Particle-mediated delivery of a DNA expression vector encoding the hemagglutinin (HA) of an H1N1 influenza virus (A/Swine/Indiana/1726/88) to porcine epidermis elicits a humoral immune response and accelerates the clearance of virus in pigs following a homotypic challenge. Mucosal administration of the HA expression plasmid elicits an immune response that is qualitatively different than that elicited by the epidermal vaccination in terms of inhibition of the initial virus infection. In contrast, delivery of a plasmid encoding an influenza virus nucleoprotein from A/PR/8/34 (H1N1) to the epidermis elicits a strong humoral response but no detectable protection in terms of nasal virus shed. The efficacy of the HA DNA vaccine was compared with that of a commercially available inactivated whole-virus vaccine as well as with the level of immunity afforded by previous infection. The HA DNA and inactivated viral vaccines elicited similar protection in that initial infection was not prevented, but subsequent amplification of the infection is limited, resulting in early clearance of the virus. Convalescent animals which recovered from exposure to virulent swine influenza virus were completely resistant to infection when challenged. The porcine influenza A virus system is a relevant preclinical model for humans in terms of both disease and gene transfer to the epidermis and thus provides a basis for advancing the development of DNA-based vaccines.

Resistance to bialaphos, a non-selective herbicide, was intro duced into cotton through genetic engineering. A gene encoding phosphinothric in acetyltransferase (bar) from Streptomyces hygroscopicus was inserted into elite varieties of cotton through particle bombardment. Based on the marker gene, β-glucuronidase (gus) expression, a total of 18 Pima (Gossypium barbadense), 45 DP50 (G. hirsutum L.), 20 Coker 312 (G. hirsutum) and 2 El Dorado (G. hirsutum) transgenic plants were recovered. Integration of the bar gene into cotton genomic DNA was confirmed by Southern blot analysis and gene expression was confirmed by northern blot and enzyme assays. Herbicide (Basta®) tolerance up to 15 000 ppm was demonstrated in greenhouse trials. The newly introduced herbicide tolerance trait is inherited in a Mendelian fashion in the progenies of germline transformants. This study demonstrates the potential for particle bombardment to introduce commerically important genes directly into elite varieties of cotton. This mode of gene transfer can expedite the introduction of transgenic cotton products into world markets

Nucleic acid immunization involves the direct in vivo administration of antigen-encoding plasmid DNA molecules that results in the de novo production of correctly folded microbial antigens at the site of DNA delivery. While this process can lead to the development of neutralizing antibody responses recognizing authentic protein conformations, in vivo antigen production also results in epitope presentation via the MHC class I antigen processing pathway, leading to the elicitation of cytotoxic cellular immune responses. Recent efforts in the authors' laboratories have focused on use of the Accell® gene delivery system (gene gun) to achieve the direct, intracellular delivery of small quantities of DNA into cells of the epidermis. The gene gun approach to nucleic acid vaccination capitalizes on the synergistic combination of an effective DNA delivery system and a target tissue that serves as a major immunological inductive site. Experimental gene gun-based nucleic acid vaccines can achieve potent humoral and cytotoxic cellular immune responses in rodent models following immunization with as little as 16 ng of DNA. Equally strong responses have also been elicited in larger animals, such as pigs and monkeys, following epidermal immunization with as little as 2 to 4 μg of DNA.

A variety-independent protocol was developed to allow the production of transgenic cotton. High velocity gold beads coated with DNA were used to deliver foreign genes directly into the meristematic tissue of excised embryonic axes. Bombarded explants were allowed to develop into plants which were subsequently screened for gus gene activity. Buds in the axils of transformed leaves were forced to develop into plants by pruning away non-transformed primary shoot tips. Plants derived from this process carried the foreign gene in one or more of their tissue layers. Transformation frequencies varied with the genotype used, but all cultivars attempted to date have yielded transgenic progeny. Molecular and genetic characterization of primary transformants and their progeny established that foreign genes were stably integrated and transmitted to progeny in a Mendelian fashion.

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.

By defining the somatic embryo developmental stage which expressed beta-glucuronidase (GUS) at a high level yet was also competent to form embryogenic callus at a high frequency under selection, we obtained transformed Picea glauca (white spruce) embryogenic callus, embryos and seedlings expressing GUS in all cells. Plasmid DNA, containing three chimeric constructs [enhanced cauliflower mosaic virus (CaMV) 35s-GUS, nopaline synthase-neomycin phosphotransferase (NPTII), and CaMV 35s-Bacillus thuringiensis (B.t.) cryIA endotoxin] was introduced into four developmental stages of white spruce somatic embryos by particle acceleration. Transient expression was observed in all of the stages of somatic embryos tested, but transformed embryogenic callus was induced only from the two most advanced. Embryogenic callus was identified by histochemical staining for GUS as early as 6 weeks following particle acceleration. All GUS positive embryogenic callus lines also showed NPTII activity. Incorporation of the introduced genes into the genome was confirmed by PCR and Southern blot analysis of embryogenic callus and regenerated transformed plants. Plants derived from several transformed embryogenic callus lines are currently undergoing acclimatization in the greenhouse. Spruce budworm (Chorisoneura fumiferana) feeding trials with embryogenic callus and transformed seedlings indicate a low, sublethal level of Bt. expression.