Particle Bombardment Research Articles

Particle bombardment and subcellular protein localization analysis in the aquatic plant Egeria densa.

Particle bombardment is a powerful and relatively easy method for transient expression of genes of interest in plant cells, especially those that are recalcitrant to other transformation methods. This method has facilitated numerous analyses of subcellular localization of fluorescent fusion protein constructs. Particle bombardment delivers genes to the first layer of plant tissue. In leaves of higher plants, epidermal cells are the first cell layer. Many studies have used the epidermal cell layer of onion bulb (Allium cepa) as the experimental tissue, because these cells are relatively large. However, onion epidermal cells lack developed plastids (i.e., chloroplasts), thereby precluding subcellular localization analysis of chloroplastic proteins. In this study, we developed a protocol for particle bombardment of the aquatic plant Egeria densa, and showed that it is a useful system for subcellular localization analysis of higher plant proteins. E. densa leaflets contain only two cell layers, and cells in the adaxial layer are sufficiently large for observation. The cells in both layers contain well-developed chloroplasts. We fused fluorescent proteins to conventional plant localization signals for the nucleus, cytosol, mitochondria, peroxisome, and chloroplast, and used particle bombardment to transiently express these fusion constructs in E. densa leaves. The plant subcellular localization signals functioned normally and displayed the expected distributions in transiently transformed E. densa cells, and even chloroplastic structures could be clearly visualized.

Benefits of energetic ion bombardment for tailoring stress and microstructural evolution during growth of Cu thin films

We have studied the development of intrinsic stress and microstructure of copper (Cu) films deposited under energetic ion bombardment. Stress evolution during growth of ∼150 nm thick Cu films on Si(001) substrates has been investigated by in situ measurements in a high power impulse magnetron sputtering (HiPIMS) process for different substrate bias voltages (from 0 to −160 V) and benchmarked with conventional direct current magnetron sputtering (DCMS). The microstructure and crystal orientation of the studied films have been examined by various ex situ methods. For HiPIMS, we found that the substrate bias voltage (energy of Cu ions) strongly affects the film continuity during the early growth stages and the compressive stress developed during the post-coalescence stage. Contrarily to common expectations, the stress magnitude can be significantly reduced despite the energy increase of the bombarding particles when using HiPIMS. These results are discussed based on a recent kinetic model taking into account the grain size-dependent defect incorporation due to energetic particle bombardment. Reversible stress relaxations are observed upon growth interrupts, with characteristic time constants of tens of seconds, which suggests that the stress and microstructure development are mainly mediated by surface diffusion processes. In addition, polycrystalline films (111)-textured are obtained for negative bias voltages from 0 to −100 V, while for even higher negative bias voltages (up to −160 V), epitaxial growth of Cu(001) is achieved. For the DCMS samples, there is no significant change in film continuity and crystal orientation when varying the bias voltage.

A novel role of the soybean clock gene LUX ARRHYTHMO in male reproductive development

The evening complex of ELF4-ELF3-LUX proteins is an integral component of a plant circadian clock. LUX ARRHYTHMO (LUX) is one of the key components of the evening complex, and that play a key role in circadian rhythms and flowering. Here, we report that diverged soybean LUX has the additional role in male reproductive development. We studied diurnal and circadian rhythms of soybean LUX (GmLUXa, GmLUXb, and GmLUXc) using qRT-PCR, and show its nuclear localisation by particle bombardment. Yeast-two hybrid (Y2H) studies indicate that both GmLUXb and GmLUXc form an evening complex with GmELF4b and GmELF3a, respectively. Ectopic expression of GmLUXb in Arabidopsis lux mutants can complement functions of AtLUX, whereas GmLUXc generates novel phenotypes of serrated leaves, stunted plants, shortened anther filament, and low seed set. Overall, our results suggest that the LUX gene has diverged in soybean where GmLUXb and GmLUXc share the role to control flowering time, but GmLUXc has evolved to regulate anther filament growth and seed set by regulating the Gibberellin hormone biosynthesis pathway.

Space-Object Charging and Its Effect on Orbit Evolution

With the increasing number of uncontrolled objects in the space environment surrounding Earth, it has become important to keep track of the orbits of these objects so as to avoid collisions with active space assets. Because of the continuous bombardment of plasma particles, a space object is often subject to charging. One of the forces affecting the trajectory of a space object is the Lorentz force, which acts when a charged body moves through Earth’s magnetosphere. Correct modeling of Lorentz force requires correct modeling of magnetosphere and the body charge, which in turn depends on correct modeling of body currents, space plasma environment, and body capacitance. This research focuses on modeling of in-space charging for space objects, which are modeled as spherical conductors. Simulations incorporating Lorentz force as an additional perturbation force to Earth’s gravity have been run to evaluate the propagation of low-area-to-mass-ratio and high-area-to-mass-ratio objects in geosynchronous and low Earth orbits. Trends in the evolution of in-track, cross-track, and radial perturbations due to Lorentz force over a four-day period are investigated. It is found that, for a low-area-to-mass-ratio object in low Earth orbit, in-track perturbations due to Lorentz force can reach an order of decimeter in a mere four-day period under high-charge plasma conditions. A high-area-to-mass-ratio object in geosynchronous orbit can exhibit decimeter level in-track perturbations under high-charge plasma conditions during the same period.

Influence of hysteresis effect on properties of reactively sputtered TiAlSiN films

This article reports on the hysteresis effect in TiAlSiN films prepared by an intermediate frequency magnetron. The discharge voltages for different metallic alloy targets varying with nitrogen flow rate were systematically investigated, under a constant pressure provided by sputtering gas. The hysteresis transition was introduced by the sudden changes in sputtering rate, fraction of compound formation, phase composition and mechanical properties. The result was shown that: the initial growth rate aD in metallic mode was 4 times faster than that in supersaturated state. The optimized stoichiometric TiAl(Si)Nx=1 films containing 50 at.% N were founded in the transition region. The discussion on the plasma characteristics caused by hysteresis process showed that the TiN(111) texture could be increased by applying higher particle bombarding energy. The hardness of TiAlSiN film was strongly influenced by the orientation, which depended on the loading history of nitrogen. The superior TiAlSiN film with hardness 33GPa could be prepared during the nitrogen unloading for same nitrogen flow rates.

A wheat NBS-LRR gene TaRGA19 participates in Lr19-mediated resistance to Puccinia triticina

Wheat leaf rust, caused by Puccinia triticina (Pt), is one of the most severe fungal diseases on wheat globally. Rational utilization of wheat leaf rust resistance (Lr) genes is still the best choice for control this disease. Wheat seedlings carrying Lr19 showed a high resistance phenotype to all Pt races in China. So far, all the cloned seedling Lr genes including Lr1, Lr10 and Lr21, encode protein with NBS-LRR domain. In this study, a wheat gene with NBS-LRR domain from previously established Lr19-resistance-related cDNA library was cloned and designated as TaRGA19. Full length of this gene was amplified by rapid amplification of cDNA ends (RACE). By blast against IWGSC wheat genome database, we have noticed that TaRGA19 was located on chromosome 2DS, which was different from Lr19 located on chromosome 7DL. Compared with susceptible Thatcher line, expression level of TaRGA19 was upregulated in wheat isogenic lines carrying Lr19 (TcLr19) after inoculation of Pt race THTS. By particle bombardment, TaRGA19-GFP fused protein was localized on plasma membrane of epidermal cells. Using virus-induced gene silencing (VIGS), TaRGA19-knockdown plants of TcLr19 showed reduced resistance and few sporulation phenotype upon Pt challenge. Further histological observation indicated that Pt hyphal growth at the infection sites was less suppressed in the TaRGA19-knockdown plants. In conclusion, we speculate this TaRGA19 gene was involved in the Lr19-mediated resistance to wheat leaf rust along with other components.

A review on advanced methods in plant gene targeting.

Plant genetic engineering is one of the most significant tools implemented in the modern molecular crop breeding techniques. The conventional approaches of plant genetic transformation include Agrobacterium tumefaciens, particle bombardment, DNA uptake into protoplast. The transgenic events derived by these methods carry the transgenes that are integrated at random sites in the plant genome. Novel techniques that mediate integration of foreign genes at specific pre-determined locations circumvent many problems associated with the existing methods of gene transfer. The recent years have witnessed the emergence of gene targeting techniques by employing zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindrome repeats (CRISPR). The present review focuses on the various approaches and their performance of plant gene targeting and suggests future directions in the important areas of plant molecular biology.

The fate of meteoric metals in ice particles: Effects of sublimation and energetic particle bombardment

The uptake and potential reactivity of metal atoms on water ice can be an important process in planetary atmospheres and on icy bodies in the interplanetary and interstellar medium. For instance, metal atom uptake affects the gas-phase chemistry of the Earth's mesosphere, and has been proposed to influence the agglomeration of matter into planets in protoplanetary disks. In this study the fate of Mg and K atoms incorporated into water-ice films, prepared under ultra-high vacuum conditions at temperatures of 110–140 K, was investigated. Temperature-programmed desorption experiments reveal that Mg- and K-containing species do not co-desorb when the ice sublimates, demonstrating that uptake on ice particles causes irreversible removal of the metals from the gas phase. This implies that uptake on ice particles in terrestrial polar mesospheric clouds accelerates the formation of large meteoric smoke particles (≥1 nm radius above 80 km) following sublimation of the ice. Energetic sputtering of metal-dosed ice layers by 500 eV Ar+ and Kr+ ions shows that whereas K reacts on (or within) the ice surface to form KOH, adsorbed Mg atoms are chemically inert. These experimental results are consistent with electronic structure calculations of the metals bound to an ice surface, where theoretical adsorption energies on ice are calculated to be −68 kJ mol−1 for K, −91 kJ mol−1 for Mg, and −306 kJ mol−1 for Fe. K can also insert into a surface H2O to produce KOH and a dangling H atom, in a reaction that is slightly exothermic.

Stress control of reactively sputtered thick NbN film on Si wafer changing the location of the substrate Si wafer against the Nb target on a magnetron cathode

We have been developing a superconducting NbN thin film coil in a spiral trench on a Si-wafer using MEMS technology. Connecting the coils on the different wafers using waferbonding process, a cylindrical wafer stack is to be formed as a unit of a compact SMES. The critical current density of our NbN film was measured to be around 1100 A/mm2. We measured critical current Ic of 47 mA for the previously fabricated coil of the film thickness tf = 0.5 μm. Ic in the spiral coil increases with tf. However, if we make the NbN film thicker, the film is apt to have higher lateral force caused by tensile or compressive stress which can cause peeling of the film from the Si substrate. It is well known that the stress of the sputtered thin films can be controlled from tensile to compressive stress by controlling the bombardment of high energy particles including argon atoms backscattered from the target surface. Based on this knowledge, a specially designed sputter-deposition apparatus was fabricated in which the substrate can be located not only at the different target-to-substrate distances but also at several different lateral distances from the central axis of the target (off-axis lateral shift). Using this apparatus, various stress conditions could be realized which contributed to fabrication of thick NbN film spiral coil in the trench. The film stress was calculated from bending analysis of the substrate Si wafer by stylus method using Stoney’s formula. The maximum compressive stress of 2.5 GPa was measured. By an off-axis lateral shift, tf could be increased from 0.5 to 1 μm. By increasing sputtering gas pressure from 0.7 to 2 Pa, the compressive stress could be mitigated and tf could be further increased from 1.0 to 3 μm. Up to now, we measured Ic of 220 mA for a NbN spiral coil at tf around 3 μm. More detailed adjustment of the deposition condition will bring further increase in tf, and hence Ic into sight.

Expression of Leafy Cotyledon 2 from Arabidopsis increased the content of lipid in Chlorella sorokiniana

The low lipid content is one of the major bottlenecks to realize the industrialization of the algae biodiesel. Improvement of lipid content through global regulation to get high-yield generating algae is a good strategy. Leafy Cotyledon 2 (LEC2) is an important transcription factor for seed maturation and oil accumulation in Arabidopsis. However, there are few reports regarding adoption of LEC2 for lipid accumulation until now. In this study, LEC2 from Arabidopsis was cloned into the plant expression vector pCIMBIA1300 and transformed into C. sorokiniana through particle bombardment. One recombinant was screened by PCR, RT-PCR and Western blot analyses. Compared with the wild type one, the total lipid content in the recombinant increased one fold, which did not show effect on cell growth, indicating that LEC2 can efficiently enhance the lipid accumulation in C. sorokiniana.

The Structural and Electrical Properties of Nanostructures ZnO Thin Films on Flexible Substrate

Zinc oxide (ZnO) thin films were deposited on Teflon substrates by radio frequency (RF) magnetron sputtering method at different substrate temperature. The dependence of residual stress on the substrate temperature was investigated in this work due to the growth process, the bombardment of energetic particles and process heating to the deposited thin films. From field emission scanning electron microscope (FESEM) images, samples that deposited at various substrate temperatures consists nano-sized particles. The obtained X-ray diffraction (XRD) results, it suggested that ZnO thin film deposited at 40 o C with highly c-axis oriented shows unstressed film compared to other thin films. Besides that, the ZnO thin films deposited at 40 o C shows improved electrical properties.

Displacement damage dose and implantation temperature effects on the trapping and release of deuterium implanted into SiC

Radiation damage to flow channel insert (FCI) materials is an important issue for the concept of dual-coolant blanket development in future fusion devices. Silicon Carbide (SiC) is one of the most suitable materials for FCI. Because of the severe radiation environment and exposure to tritium during operation it is of fundamental importance to study hydrogen isotope trapping and release in these materials. Here the trapping, detrapping, and diffusion of deuterium implanted into SiC is studied in correlation with pre- and post-damage induced under different conditions. For this, SiC samples are pre-damaged with 50 keV Ne+ ions at different temperatures (20, 200, 450, 700 °C) to different damage doses (1, 3.6, 7 dpa). Next, deuterium is introduced into the samples at 450 °C by ion implantation at 7 keV. The implanted deuterium retained in the sample is analysed using secondary ion mass spectrometry (SIMS) and thermo-stimulated desorption (TSD) measurements. The results indicate that with increasing neon damage dose, the maximum deuterium desorption occurs at higher temperatures. In contrast, when increasing neon implantation temperature for a fixed dose, the maximum deuterium desorption release temperature decreases. It is interpreted that the neon bombardment produces thermally stable traps for hydrogen isotopes and the stability of this damage increases with neon pre-implantation dose. A decrease of the trapping of implanted deuterium is also observed to occur due to damage recovery by thermal annealing during pre-implantation at the higher temperatures. Finally, direct particle bombardment induced deuterium release is also observed.

Transformation of Cardamom with the RNA Dependent RNA Polymerase Gene of Cardamom mosaic virus

Cardamom (Elettaria cardamomum Maton) is an important spice crop. It is affected by Cardamom mosaic virus (CdMV). In order to make cardamom plants resistant to CdMV by the pathogen-derived resistance approach, the RNA dependent RNA polymerase gene (NIb) of CdMV in the plant expression vector pAHC17, was introduced into cardamom embryogenic calli along with GFP-BAR by particle bombardment. Transformants were selected on a medium containing bialaphos and the presence of NIb and gfp genes in cardamom plants were confirmed by PCR, Southern hybridization and GFP expression.

Immobilisation of barley aleurone layers enables parallelisation of assays and analysis of transient gene expression in single cells

The barley aleurone layer is an established model system for studying phytohormone signalling, enzyme secretion and programmed cell death during seed germination. Most analyses performed on the aleurone layer are end-point assays based on cell extracts, meaning each sample is only analysed at a single time point. By immobilising barley aleurone layer tissue on polydimethylsiloxane pillars in the lid of a multiwell plate, continuous monitoring of living tissue is enabled using multiple non-destructive assays in parallel. Cell viability and menadione reducing capacity were monitored in the same aleurone layer samples over time, in the presence or absence of plant hormones and other effectors. The system is also amenable to transient gene expression by particle bombardment, with simultaneous monitoring of cell death. In conclusion, the easy to handle and efficient experimental setup developed here enables continuous monitoring of tissue samples, parallelisation of assays and single cell analysis, with potential for time course studies using any plant tissue that can be immobilised, for example leaves or epidermal peels.

Monte Carlo simulation of ion–material interactions in nuclear fusion devices

ABSTRACTOne of the key aspects regarding the technological development of nuclear fusion reactors is the understanding of the interaction between high-energy ions coming from the confined plasma and the materials that the plasma-facing components are made of. Among the multiple issues important to plasma–wall interactions in fusion devices, physical erosion and composition changes induced by energetic particle bombardment are considered critical due to possible material flaking, changes to surface roughness, impurity transport and the alteration of physicochemical properties of the near surface region due to phenomena such as redeposition or implantation. A Monte Carlo code named MATILDA (Modeling of Atomic Transport in Layered Dynamic Arrays) has been developed over the years to study phenomena related to ion beam bombardment such as erosion rate, composition changes, interphase mixing and material redeposition, which are relevant issues to plasma-aided manufacturing of microelectronics, components on object exposed to intense solar wind, fusion reactor technology and other important industrial fields. In the present work, the code is applied to study three cases of plasma material interactions relevant to fusion devices in order to highlight the code’s capabilities: (1) the Be redeposition process on the ITER divertor, (2) physical erosion enhancement in castellated surfaces and (3) damage to multilayer mirrors used on EUV diagnostics in fusion devices due to particle bombardment.

Development and drought tolerance assay of marker-free transgenic rice with OsAPX2 using biolistic particle-mediated co-transformation

Abiotic stresses such as drought, salinity, and low temperature cause–losses in rice production worldwide. The emergence of transgenic technology has enabled improvements in the drought resistance of rice plants and helped avert crop damage due to drought stress. Selectable marker genes conferring resistance to antibiotics or herbicides have been widely used to identify genetically modified plants. However, the use of such markers has limited the public acceptance of genetically modified organisms. Marker-free materials (i.e., those containing a single foreign gene) may be more easily accepted by the public and more likely to find common use. In the present study, we created marker-free drought-tolerant transgenic rice plants using particle bombardment. Overall, 842 T0 plants overexpressing the rice ascorbate peroxidase-coding gene OsAPX2 were generated. Eight independent marker-free lines were identified from T1 seedlings using the polymerase chain reaction. The molecular characteristics of these lines were examined, including the expression level, copy number, and flanking sequences of OsAPX2, in the T2 progeny. A simulated drought test using polyethylene glycol and a drought-tolerance test of seedlings confirmed that the marker-free lines carrying OsAPX2 showed significantly improved drought tolerance in seedlings. In the field, the yield of the wild-type plant decreased by 60% under drought conditions compared with normal conditions. However, the transgenic line showed a yield loss of approximately 26%. The results demonstrated that marker-free transgenic lines significantly improved grain yield under drought-stressed conditions.

Viral Vectors for Plant Genome Engineering.

Recent advances in genome engineering (GE) has made it possible to precisely alter DNA sequences in plant cells, providing specifically engineered plants with traits of interest. Gene targeting efficiency depends on the delivery-method of both sequence-specific nucleases and repair templates, to plant cells. Typically, this is achieved using Agrobacterium mediated transformation or particle bombardment, both of which transform only a subset of cells in treated tissues. The alternate in planta approaches, stably integrating nuclease-encoding cassettes and repair templates into the plant genome, are time consuming, expensive and require extra regulations. More efficient GE reagents delivery methods are clearly needed if GE is to become routine, especially in economically important crops that are difficult to transform. Recently, autonomously replicating virus-based vectors have been demonstrated as efficient means of delivering GE reagents in plants. Both DNA viruses (Bean yellow dwarf virus, Wheat dwarf virus and Cabbage leaf curl virus) and RNA virus (Tobacco rattle virus) have demonstrated efficient gene targeting frequencies in model plants (Nicotiana benthamiana) and crops (potato, tomato, rice, and wheat). Here we discuss the recent advances using viral vectors for plant genome engineering, the current limitations and future directions.

Production of eicosapentaenoic acid (EPA, 20:5n-3) in maize (Zea mays L.) through the alternative ∆8 desaturation pathway mediated by particle bombardment

Very long chain polyunsaturated fatty acids (VLCPUFAs), especially eicosapentaenoic acid (EPA, 20:5∆5,8,11,14,17) and docosahexaenoic acid (DHA, 22:6∆4,7,10,13,16,19) have demonstrated important roles in a number of aspects of human health. Currently, our primary dietary sources for these fatty acids are from marine fish. Producing VLCPUFAs in oilseed crop by metabolic engineering was considered to provide an alternative, sustainable sources. Here, three heterologous genes, ∆9 elongase (∆9-Elo) of Isochrysis galbana, ∆8 desaturase (∆8-Des) of Euglena gracilis, ∆5 desaturase (∆5-Des) of Mortierella alpina, were co-transformed into maize inbred line Qi319 using a particle bombardment transformation method to produce EPA through the alternative ∆8 desaturation synthetic pathway. A total of 144 herbicide resistant lines were obtained with an average transformation efficiency of 7.95%, of which 98 lines contain the Bar genes with the positive transformation efficiency of 4.74%. In addition, 60 of the 98 positive transgenic lines were identified to contain all three transgenes, ∆9-Elo, ∆8-Des, and ∆5-Des. The fatty acid composition of the leaves from the 60 transgenic lines were subjected to gas liquid chromatography analysis and the results showed that the amounts of EPA reached 1.99% in an individual leaf. These data demonstrate the feasibility for the heterologous production of EPA in maize and this will lay a foundation for the production of VLCPUFAs, including EPA and DHA, in maize by metabolic engineering in the future.

Improved method for in situ biolistic transformation to analyze barley–powdery mildew interactions

Although the use of stable transformants is indispensable to elucidate mechanisms underlying molecular plant–pathogen interactions, this approach remains difficult to apply to crops. Alternatively, biolistic transformation has often been used as a transient expression method in various plants. In this study, we developed a method for in situ biolistic transformation without separating leaves from barley seedlings by using a hand-held particle bombardment system because unwounded leaves are preferable for analyzing interactions between barley and Blumeria graminis f. sp. hordei which requires healthy living cells. As a result, we found that the infection rate in intact leaves was higher than in separated leaves and that the transformation efficiencies in leaves were higher when plants were grown in vermiculite rather than in culture soil. Furthermore, we determined the appropriate inoculation time after bombardment to analyze the incompatible interaction and successfully monitored the gradual occurrence of cell death over time. Our system was suitable for relatively long-term follow-up analysis of the fate of each single cell during plant–pathogen interactions.

Optimization of a protocol for the production of transgenic lily plants via particle bombardment

유전자총을 이용한 형질전환 체계와 PPT (D-L-phosphinothricin) 선발을 통하여 나리 인편조직으로부터 형질전환 식물체가 획득되었다. 본 연구에서 나리 '레드플레임' 품종의 인편조직에 선발유전자로 제초제저항성 유전자인 bar 유전자 그리고 내염성과 내건성의 복합환경저항성을 나타내는 AtSIZ 유전자를 목적유전자로 ...