Particle Gun Method Research Articles

Expression of a Bacterial Chitinase (<i>ChiB</i>) Gene Enhances Resistance against <i>Erysiphae polygoni</i> Induced Powdery Mildew Disease in the Transgenic Black Gram (<i>Vigna mungo</i> L.) (cv. T9)

To enhance the antifungal response of blackgram (Vigna mungo L.), transgenic plants were generated by transferring bacterial chitinase gene with a CaMV 35S promoter. The chopped multiple shoot cells developed on the cotyledonary node were transformed by Particle gun method. Thecalli were raised on the Murashige and Skoog (MS) modified media supplemented with 50 m·gl-1 kanamycin. The transformation efficiency was 13% approximately. The resultant shoot buds were selected and the antibiotic resistant transgenic plantlets were regenerated. The development of the transgenic plants from the shoot buds took about four to six months. The integration of the transgene was confirmed by PCR, RT-PCR, Southern and western blot analyses. The transgenic plants exhibited higher chitinase activity than the non-transformed ones. The chitinase activity was examined by native polyacrylamide in-gel assay. The transgenic plants showed fungal tolerance as evidenced by the delayed onset of the disease and smaller lesions following an in vitro inoculation of the powdery mildew pathogen (Erysiphae polygoni DC). The transgenic plants adapted well to the greenhouse and did not show any phenotypic alterations.

Stable plastid transformation in Scoparia dulcis L.

In the present investigation we report stable plastid transformation in Scoparia dulcis L., a versatile medicinal herb via particle gun method. The vector KNTc, harbouring aadA as a selectable marker and egfp as a reporter gene which were under the control of synthetic promoter pNG1014a, targets inverted repeats, trnR/trnN of the plastid genome. By use of this heterologous vector, recovery of transplastomic lines with suitable selection protocol have been successfully established with overall efficiency of two transgenic lines for 25 bombarded leaf explants. PCR and Southern blot analysis demonstrated stable integration of foreign gene into the target sequences. The results represent a significant advancement of the plastid transformation technology in medicinal plants, which relevantly implements a change over in enhancing and regulating of certain metabolic pathways.

Effect of Seeding and pH Conditions on the Size and Shape of Au Nanoparticles in Reduction Crystallization

AbstractEfficient and simple methods for gene integration into various plants have been studied by many researchers. The particle gun method using fine metal particles is one of the most potent technologies. However, it is difficult to control the particle size distribution and shape of the metal particles and, thus, to design and create adequate particles of suitable quality for the gene‐supporting media. Here, reduction crystallization of nanometer‐ and submicron‐sized Au was investigated in order to clarify the effects of the seeding and reaction conditions on the sizes and shapes of Au particles. The seed size has an influence on the surface roughness of the Au nanoparticles. Experiments to form Au particles were also performed using pH‐controlled ascorbic acid. The pH has a great influence on the size and shape of nano/submicron Au particles and the mechanism of reduction crystallization.

GM foods - a controversy between tradition and modernity

Genetically engineered plants are developed in a laboratory by altering their genetic makeup and are evaluated in the laboratory for desired qualities. This is accomplished by adding one or few genes to a native or a local genome using genetic engineering techniques. Genetically modified plants are generated by the biolistic particle gun method or by Agrobacterium tumefaciens mediated transformation. When plants of desired quality are produced, sufficient seeds are multiplied and the companies producing them have to apply for regulatory approval for field trials.

Enhanced single copy integration events in corn via particle bombardment using low quantities of DNA

Transgene copy number is an important criterion for determining the utility of transgenic events. Single copy integration events are highly desirable when the objective is to produce marker free plants through segregation or when it is necessary to introgress different transgenes into commercial cultivars from different transgenic events. In contrast multi-copy events are advocated by several authors for higher expression of the transgene. Till recently, it was thought that employment of the particle gun for transformation results in the production of a high proportion of multi-copy events often with complex integration pattern when compared to Agrobacterium-mediated transformation. However, it has been demonstrated that usage of cassette DNA for bombardment in place of whole plasmids would result in simple insertion pattern of the transgenes. While investigating the effect of varying the cassette DNA amount on stable transformation, the frequency of occurrence of low copy events was observed to increase when lower doses of cassette DNA was employed for bombardment. Large scale experimentation with rigorous statistical analysis performed to verify the above observations employing Helium gun and the Electric discharge gun for gene delivery confirmed the above observations. Helium gun experiments involving production of more than 1,600 corn events consistently yielded single copy events at higher frequencies at lower cassette DNA load (46% at 2.5 ng/shot) as compared to higher cassette DNA load (29% at 25 ng/shot) across 18 independent experiments. Results were nearly identical with the Electric discharge particle gun device where single copy events were recovered at frequencies of 54% at 2.5 ng cassettes DNA per shot as compared to 18% at 25 ng cassette DNA per shot. The transformation frequency declined from 41 to 34% (Helium gun) and from 48 to 31% (Electric discharge gun) with reduction in cassette DNA quantity from 25 to 2.5 ng per shot. This reduction in the transformation frequency is more than compensated by the savings in time and effort involved in the production and screening of events if the desired outcome is single copy events. These results demonstrate the flexibility of the particle gun method for controlling the frequency of production of either low copy or high copy events by altering the quantity of cassette DNA used for bombardment. The transgene expression levels over generations in relation to its integration need further investigations.

Stickiness of skim milk powder using the particle gun technique

The particle gun method for investigating the initiation of stickiness of dairy powders was examined using skim milk powder (SMP). The point at which significant stickiness occurred was at a constant temperature above the glass transition temperature ( T g) of amorphous lactose regardless of the temperature and relative humidity (RH) conditions of the air in the particle gun. This point has been called ( T– T g) critical. The initial water activity of SMP did not significantly affect ( T– T g) critical, confirming that stickiness is a surface phenomenon. Changing the particle feed rate did not effect ( T– T g) critical or the rate at which the powder accumulated on the target plate. The errors in measuring the value of ( T– T g) critical and the rate of stickiness development [slope of the deposition line versus ( T– T g)] for SMP were found to be 33.6 ± 0.8 °C and 3.1 ± 0.7% deposition °C −1, respectively, after standardisation of the ambient air conditions around the powder feeder and the initial water activity of the SMP.

Gene transfer for Japanese flounder fertilized eggs by particle gun bombardment

Fish transgenesis has progressed considerably. However, the technique of gene transfer for most marine aquaculture species has not been established. A method to introduce foreign genes into fertilized eggs of Japanese flounder Paralichthys olivaceus by particle gun bombardment was developed by the authors. A recombinant plasmid which contains the Japanese flounder keratin gene promoter linked to the green fluorescence protein (GFP) gene was introduced into fertilized eggs by particle gun bombardments twice at 250 psi. In each experimental group, 25000–35000 eggs were treated. However, the survival rate was 12.8% which is lower than that of the control groups (56.8%). Of 2606–5205 the embryos survived for 24h, 43–61 GFP positive embryos were obtained in one experiment, giving a final gene transfer efficiency of 1.4%. All GFP-positive embryos developed and hatched normally. GFP expression was observed in epithelial tissue throughout the developmental stages. At 3 months after gene transfer, foreign DNA was detected by genome polymerase chain reaction analysis in 37 of 69 fry (53.6%). These results suggest that the particle gun method is an effective method to use with fertilized Japanese flounder eggs.

A transgene and its expression profile are stably transmitted to offspring in transgenic medaka generated by the particle gun method.

A particle gun is used in a potential method for introducing foreign genes into fish. In this paper, we report on the stable transmission of a transgene and its expression profile of the F4 generation in the transgenic medaka (Oryzias latipes). We established four transgenic strains, which contained a green fluorescent protein (GFP) gene controlled by a medaka beta-actin promoter, using a particle gun. One more transgenic strain was also generated by microinjection for comparison. In all five strains, the founder was discovered to be mosaic for the transgene. However, from the F1 to F4 generations, transgenes and their expression profiles were stably inherited in the Mendelian manner. The expression profile was common among the five strains regardless of the method for gene transfer: GFP fluorescence became detectable at an early neurula stage. In this stage, the fluorescence was observed ubiquitously in most tissues. As somite developed, GFP fluorescence became intense only in the skeletal muscle and lens but it decreased in other tissues. In adult fish, an intense fluorescence was restricted in the skeletal muscle and lens, while a considerably weak fluorescence was observed in the brain, gill, heart, kidney, spleen, and ovary. From these results, it was concluded that the transgene and its expression profile were stably transmitted to offspring, and thus the particle gun is an effective method for transgenesis in spite of its easiness.

Utilization of a particle gun DNA introduction system for the analysis of cis-regulatory elements controlling the spatial expression pattern of the arylsulfatase gene (HpArs) in sea urchin embryos.

We applied a particle gun method to introduce DNA into fertilized sea urchin eggs for the analysis of cis-regulatory elements responsible for spatial gene expression during development. We introduced HpArs (sea urchin arylsulfatase gene) -GFP and HpArs-LacZfusion constructs into the fertilized eggs and obtained high expression levels of the fusion genes. Using this assay system, we demonstrated that a fragment of HpArs (-3,484 to +4,636) is sufficient for aboral ectoderm-specific expression, and that the region in the first intron from +406 to +1,993 contains the control elements responsible for the repression of the HpArs promoter activity in secondary mesenchyme cells.

Expression of Sugar Transporters by In Vivo Electroporation and Particle Gun Methods in the Rat Liver: Localization to Specific Membrane Domains.

To see the cellular localization mechanism of membrane proteins in the hepatocyte in situ, we introduced cDNAs of facilitated-diffusion sugar transporters, GLUT1, GLUT3, GLUT4, and GLUT5, and a Na+-dependent active sugar transporter SGLT1 into the rat liver by the in vivo electroporation method and the particle gun method, and the localization of their products was analyzed by immunofluorescence microscopy. SGLT1 was strictly restricted to the bile canalicular (apical) domain, whereas GLUT1 was found in the sinusoidal (basolateral) membrane of hepatocytes. GLUT3 and GLUT5 were present along the whole aspects of the plasma membrane with a tendency for the bile canalicular membrane to be enriched with transporters as compared with the sinusoidal membrane. GLUT4 remained in the intracellular compartments. Simultaneous expression of two of the transporters confirmed these results. Compared with membrane localization of sugar transporters in MDCK cells, GLUT1, GLUT4 and SGLT1 exhibited a similar localization pattern. On the other hand, localization of GLUT3 and GLUT5 was different from that in MDCK cells. These observations suggest that hepatocytes in situ may have a different localization mechanism for sugar transporters from that in MDCK cells. In addition, the in vivo electroporation and the particle gun methods seem to be useful tools for the introduction and analysis of foreign genes in the liver in situ.

Inheritance and expression of multiple disease and insect resistance genes in transgenic rice

2–3 anti-fungal disease genes are coinserted with hygromycin phosphotransferase in the same vector. Two insecticidal genes and PPT acetyl transferase genes are placed in another one. The vectors are co-delivered to rice embryonic cellus tissue at a molar ratio of 1 : 1 using the particle gun method. 55 independent regenerated lines have been obtained through screening for hygromycin resistance. Of these, 70% transgenic plants harbor 6–7 foreign genes. The genes on the same vectors are always co-delivered to rice plant. Northern blot analysis has indicated that the multiple foreign genes give stable expression. In the 6 transgenic plants carrying 6–7 foreign genes, multiple foreign genes tend to integrate in 1 or 2 genetic loci. Progeny segregation is consistent with Mendel’s 3 : 1 segregation law. 8 homozygous R1 transgenic plants harboring 2–3 anti-fungal and 2 insecticidal genes are selected from large number of transgenic progeny screening for hygromycin and Basta resistance.

Introduction of a foreign gene into medakafish using the particle gun method

Introduction of a foreign gene into medakafish using the particle gun method

Introduction of a foreign gene into medakafish using the particle gun method

We developed a procedure to introduce a foreign gene into fertilized eggs of medakafish (Oryzias latipes) using the particle gun method, which is one of the easiest and most reliable techniques for gene transfer. A plasmid construct with the green fluorescence protein (GFP) gene driven by the madakafish beta-actin gene promoter was successfully introduced into eggs, and the expression of GFP was observed in 20% of the primary transfectant (chimera) fish. In addition, germ line transmission of GFP was observed in 13% of the GFP-positive primary transfectant fish. The new application described here should enable us to investigate gene expression using the fish model on a routine basis without high technical sophistication. J. Exp. Zool. 287:285-293, 2000.

Perspectives on the application of biotechnology to assist the genetic enhancement of plantain and banana (Musa spp.)

Bananas and plantains (Musa spp.) are the most important tropical fruit crops. They form an integral component of the farming systems in the humid agroecological zones of the tropics. A broad array of applied cell and molecular techniques are increasingly being used worldwide to facilitate and enhance the handling and improvement of plantain and banana germplasm. Tissue culture is used for germplasm exchange, conservation and rapid multiplication, while in vitro seed germination (based on embryo culture or rescue) plays a critical role in generating hybrid plants. DNA marker systems have been developed in Musa to assist germplasm management, selection within the breeding pool or gene introgression from wild species, and for disease diagnosis. Likewise, genetic transformation using the particle gun method or through Agrobacterium co-cultivation shows potential for the genetic betterment of the crop. This article discusses the applications of biotechnology for the genetic enhancement of banana and plantain. It highlights current advances by research teams across the world and reviews progress in molecular breeding of Musa by the International Institute of Tropical Agriculture and its collaborators. Plantains and bananas (Musa spp.) are staple foods for rural and urban consumers in the humid tropics and an important source of rural income, particularly in some locations where smallholders produce them in compound or home gardens. World Musa production is around 85.5 million tonnes annually (FAO 1998), of which bananas cultivated for the export trade account for only 10%. Hence, fruit harvested from bananas and plantains are important components of food security in the tropical world, and provide income to the farming community through local and international trade. Most banana cultivars and all plantain landraces have 33 chromosomes (2n = 3x). These triploid genotypes are virtually or completely sterile and develop their fruit by vegetative parthenocarpy. Diploid landraces and tetraploid cultivars (mostly artificial hybrids) are also cultivated. The

Gene insertion into Hevea brasiliensis

A transformation system has been developed for Hevea brasiliensis using the particle gun method. Anther derived calluses were transformed with vectors harbouring the ß-glucuronidase (gus) gene, the neomycin phosphotransferase (nptII) gene, and the chloramphenicol acetyl transferase (cat) gene. Gene transfer was determined by histochemical staining and fluorometric assay for ß-glucuronidase activity, enzyme linked immunosorbent assay for detecting neomycin phosphotransferase II gene and direct enzyme assay for detection of expression of the cat gene. These independent assays all showed a several-fold increase, compared to control values, in gene product level and enzyme activity in extracts from transformed callus and embryoids of Hevea. These results were confirmed using polymerase chain reaction with primers designed to amplify an internal gus fragment. Together, the results show the feasibility of the particle gun method for the introduction of foreign genes into Hevea.