Post Bombardment Research Articles

Development of an Efficient Particle Bombardment Transformation System for the Endemic Orchid, Phalaenopsis bellina

Phalaenopsis bellina is an important indigenous orchid with high commercial value. In this study, we established an efficient particle bombardment transformation system for P. bellina using the protocorm-like-bodies (PLBs) as target tissues. Leaf derived PLBs were proliferated on ½ strength Murashige and Skoog (MS) medium supplemented with 0.8 μM 2,4 dichlorophenoxyacetic acid (2, 4-D). Both physical and biological parameters affecting the transformation system were optimised using the green-fluorescent protein (GFP) and β-glucuronidase (GUS) as reporter systems. Optimal bombardment conditions with 6 cm target tissues distance, 1100 psi acceleration pressure, 1.0 μm gold particle size, 27 mmHg chamber vacuum pressure, single bombardment time, spermidine as DNA precipitation agent, 72 h post bombardment incubation time, 2 μg plasmid DNA in 0.15:0.12 pmol ratio (pSMCHS:p35SGFP) were successfully determined. Surviving PLBs transformants were successfully recovered from the hygromycin selection medium and verified using genomic PCR analysis. The established system is not only useful for a simple and reliable transient gene analysis but as well as generating stable transformants for selective traits improvement in orchids.

Modification of the Electrochemical Behavior of InP Electrodes Induced By Ionic Bombardment: The Case of the Response of the Ce4+/Ce3+ Couple

The concept of modified electrode is very important for a lot of topics related to interfacial electrochemistry. It is particularly true when semiconductor (SC) interfacial electrochemistry is considered. Due to the specificities of the SC/liquid interfaces, modified SC electrodes have to be examined from different angles. The external part of the surface can be strongly modified due to the variations of surface /solution equilibrium inducing drastic transformations of the energy diagram at the interface. A direct consequence is a possible alteration of electronic transfers. Moreover the modification of the SC electrode can also concern deeper region through variation of the depletion layer distribution even of buried diffusion layer. Evolution of these parameters could again modify the energy diagram and the related electronic transfer abilities. Finally surface modifications can induced also evolution of the photoelectrochemical properties through evolution of the optical properties of the SC surface. All this different parameters are more or less related to the global response of the interface. It is in this context that our paper is being taken. For this study, our tool of surface modification is based on ion bombardment through mono-atomic or cluster Argon beams. Modulation of the perturbation induced by ion / SC surface interaction can be largely modulated by using mono or cluster Argon beam conditions [1]. In UHV the level and the nature of the perturbation can be easily in-situ evaluated from XPS responses evolutions from the core levels and also from the valence band. At this in-situ approach can be also associated optical responses such as ex-situ Ellipsometric Spectroscopy giving a global evaluation of the SC modification. As supporting SC material we choose InP whose surface, optical and interfacial electrochemical responses are well known in their initial stage before ionic bombardment. Based on well characterized InP (-n or -p) electrodes effect of ionic bombardment can be explored through a lot of electrochemical responses. Among the more attractive modification towards electronic transfer on InP, one can consider the addition in the electrolyte of a redox probe which acts as a perfect tool giving quantitative information for post bombardment. Ce4+/Ce3+ couple is particularly efficient due to its transfer specificities when its oxidant state (Ce4+) is only present [2-6]. Indeed pure holes injection inside the valance band (BV) is observed during the Ce4+ reduction mechanism which is only governed by the Ce4+ diffusion at the surface. As a direct consequence, the evolution of the reduction rate of Ce4+ can be easily measured vs the level of ionic bombardment. Moreover when InP is properly polarized all the holes injected are used in the anodic dissolution process with a very attractive and unusual conduction band (BC) injection phenomenon due to corrosion intermediates [6]. This is again a very sensitive probe to the level of modification of the InP surface after ionic bombardment. Note that this induced anodic process as a very similar potential distribution as the one obtained under illumination for which the holes creation is nevertheless only associated to the photon absorption. Common and difference points are also a subject of discussion for a characterization of the induced modifications. Finally we will discuss how an anodic and/or a chemical etching are able to regenerate the initial chemical state of the surface. The underlying question is how the anodic process linked to Ce4+/Ce3+ is sufficient to perform the surface regeneration even if it is initially modified by the ionic bombardment or if it requires the assistance of high positive polarization of the modified InP electrode.

New Selectable Markers for Volvox carteri Transformation

Volvox carteri is an excellent model for investigating the evolution of multicellularity and cell differentiation, and the rate of future progress with this system will depend on improved molecular genetic tools. Several selectable markers for nuclear transformation of V. carteri have been developed, including the nitrate reductase (nitA) gene, but it would be useful to have additional markers to multiplex transgenes in this species. To further facilitate molecular genetic analyses of V. carteri, we developed two new selectable markers that provide rapid, easily selected, and stable resistance to the antibiotics hygromycin and blasticidin. We generated constructs with Volvox-specific regulatory sequences and codon-optimized hygromycin (VcHyg) and blasticidin (VcBlast) resistance genes from Coccidioides posadasii and Bacillus cereus, respectively. With these constructs, transformants were obtained via biolistic bombardment at rates of 0.5-13 per million target cells bombarded. Antibiotic-resistant survivors were readily isolated 7days post bombardment. VcHyg and VcBlast transgenes and transcripts were detected in transformants. Co-transformation rates using the VcHyg or VcBlast markers with unselected genes were comparable to those obtained with nitA. These results indicate that the pVcHyg and pVcBlast plasmids are highly efficient and convenient for transforming and co-transforming a broad range of V. carteri strains.

Transient expression of co-bombarded β-glucuronidase and β-1, 3-glucanase genes in immature cotton embryos

The immature embryos of Gossypium arboreum cv. RG8 were co-bombarded with two plasmids H1Z and pBI121 using Biolistic® PDS-1000/He Particle Delivery System. The two plasmids were coated onto tungsten particles in the ratio 1:1 at a concentration of 100ng/μl which was 10 times less than recommended in the standard protocol. A total of 1827 embryos were bombarded in 12 different experiments. The representative embryos from each bombardment experiment were analysed 48 hr post bombardment through beta glucuronidase assay called GUS assay and PCR. The histochemical results showed indigo colouration in the representative embryos from 11 experiments (91.6%). The gene specific PCR analysis on genomic DNA isolated 48 hr post bombardment from 12 representative bombarded embryos revealed 1764bp amplicon corresponding to β-1, 3-glucanase ORF in nine (75%) bombarded embryos. The results confirmed successful co-bombardment of two transgenes despite using ten times less concentration of plasmid DNA.

Factors Affecting Transient Gene Expression in Mango Tissue Using Particle Bombardment

Particle bombardment seems to be a promising method for genetic transformation of mango and the information on various factors affecting transient gene expression is inconclusive. Pro-embryonic masses (PEMs) of mango (Mangifera indica L. cv. Kensington Pride) used for particle bombardment were established and maintained in vitro using various liquid and semisolid media containing 2,4-D. Various factors affecting the biolistic transformation efficiency in mango and subsequent transient gene expression were optimized using β-glucuronidase (GUS) as a reporter gene driven by CaMV 35S promoter. Our results show that bombardment pressure significantly affects transient gene expression with the best results obtained from PEMs bombarded at 1200 kPa of helium pressure under vacuum. The application of osmoticum pre and post-bombardment also significantly increased the transient gene expression in the PEMs as compared to the controls. Mannitol (0.2 m) proved to be the best osmoticum in improving transient GUS expression as compared to sorbitol. The duration of exposure of PEMs to mannitol (0.2 m) both pre and post-bombardment also played a significant role in improving transient gene expression. The transient GUS expression was significantly highest with a pre-treatment of 0.2 m mannitol for 4 hours as compared to 0, 8 and 12 hours. The post-bombardment treatment of 0.2 m mannitol for 16 hours resulted in significantly highest transient gene expression as compared to 0, 4, 8, 12, 20 and 24 hours. In conclusion, PEMs of `Kensington Pride' mango bombarded at 1200 kPa, which were exposed to mannitol (0.2 m) for 4 and 16 hours pre and post bombardment respectively, resulted in the highest transient GUS expression (25.1 GUS foci/mg PEMs).

Assessment of DNA vaccine potential for juvenile Japanese flounder Paralichthys olivaceus, through the introduction of reporter genes by particle bombardment and histopathology

Genetic immunisation potential, following DNA bombardment for juvenile Japanese flounder, Paralichthys olivaceus was examined. GFP plasmids bombarded at two pressures, 150 and 300psi were sampled at 1, 7, 14 and 28 days, greater immunofluorescence was observed at the higher bombardment pressure. Histopathology, at 3 h post bombardment showed considerable damage to fish epithelial and dermal tissues when bombarded at pressures greater than 200 psi, with many DNA-coated gold particles present. At 150psi there was little pathology and no DNA-coated particles. Histopathology, up to 28 days again showed little pathology at 150 psi with few DNA-coated particles, whereas at 300 psi there was significant pathology observed with many DNA-coated particles seen in conjunction with the cytoplasm of inflammatory cells. By day 28 epithelial coverage was observed with tissue damage restricted to the dermal layer. Chloramphenicol acetyltransferase (CAT) assay showed long term and stable expression of the CAT protein from day 1 to day 60. The transcription activity of two promoters; pCMV-CAT and pSV2-CAT showed greater activity in the former. It was concluded that DNA vaccination potential for juvenile flounder is a viable option.

Preparation of Si Nanocrystals Using Anodic Porous Alumina Template Formed on Silicon Substrate

A novel technique to extend template application of anodic porous alumina to Si has been reported. First, porous alumina template about 400 nm thick was prepared on silicon substrate by anodizing thin aluminum film with high purity of 99.99% in 15 wt.% sulfuric acid under a constant voltage of 20 V and at an electrolyte temperature of 5°C. Then, amorphous Si layer approximately 50 nm in thickness was deposited onto the surface of template by using electron beam evaporation technique followed by an Xe ion beam bombardment, upon which as-coated Si layer at the pore mouth could be removed into pores smoothly. Three runs were performed by repeating above process of deposition and post bombardment. Finally, samples were annealed at 800°C for 30 min in nitrogen. Transmission electron microscopy and x-ray diffraction analysis reveal Si nanocrystals with a size of 15-20 nm being formed in the pores of template.

Ion beam synthesis of Au and Cu nanoclusters in MgO

Implantation of 2 MeV ions of Au or Cu into single crystals of MgO (1 0 0) at room temperature results in the spontaneous formation of metallic nanoclusters for implantation fluences above a threshold value. The formation and growth of these clusters may be induced by post implantation bombardment with silicon ions which stop beyond the implantation layer, as well as by thermal annealing. We have compared spontaneous nanocluster formation, ion bombardment induced cluster formation and post thermal annealing. The size and number of nanoclusters were studied by optical absorption spectroscopy and using the Mie and Doyle theories which relate the optical absorption bands to the formation of nanoclusters. The post bombardment of the gold implanted samples was made with 1.2, 2.0 and 5.0 MeV silicon ions. The depth and distribution of the gold and silicon were observed using RBS.

Post bombardment enhanced optical absorption in gold implanted silica

We have demonstrated that the post bombardment of 3.6 MeV Au implanted suprasil by 1.2 MeV boron, 2.0 MeV boron and 0.9 MeV alpha particles can induce the formation of Au nanoclusters. We have also demonstrated that post bombardment increases the annealing temperatures necessary for Au cluster formation beyond 1000°C. The optical absorption band characteristic of the Au implanted suprasil was used to monitor the formation of the metallic clusters before and after post bombardment as well as at each annealing temperature.

Effects of He and Ar post-bombardment on carbonitrides formed in a Cr-rich commercial steel

Cr-rich steel samples were implanted with nitrogen ions and post-irradiated with light particles (alphas and argon ions). The thermal evolution of the formed carbonitrides was followed by Conversion Electron Mossbauer Spectroscopy. The nitrogen profiles were determined through the nuclear reaction p(1αN, αγ)1αC also as a function of the annealings. The present results are compared with previous post bombardment data for the 1020 carbon steel.

Sputtering from Binary Alloys - A Technique for Evaluating Change in Surface Binding Energy

In recent years computer simulations, experimental observations and analyses of the collected sputtered products from the interaction of ion beams on solid materials have demonstrated the sensitivity of the sputtering process to the surface characteristics of the material being investigated or modified. The usual characterization of the target material has been a post bombardment analysis of the surface for the relative concentration of the implanted atoms compared with the initial target components coupled with an examination of the profile of the implanted atoms in the surface region. Recent experiments have collected sputtered products during ion implantation and have compared the distribution of sputtered particles with theory. Techniques have been developed to measure the partial and net sputtering yields from the amount of material collected on the foils in these experiments. Typically the evaluation of the target material and the collected sputtered products has been reported for the steady state conditions during ion implantation. Our experimental group has chosen to evaluate the sputtering process from the onset of ion bombardment through the steady state condition. From these experiments models have been proposed for the behavior of the surface composition and the sputtering yields. Techniques developed for monitoring of the surface have provided data supporting the concept that the average surface binding energy of the bombarded target material is sensitive to the sputtering process. Calculating the manner in which the surface binding energy will change with the developing atomic surface fraction can qualitatively lead to more precise prediction of implantation requirements.