Electric Discharge Particle Acceleration Research Articles

Genetic Transformation Via Biolistic Gene Gun Method and Regeneration of Common Bean

The objective of the present research was to utilize biotechnology to improve agronomical traits such as drought stress tolerance in common bean (Phaseolus vulgaris L.) as important recalcitrant crop species via a particle gun “Genebooster” for the bombardment of plant tissues with DNA. The process involves the high velocity acceleration of microprojectiles carrying foreign DNA, penetration of the cell wall and membrane by microprojectiles, and delivery of the DNA into plant cells. The initial phase of the two common bean varieties “Fonix” and “Maxidor” focused on the development of a highly regenerable tissue culture procedure amenable to the particle gun physical method. The results showed that MS medium supplemented with 1 mg/l BA and 0.1 mg/l NAA were the optimal for shoot induction from meristematic ring of cotyledonary node explants in the both bean varieties. The author has developed a tissue culture protocol that enables the “meristematic ring” that develops below axillary shoots on nodal bean explants to produce shoot buds, and developed an electric-discharge particle acceleration procedure to produce transgenic plants in the two common bean varieties. The plasmid pFF19K, harboring the kanamycin resistance gene nptII as a selectable marker and pFF19-mtlD harboring the mtlD gene which confers mannitol (drought stress) tolerance were used for adapting transformation in the two common bean varieties. The results indicated that the successful delivery of plasmids DNA and efficient gene transfer into common bean meristematic tissues included 20 bar nitrogen pressure, 135 mm shooting distance, 1.1 μm diameter size of microprojectiles, and twice bombarding of the target tissues. Multiple shoots are then generated and screened to recover transgenic plants at a rate of 7.6% and 8.4% in “Fonix” and “Maxidor” bean varieties, respectively. Transgenic plants were recovered using both kanamycin (100 mg/l) and mannitol (1.2 M/l) screening to introduce the neomycin phosphotransferase II (nptII) and the mannitol-1-phosphate dehydrogenase (mtlD) genes into the two common bean varieties. The introduced nptII and mtlD genes have been shown to confer strong resistance and tolerance in transgenic beans to kanamycin and mannitol stress applications. The transgenic bean plants were healthy and the method has proven to be applicable to common bean crop. To my knowledge, this is the first report on the production of common bean transgenic plants with mtlD gene using biolistic gun-mediated gene transfer system.

Electric discharge particle acceleration (Accell ®) technology for the creation of transgenic plants with altered characteristics

Recent advances in cell biology in combination with the development of direct DNA transfer techniques into organized tissue have resulted in a revolution in agricultural biotechnology. Transgenic plants, from almost all of the major agronomic crops, have been recovered and subjected to exhaustive molecular and genetic characterization. Numerous field trials have been carried out and we are about to witness release of the first products derived from recombinant DNA technology in the very near future. In this paper I will describe one technique, electric discharge particle bombardment, which has resulted in the recovery of transgenic plants, often in a cultivar-independent fashion. Transgenic cereals, legumes and woody species will be used as examples to illustrate advantages, as well as limitations, of this and alternative technologies. In addition, application of this and other relevant techniques to the engineering of secondary metabolites in medicinal and other plants will be discussed.

Recovery of Chimeric Rice Plants from Dry Seed using Electric Discharge Particle Acceleration

The development of an efficient system for the creation of transgenic rice plants from immature embryo tissue was described previously. That system resulted in the generation of clonal plants, probably derived from a single cell or a very small number of cells. Creation of chimeric cereal plants is of interest in studies directed towards the elucidation of developmental pathways. We were able to take advantage of the relative ease with which rice axillary buds, in a germinating seedling, can from embryogenic callus or be induced to form multiple tillers. Particle bombardment of such tissues, at various stages of development, resulted in the recovery of transgenic plants that were chimeric. The creation, origin and development of chimeric cereal plants can thus be studied and new insight into fundamental aspects of early plant development can be obtained. This regeneration/transformation system is compared with a previously developed protocol involving gene transfer into immature embryos which resulted in the exclusive recovery of clonal plants. It is also compared and contrasted with a system described earlier for soybean engineering that resulted on the recovery both chimeric and clonal plants.

Parameters Influencing Stable Transformation of Rice Immature Embryos and Recovery of Transgenic Plants using Electric Discharge Particle Acceleration

Recovery of transgenic rice plants from elite varieties was reported from the author's laboratory. By using electric discharge particle acceleration, recombinant technology was extended to commercially important japonica and indica rice cultivars not amenable to conventional transformation methodologies. Critical parameters influencing the recovery of transformed embryogenic callus from which transgenic plants were recovered have been defined. Such parameters included the physiological condition of explants prior to bombardment, DNA and gold particle loading rates, accelerating voltage, and depth of particle penetration. Selection for recovery of stable transformants was not critical in this transformation/regeneration system; both selected and non-selected tissues yielded transformed embryogenic callus and plants at approximately similar frequencies.

Molecular and genetic characterization of elite transgenic rice plants produced by electric-discharge particle acceleration

The recovery of transgenic rice plants expressing a number of exogenous genes was reported previously. Using immature embryo explants as the target tissue, plasmids containing both selectable and screenable marker genes were introduced into elite rice varieties via electric-discharge particle acceleration. Co-integration, copy number, expression, and inheritance of these genes were analyzed. A 100% co-integration frequency was confirmed by Southern-blot analyses of R0 plants. The majority of transgenic plants contained between one and ten copies of exogenous DNA and molecular and genetic analyses of progeny indicated that all copies in almost all R0 plants were inherited as a single dominant hemizygous locus. Co-expression of unselected genes ranged from 30-66% for gus/hmr constructs, depending on the promotor used, and up to 90% for bar/hmr constructs. The integrative structures of two unlinked transgenic loci of a rare R0 plant were analyzed in detail by Southern-blot analysis of its progeny.

The impact of selection parameters on the phenotype and genotype of transgenic rice callus and plants

Transgenic rice embryogenic callus and plants were recovered from experiments involving electric discharge particle acceleration (AccellTM technology). Critical parameters influencing successful delivery and stable integration of exogenous DNA into organized rice tissue had been identified previously. We report here on the effects of one selective agent (hygromycin B) on the phenotype and genotype of recovered callus and plants. The nature, timing and culture practices appeared to be more critical for the successful recovery of transgenic callus and plants than the level of selection used. By utilizing the procedures described in this report, transformation frequencies well in excess of 10% were obtained routinely in all varieties of rice tested. The combination of AccellTM technology with a selectable and prolific regeneration culture system resulted in the development of a variety-independent and highly efficient method for the routine introduction of any gene into any rice variety.

Whitefly Transmission and Efficient ssDNA Accumulation of Bean Golden Mosaic Geminivirus Require Functional Coat Protein

Bean golden mosaic geminivirus (BGMV) has a bipartite genome composed of two circular ssDNA components (DNA-A and DNA-B) and is transmitted by the whitefly, Bemisia tabaci . DNA-A encodes the viral replication proteins and the coat protein. To determine the role of BGMV coat protein in systemic infection and whitefly transmission, two deletions and a restriction fragment inversion were introduced into the BGMV coat protein gene. All three coat protein mutants produced systemic infections when coinoculated with DNA-B onto Phaseolus vulgaris using electric discharge particle acceleration "particle gun." However, they were not sap transmissible and coat protein was not detected in mutant-infected plants. In addition, none of the mutants were transmitted by whiteflies. With all three mutants, ssDNA accumulation of DNA-A and DNA-B was reduced 25- to 50-fold and 3- to 10-fold, respectively, as compared to that of wild-type DNA. No effect on dsDNA-A accumulation was detected and there was 2- to 5-fold increase in dsDNA-B accumulation. Recombinants between the mutated DNA-A and DNA-B forms were identified when the inoculated coat protein mutant was linearized in the common region.

Direct DNA transfer using electric discharge particle acceleration (ACCELL™ technology)

Direct DNA transfer methods based on particle bombardment have revolutionized plant genetic engineering. Major agronomic crops previously considered recalcitrant to gene transfer have been engineered using variations of this technology. In many cases variety-independent and efficient transformation methods have been developed enabling application of molecular biology techniques to crop improvement. The focus of this article is the development and performance of electric discharge particle bombardment (ACCELL™) technology. Unique advantages of this methodology compared to alternative propulsion technologies are discussed in terms of the range of species and genotypes that have been engineered, and the high transformation frequencies for major agronomic crops that enabled the technology to move from the R&D phase to commercialization.

Stable transformation of Phaseolus vulgaris via electric-discharge mediated particle acceleration

Transgenic Phaseolus vulgaris or common bean has been produced using electric-discharge particle acceleration. The method uses particle acceleration to introduce DNA into bean seed meristems. Multiple shoots are then generated and screened to recover transgenic plants at a rate of 0.03% germline transformed plants/shoot. We have been able to recover transgenic plants using both GUS and herbicide screening to introduce the gus, bar, and bean golden mosaic virus coat protein genes into the navy bean cultivar, Seafarer. The transgenic plants have been characterized over 5 generations of self-fertilization with no loss of introduced genes or expression. In addition, several families have been crossed with non-transgenic parents and these plants also show expected inheritance patterns. The introduced bar gene has been shown to confer strong resistance in transgenic beans to basta herbicide application in the greenhouse.

FOREIGN GENE EXPRESSION IN TRANSGENIC CRANBERRY PLANTS

Genetically transformed Vaccinium macrocarpon `Stevens' and `Pilgrim' plants have been obtained using electric discharge particle acceleration. Three foreign genes, kan encoding a selectable marker, gus a reporter gene, and B.t.k. conferring lepidopteran resistance, were incorporated into the genome. Expression of kan was assayed by culturing shoots in vitro on media with several concentrations of kanamycin. Expression among transformed clones (transclones) varied from high resistance (normal growth at 300 mg/L kan) to no resistance. Histochemical analyses for gus expression revealed variability among transclones. Some transclones exhibited no gus expression, others had consistent area-specific expression while others displayed random expression. In preliminary feeding trials with blackheaded fireworm larvae, B.t.k. expression was found to be ineffective at controlling insect development. We have recovered plants transformed with a different promoter driving the B.t.k. gene in an effort to enhance expression.

Prediction of germ‐line transformation events in chimeric Ro transgenic soybean plantlets using tissue‐specific expression patterns

SummaryChimeric soybean plants derived from electric discharge particle acceleration experiments were used to develop relationships between tissue‐specific expression patterns of a marker gene and germ‐line transformation events in Ro plants. Using the GUS histochemical assay the likelihood of germ‐line transformation was correlated to the localization of enzyme activity in specific tissues of the parent plant. The expression patterns of the 35S‐GUS marker gene in specific tissue types in Ro plants and correlation with transmission of the introduced gene to progeny allowed the production of a clear picture of the development of these plants and their germ‐line cells. Advances reported in this article resulted in the development of a commercial process for the genetic improvement of this important staple crop which until recently was very recalcitrant to conventional transformation methods.

Gene Transfer Using Electric Discharge Particle Bombardment and Recovery of Transformed Cranberry Plants

Genetic transformation of the American cranberry, Vaccinium macrocarpon Ait., was accomplished using electric discharge particle acceleration. Plasmid DNA containing the genes GUS (β-glucuronidase), NPTII (neomycin phosphotransferase II), and BT (Bacillus thuringiensis subsp. kurstaki crystal protein) was introduced into stem sections, derived from in vitro cultures, that had been induced to form adventitious buds. The stage of development of these adventitious buds was critical for efficient initial expression. After exposure to electric discharge particle acceleration, stem sections were cultured on a solid-phase bud-inducing medium containing 300 mg kanamycin/liter. In addition, a thin overlay of 300 mg kanamycin/liter in water was added to inhibit growth of nontransformed cells. Within 7 weeks, green shoots emerged amidst kanamycin-inhibited tissue. No escape (nontransformed) shoots were recovered, and 90% of the transformed shoots were shown through PCR and Southern blot analysis to contain all three introduced genes. GUS expression varied markedly among various transformed plants. Preliminary bioassays for efficacy of the BT gene against the feeding of an economically important lepidopteran cranberry pest have shown no consistently effective control. Potential problems with the expression of the BT and GUS genes are discussed

Production of Transgenic Rice (Oryza Sativa L.) Plants from Agronomically Important Indica and Japonica Varieties via Electric Discharge Particle Acceleration of Exogenous DNA into Immature Zygotic Embryos

We have recovered transgenic rice plants from a number of commercially important cultivars, including until now recalcitrant Indica varieties, using electric discharge particle acceleration. Immature embryos from greenhouse–grown plants were bombarded with gold particles carrying DNA, and transgenic plants were recovered following a simple culture protocol. Mendelian segregation of foreign genes was observed in R1 progeny and stable integration was demonstrated by Southern blot analysis of genomic DNA isolated from progeny plants. Alternative transformation protocols that are dependent on the development of protoplast and suspension culture systems are no longer necessary as we have shown that a wide variety of diverse cultivars can be transformed. Transgenic plants expressing agronomically useful traits such as herbicide resistance have been obtained and are currently undergoing further evaluation. This report also demonstrates that it is possible to produce transgenic monocoty–ledonous plants by transforming scutellar tissue of immature embryos.

Stable transformation of Populus and incorporation of pest resistance by electric discharge particle acceleration

Three different target tissues (protoplast-derived cells, nodules, and stems) and two unrelated hybrid genotypes of Populus (P. alba x P. grandidentata 'Crandon' and P. nigra 'Betulifolia' x P. trichocarpa) have been stably transformed by electric discharge particle acceleration using a 18.7 kb plasmid containing NOS-NPT, CaMV 35S-GUS, and CaMV 35S-BT. Four transformed plants of one hybrid genotype, NC5339, containing all 3 genes were recovered and analyzed. Two expressed GUS and one was highly resistant to feeding by 2 lepidopteran pests (the forest tent caterpillar, Malacosoma disstria, and the gypsy moth, Lymantria dispar.) Pretreatment of the target tissues, fine-tuning of the bombardment parameters, and the use of a selection technique employing flooding of the target tissues were important for reliable recovery of transformed plants.

Cloning of the Complete DNA Genomes of Four Bean-Infecting Geminiviruses and Determining Their Infectivity by Electric Discharge Particle Acceleration

Geminiviruses are small plant viruses that encapsidate a single-stranded circular DNA and form double-stranded DNAs in infected plants. Infection of economically important hosts with cloned DNAs of geminiviruses frequently has been difficult (...)

Morphological Description of Transgenic Soybean Chimeras Created by the Delivery, Integration and Expression of Foreign DNA Using Electric Discharge Particle Acceleration

Des plants transgeniques de soja sont obtenus par bombardement de particules sur des explants. Les explants transformes montrent differents degres de chimerisme suivis par l'expression du gene de la β glucuronidase (gus). Son expression est observee par la technique des spots bleus dans les feuilles ou les tiges des transformants. La presence des genes est confirmee par analyse en Southern blot

STABLE GENETIC TRANSFORMATION OF CRANBERRY USING ELECTRIC DISCHARGE PARTICLE ACCELERATION

Electric discharge particle acceleration was used to introduce three foreign genes into the American cranberry (Vaccinium macrocarpon Ait.). These genes were NPTII (conferring resistance to the antibiotic, kanamycin), GUS (allowing for visual verification), and B.t. (conferring resistance to lepidopteran insects). Adventitious buds were induced on stem sections prior to bombardment with DNA-coated gold pellets. Bombarded stem sections were then transferred to a selection medium containing kanamycin. The surface of the medium was overlaid with a thin layer of kanamycin solution. Approximately 35 days after blasting, proliferating cell masses and elongating shoots were observed amidst the mass of kanamycin-inhibited tissue. Seven weeks after blasting, a histochemical assay verified GUS expression in these tissues, and polymerase chain reaction was used to confirm the presence of the introduced genes.

Soybean transformation by electric discharge particle acceleration

By the use of a direct DNA‐delivery system based on electric discharge particle acceleration we have been able to obtain transgenic soybean plants expressing the neomycin phosphotransferase II and the β‐glucuronidase genes. Techniques for efficient introduction of foreign genes into agronomically important crop plants have been limited by the inability to regenerate fertile plants from single cells or by the limited hostrange of Agrobacterium vectors. In the case of soybean (Glycine max) the problem has been compounded due to the lack of a regeneration method compatible with existing transformation technology. In a commercial genetic engineering program the following points need to be considered: (a) not all crops/cultivars can be regenerated from transformed tissues, (b) long time frames are required for the regeneration of transgenic plants from callus, (c) Agrobacterium has a rather limited host specificity and (d) problems associated with somaclonal variation.

Soybean transformation by electric discharge particle acceleration

Soybean transformation by electric discharge particle acceleration

Cell type specific expression of a CaMV 35S‐GUS gene in transgenic soybean plants

AbstractA number of independently derived transgenic soybean plants expressing a chimeric β‐glucuronidase (GUS) gene under the control of the 355 CaMV promoter and a nopaline synthase polyadenylation signal were recovered using direct DNA transfer via electric discharge particle acceleration. Expression of GUS in R, plants was localized using thin tissue sections. Many tissue types expressed GUS at various levels. Pericycle cells in root, parenchyma cells in xylem, and phloem tissues of stem and leaf had high levels of enzyme activity. Procambium, phloem, and cortex cells in root, vascular cambium cells in stem, and the majority of cortex cells in leaf midrib, expressed low or no GUS activity. Intermediate levels of GUS activity were detected in leaf mesophyll cells, certain ground tissue cells in stem and leaf midrib, and in trichome and epidermal guard cells. Thus, we conclude that the 35S CaMV promoter is cell‐type specific and is developmentally regulated in soybean.