Improved domestic animal productivity is necessary in order to provide for an increasing world population over the next two to three decades and such improvement would be aided by an increase in the efficiency of nutrient utilization. This can be achieved by conventional genetic selection protocols but progress by this approach is slow. A more rapid but as yet largely unproven technique is the direct modification of the genome which can be achieved by the transfer of recombinant DNA to the nuclei of early embryos. This new technology is potentially powerful because it allows the direct transfer of genes without regard to inter-species barriers to breeding. However, it raises a new set of problems associated with the integration and expression of the foreign genetic information in the new genome. In this review the application of the technology to increasing nutrient utilization and increased productivity are discussed. Two areas have received substantial attention in the 15 years since the technique was first applied to domestic animals. First, the current status of the modification of growth hormone levels to improve productivity and feed utilization efficiency is reviewed, with current results suggesting that several of the projects may soon be approaching field trial status. Second, the introduction of novel biochemical pathways to domestic animals to provide them with different sources of the substrates required for growth and production is discussed. Recent results obtained in the introduction of a cysteine biosynthetic pathway to animals is reviewed. While this line of research remains some distance from commercial application, it provides a useful example of the powerful possibilities inherent in the new technology. However, it also serves to highlight some of the difficulties that might be expected as new genes are expressed to produce enzymes that must fit compatibly with existing animal biochemistry.

Increasing the growth rate is especially important for low-quality wood applications, so this has become an important goal in poplar breeding. The present study describes the transfer of Vitreoscilla haemoglobin (VHb) gene (vgb) driven by constitutive promoters, by Agrobacterium tumefaciens into poplar (Populus alba×P. glandulosa). From about 450 leaf discs used for transformation, 60 Kan-resistant plants were obtained, and 52 proved to be true transgenic plants. The transgenic nature of these plants was confirmed by polymerase chain reaction (PCR) amplification and Southern dot blot hybridization. The expression of vgb gene in transgenic plants was confirmed by reverse transcriptase-PCR (RT-PCR). The performance of the transgenic lines was evaluated during the first year of growth in a greenhouse. These plants showed no significant stable morphological differences from the untransformed plants. Among them, three vgb-transgenic lines exhibited noticeably higher growth rates in terms of height and diameter.

Previous studies have shown that the widely used plant transformation vector Agrobacterium tumefaciens can persist in genetically engineered plants in vitro and in transgenic greenhouse-grown plants, despite the use of counter-selective antibiotics. However, little is known regarding Agrobacterium persistence in tree species. To understand the kinetics of A. tumefaciens decline and persistence in transformation experiments, we assayed for the presence of A. tumefaciens in spruce and pine embryogenic tissue for up to 10 weeks post-transformation. The A. tumefaciens populations declined rapidly in the first five days post-cocultivation but generally declined more slowly in pine, relative to spruce. No bacteria were detected in spruce embryogenic tissue beyond four weeks after cocultivation, however in pine there were ~100 colony forming units per g tissue at 10 weeks post-cocultivation. We present evidence that the detection limit for PCR using virD2 primers to detect A. tumefaciens in a background of pine needle DNA was approximately 109–1010 A. tumefaciens cells per g of tissue. We also assayed for A. tumefaciens in transgenic pine and spruce embryogenic tissue and from needles, branches, stems and roots of transformed plants, up to four years post-inoculation. Occasionally A. tumefaciens was detected in embryogenic tissue up to 12 months post-inoculation. A. tumefaciens was never detected in cultured embryogenic tissue more than twelve months after inoculation, nor in developing somatic embryos or germinating plantlets, nor any of the parts of greenhouse-grown plants. From these data we conclude that if A. tumefaciens persists in transgenic conifers, it does so beneath our ability to detect it.

Cassava is an important subsistence crop grown only in the tropics, and represents a major source of calories for many people in developing countries. Improvements in the areas of resistance to insects and viral diseases, enhanced nutritional qualities, reduced cyanogenic content and modified starch characteristics are urgently needed. Traditional breeding is hampered by the nature of the crop, which has a high degree of heterozygosity, irregular flowering, and poor seed set. Biotechnology has the potential to enhance crop improvement efforts, and genetic engineering techniques for cassava have thus been developed over the past decade. Selectable and scorable markers are critical to efficient transformation technology, and must be evaluated for biosafety, as well as efficiency and cost-effectiveness. In order to facilitate research planning and regulatory submission, the literature on biosafety aspects of the selectable and scorable markers currently used in cassava biotechnology is surveyed. The source, mode of action and current use of each marker gene is described. The potential for toxicity, allergenicity, pleiotropic effects, horizontal gene transfer, and the impact of these on food or feed safety and environmental safety is evaluated. Based on extensive information, the selectable marker genes nptII, hpt, bar/pat, and manA, and the scorable marker gene uidA, all have little risk in terms of biosafety. These appear to represent the safest options for use in cassava biotechnology available at this time.

This study was aimed at developing a hormonal treatment protocol in order to optimize the proportion of pronuclear-stage embryos to be used for DNA microinjection in a goat transgenic founder production programme. A total of 46 adult BELE® and 47 adult standard goats (1–5 years old) were used as donors and recipients, respectively. They were heat-synchronized using intravaginal sponges containing 60 mg medroxyprogesterone acetate for 10 days with an injection of 125 μg cloprostenol on the morning of the eighth day. Recipients were injected with 400 IU eCG at the time of sponge removal while donors received a total of 133 mg NIH-FSH-P1 (Folltropin-V) given twice daily in decreasing doses over 3 days starting 48 h before sponge removal. Ovulation was induced in donors by injecting 100 μg of GnRH at 24 h (GnRH24) or 36 h (GnRH36) after sponge removal. Embryo recovery was performed by oviduct flushing following a standard mid-ventral laparotomy procedure. The proportion of embryos in the pronuclear stage of development was higher in the GnRH36 group (90% vs 34%, p<0.01). Embryos were microinjected with a DNA expression cassette followed by transfer to the oviduct of synchronized recipients. A higher, yet not statistically significant, pregnancy rate was found in the recipients transferred with pronuclear-stage embryos compared with those transferred with 2-cell-stage embryos (64% vs 37%, chi-square p=0.06). One transgenic female founder was produced from the group of recipients transferred with pronuclear-stage microinjected embryos.

We had previously developed a methodology to introduce foreign DNA into mouse eggs and embryos using cationic lipids as vectors. In this report we use this technique to produce transgenic animals. Mouse embryos at the pronuclear stage were transfected using a mixture of a plasmid DNA, encoding for a nuclear form of β-galactosidase, and a commercial lipid transfection reagent. Embryos were washed and incubated overnight. Those that cleaved and develop to the 2-cell stage of normal appearance were transferred to the Fallopian tubes of pseudopregnant foster mothers. We analysed a total of 158 offspring and found two, a female and a male, to be transgenic (1.27% of the total). Integration of the foreign DNA in the female was showed by Southern blot. Both animals expressed the lacz in several organs, but none of them either displayed expression in the germ cells or transmitted the transgene to their offspring. Taken together our results show that lipid transfection can generate transgenic mice, but the efficiency needs to be improved for this method to be widely applied.