would also decrease sharply. It was also noticed in this paper that the intensity of the ion line depended on the detective location in the chamber, namely at the bottom of the chamber the line was more intense than that in the middle of the chamber, but less intense than at the top, which is considered to be related to the capacitance coupling ability of the ICP plasma in different discharge areas.     

Improvement of Vitamin K2 Production by Escherichia sp.with Nitrogen Ion Beam Implantation Induction

Low-energy ion implantation as a novel mutagen has been increasingly applied in the microbial mutagenesis for its higher mutation frequency and wider mutation spectra.In this work,N~+ ion beam implantation was used to enhance Escherichia sp.in vitamin K_2 yield.Optimization of process parameters under submerged fermentation was carried out to improve the vitamin K_2yield of mutant FM5-632.The results indicate that an excellent mutant FM5-632 with a yield of 123.2±1.6 μg/L,that is four times that of the original strain,was achieved by eight successive implantations under the conditions of 15 keV and 60×2.6×l0~(13)ions/cm~2.A further optimization increased the yield of the mutant by 39.7%,i.e.172.1±1.2 μg/L which occurred in the mutant cultivated in the optimal fermentation culture medium composed of(per liter):15.31 g glycerol,10 g peptone,2.89 g yeast extract,5 g K2HPO_4,1 g NaCl,0.5 g MgSO_4·7H_2O and 0.04 g cedar wood oil,incubated at 33℃,pH 7.0 and 180 rpm for 120 h.     

Nitrogen Deposition Via N^＋ Implantation： Implications for Primordial Amino Acids Synthesis Revisited

In this paper amino acids synthesis in aqueous solution induced by ion implantation, which was possibly ubiquitous on primitive Earth, is investigated. As a discharge using a graphite rod as the anode under a nitrogen atmosphere was performed against ammonia water, it was found that three kinds of amino acids were produced. They were glycine, serine and alanine. By introducing ion implantation into the carboxylate solution, ammonia and amino acids were also formed via nitrogen deposition/fixation. Another isotopic experiment showed that both OH and H radicals played a crucial role in the arc-discharge-promoted reactions in aqueous solution Therefore, we believe that the impact of ions in the original atmospheric conditions might have functioned as a promoter in the chemical origin and evolution of life.     

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.     

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.     

Damaging Effect of Low Energy N＋ Implantation on Aspergillus niger Spores

The mutant effects of a keV range nitrogen ion （N＋） beam on enzyme-producing probiotics were studied, particularly with regard to the induction in the genome. The electron spin resonance （ESR） results showed that the signal of ESR spectrum existed in both implanted and non-implanted spores, and the yields of free radicals increased in a dose-dependent manner. The ionic etching and dilapidation of cell wall could be observed distinctly through the scanning electron microscope （SEM）. The mutagenic effect on genome indicated that N＋ implantation could make base mutation. This study provided an insight into the roles low-energy ions might play in inducing mutagenesis of micro-organisms.     

Improved Double-Probe Technique for Spatially Resolved Diagnosis of Dual-Frequency Capacitive Plasmas

The conventional double-probe technique was improved with a combination of selfpowering and radio-frequency(RF) choking.RF perturbations in dual-frequency capacitively coupled discharge were effectively eliminated,as judged by the disappearance of self-bias on the probes.The improved technique was tested by spatially resolved measurements of the electron temperature and ion density in both the axial and radial directions of a dual-frequency capacitive plasma.The measured data in the axial direction were compared with simulation results,and they were excellently consistent with each other.The measured radial distributions of the ion density and electron temperature were influenced significantly by the lower frequency(LF) power.It was shown that superposition of the lower frequency to the higher frequency(HF) power shifted the maximum ion density from the radial center to the edge region,while the trend for the electron temperature profile was the opposite.The changing feature of the ion density distribution is qualitatively consistent with that of the optical emission intensity reported.     

ION IMPLANTATION OF DIAMOND-LIKE CARBON FILMS

Diamond-like carbon (DLC) films (a-C:H) were implanted by 140 keV and 110 keV Ar ion beams. The resistivities of the implanted films decreased dramatically under a dose of 2×1016 Ar/cm2 . IR spectra and optical gap Eopt were measured. It was found that the sp2 and sp3 components decreased due to the loss of hydrogen during implantation, and the ratio of components sp bonds to sp bonds increased with the ion dose. And the optical gap Eopt decreased from 1.46 eV to 0.83 eV. The hydrogen (bonded and unbonded) contents in the films were measured with the nuclear resonant reaction 1H(19 F, x 7) 16O. It is shown that hydrogen plays an important role in affecting some properties of DLC fims.     

Ion Implantation Hampers Pollen Tube Growth and Disrupts Actin Cytoskeleton Organization in Pollen Tubes of Pinus thunbergii

Pollen grains of Pinus thunbergii Parl. （Japanese black pine） were implanted with 30 keV nitrogen ion beams and the effects of nitrogen ion implantation on pollen tube growth in vitro and the organization of actin cytoskeleton in the pollen tube cell were investigated using a confocal laser scanning microscope after fluorescence labeling. Treatment with ion implantation significantly blocked pollen tube growth. Confocal microscopy showed that ion implantation disrupted actin filament cytoskeleton organization in the pollen tube. It was found that there was a distinct correlation between the inhibition of pollen tube growth and the disruption of actin cytoskeleton organization, indicating that an intact actin cytoskeleton is essential for continuous pollen tube elongation in Pinus thunbergii. Although the detailed mechanism for the ion-implantation-induced bioeffect still remains to be elucidated, the present study assumes that the cytoskeleton system in pollen grains may provide a key target in response to ion beam implantation and is involved in mediating certain subsequent cytological changes.     

EXPERIMENTS OF MeV ION BEAM INDUCED ATOMIC MIXING

An experimental apparatus for studies of MeV ion beam modification of materials has been established on a 3 MV tandem accelerator at Fudan university. A system of X-Y electrostatic scanning implantation of MeV heavy ions and in situ Rutherford. backscattering analysis was included in it. The uniformity of scanning implantation was checked by the RBS measurement of a Si wafer implanted with 1 MeV Au ions. MeV ion beam mixing of Au/Si, Au/Ge and Ag/Si systems was preliminarily studied. The samples were irradiated by certain fluences of 1 MeV Ag ions at room temperature. The mixed layers were analyzed in situ using the glancing RBS technique with 2 MeV 4He+ ions. For Au/Si system, a uniformly mixed layer with a defined composition is obtained, and the intermixing is much less for Ag/Si system than for Au/Si system.