EPA and DHA are required for normal cell function and can also induce health benefits. Oily fish are the main source of EPA and DHA for human consumption. However, food choices and concerns about the sustainability of marine fish stocks limit the effectiveness of dietary recommendations for EPA + DHA intakes. Seed oils from transgenic plants that contain EPA + DHA are a potential alternative source of EPA and DHA. The present study investigated whether dietary supplementation with transgenic Camelina sativa seed oil (CSO) that contained EPA and DHA was as effective as fish oil (FO) in increasing EPA and DHA concentrations when consumed as a dietary supplement in a blinded crossover study. Healthy men and women (n 31; age 53 (range 20–74) years) were randomised to consume 450 mg/d EPA + DHA provided either as either CSO or FO for 8 weeks, followed by 6 weeks washout and then switched to consuming the other test oil. Fasting venous blood samples were collected at the start and end of each supplementation period. Consuming the test oils significantly (P < 0·05) increased EPA and DHA concentrations in plasma TAG, phosphatidylcholine and cholesteryl esters. There were no significant differences between test oils in the increments of EPA and DHA. There was no significant difference between test oils in the increase in the proportion of erythrocyte EPA + DHA (CSO, 12 %; P < 0·0001 and FO, 8 %; P = 0·02). Together, these findings show that consuming CSO is as effective as FO for increasing EPA and DHA concentrations in humans.

EPA and DHA are important components of cell membranes. Since humans have limited ability for EPA and DHA synthesis, these must be obtained from the diet, primarily from oily fish. Dietary EPA and DHA intakes are constrained by the size of fish stocks and by food choice. Seed oil from transgenic plants that synthesise EPA and DHA represents a potential alternative source of these fatty acids, but this has not been tested in humans. We hypothesised that incorporation of EPA and DHA into blood lipids from transgenic Camelina sativa seed oil (CSO) is equivalent to that from fish oil. Healthy men and women (18–30 years or 50–65 years) consumed 450 mg EPA + DHA from either CSO or commercial blended fish oil (BFO) in test meals in a double-blind, postprandial cross-over trial. There were no significant differences between test oils or sexes in EPA and DHA incorporation into plasma TAG, phosphatidylcholine or NEFA over 8 h. There were no significant differences between test oils, age groups or sexes in postprandial VLDL, LDL or HDL sizes or concentrations. There were no significant differences between test oils in postprandial plasma TNFα, IL 6 or 10, or soluble intercellular cell adhesion molecule-1 concentrations in younger participants. These findings show that incorporation into blood lipids of EPA and DHA consumed as CSO was equivalent to BFO and that such transgenic plant oils are a suitable dietary source of EPA and DHA in humans.

Transgenic Nicotiana tabacum with tolerance to 2,4-D has previously been produced using a bacterial 2,4-D-dioxygenase gene (tfdA) driven by the 35S promoter of cauliflower mosaic virus. Using promoters from the Pisum sativum plastocyanin gene (petE) and an Arabidopsis thaliana histone gene (H4A), we demonstrate that similar protection from 2,4-D can be obtained in transgenic N. tabacum by targeting expression of tfdA to either meristematic tissues or chloroplast-containing tissues. As with the 35S promoter constructs, the plants are tolerant but not completely resistant; very young seedlings in particular are only slightly protected. However, the levels of tolerance observed could offer a useful degree of protection from accidental spray drift.

Rapid strides are being made in understanding the fundamental regulation of plant growth, development, and responses to the environment due to recent advances in molecular biology. Current questions in weed science such as herbicide mechanisms of action, biodegradation, and mechanisms of weed resistance are equally approachable using such methodology. Efforts to introduce herbicide resistance into agronomically important crops are possible because of successful isolation and transfer of genes. Investigations of weed survival and competitive strategies based on developmental processes, such as seed dormancy, are currently underway using techniques designed to monitor and characterize differential gene expression. Molecular methodology also plays a key role in taxonomic studies of weed populations using restriction fragment length polymorphism (RFLP) mapping. The future potential for these and other techniques such as nucleic acid hybridization, polymerase chain reaction (PCR), gene transfer, and the use of transgenic plants is described.

Knowledge of the vertical and horizontal distribution of Aphis gossypii Glover (Hemiptera: Aphididae) on genetically modified cotton plants over time could help optimize decision-making in integrated cotton aphid management programs. Therefore, the aim of the present study was to determine the vertical and horizontal distribution of A. gossypii in non-transgenic Bt cotton and transgenic Bt-cotton over time during two cotton seasons by examining plants throughout the seasons. There was no significant interaction between years and cotton cultivar treatments for apterous or alate aphids. Considering year-to-year data, analyses on season-long averages of apterous or alate aphids showed that aphid densities per plant did not differ among years. The number of apterous aphids found per plant for the Bt transgenic cultivar (2427 apterous aphids per plant) was lower than for its isoline (3335 apterous aphids per plant). The number of alate aphids found per plant on the Bt transgenic cultivar (12.28 alate aphids per plant) was lower than for the isoline (140.56 alate aphids per plant). With regard to the vertical distribution of apterous aphids or alate aphids, there were interactions between cotton cultivar, plant age and plant region. We conclude that in comparison to non-Bt cotton (DP 4049), Bt cotton (DP 404 BG (Bollgard)) has significant effects on the vertical, horizontal, spatial and temporal distribution patterns of A. gossypii, showing changes in its distribution behaviour inside the plant as the cotton crop develops. The results of our study are relevant for understanding the vertical and horizontal distribution of A. gossypii on Bt cotton cultivar (DP 404 BG (Bollgard)) and on its isoline (DP 4049), and could be useful in decision-making, implementing controls and determining the timing of population peaks of this insect.

Different techniques to assess bacterial community structure and diversity were evaluated in silages prepared with four different maize cultivars, three conventional and one transgenic (cv. Tundra, event Bt-176). Plants were cultivated in the greenhouse and harvested after 30 days of growth. Silage samples were collected at successive times during fermentation and analyzed for bacterial counts and by various DNA-based fingerprinting techniques. Bacterial counts were similar between cultivars for the total culturable bacteria, sporeforming, and mesophilic and thermophilic lactic acid bacteria (LAB). Further analysis of the species composition of 388 LAB strains by intergenic transcribed spacer (ITS) PCR followed by sequencing of 16S rRNA gene did not reveal differences between cultivars. In contrast, molecular fingerprinting methods targeting whole bacterial communities, such as automated ribosomal intergenic spacers analysis (ARISA) and 16S rRNA gene length heterogeneity-PCR (LH-PCR), indicated that different maize silage batches or cultivars hosted different bacterial communities. Thus, ARISA and LH-PCR fingerprinting techniques offer a fast and sensitive method to compare bacterial communities, and to detect differences in silage bacterial communities.

Risk-assessment studies of virus-resistant transgenic plants (VRTPs) focussing on recombination of a plant virus with a transgenic sequence of a different virus should include a comparison of recombination frequencies between viruses in double-infected non-transgenic plants with those observed in singly infected transgenic plants to estimate recombination incidence in VRTPs. In this study, the occurrence of recombination events was investigated in non-transgenic plants double-infected with two different potyviruses, as well as in potyviral genomes in singly infected transgenic plants expressing potyvirus sequences. Different potyviruses, namely Potato virus A (PVA), Tobacco vein mottling virus (TVMV), two strains of Potato virus Y (PVY-O, PVY-H) and two strains of Plum pox virus (PPV-NAT, PPV-SK68), were used in three combinations for double infection of a common host. Furthermore, transgenic plants expressing either potyviral coat protein (CP), helicase (CI) or polymerase (NIb) coding sequences (PPV-NAT-CP, PVY-CI, PVY-NIb) were singly-infected with a heterologous potyvirus, which was not targeted by the respective transgenic resistance. To identify recombinant potyviral sequences, a sensitive RT-PCR was developed to detect up to one recombinant molecule out of 106 parental molecules. In 304 mixed infected non-transgenic plants, 92 mixed and 164 single infected transgenic plants screened for recombinant sequences no recombinant potyviral sequence was found. These results indicate that recombination events between different potyviruses in mixed infections and between a potyvirus infecting a potyvirus-resistant transgenic plant are likely to be very infrequent.

Crops transformed to express Bacillus thuringiensis (Bt) toxins can cause close to 100% mortality of certain target pest species. This study assessed the effect of target pest reduction on the predatory insect Chrysoperla carnea (Stephens) in the presence of alternative prey. Numbers of lacewings recovered from Bt oilseed rape (cultivar Oscar, event O52) did not differ significantly from numbers of lacewings recovered from conventional oilseed rape in cage experiments with the target pest Plutella xylostella (Linnaeus) and the non-target pest Myzus persicae (Sulzer) when aphid densities were high. However, significantly fewer lacewings were recovered from Bt plants as aphid densities were lowered. Lacewing weights were not affected by plant type.

There is now considerable evidence of the importance of n-3 long-chain PUFA in human health and development. At the same time, the marine fish stocks that serve as the primary sources of these fatty acids are threatened by continued over-exploitation. Thus, there is an urgent need to provide a sustainable alternative source of the n-3 long-chain PUFA normally found in fish oils. The possibility of using transgenic plants genetically engineered to synthesise these important fatty acids has recently been demonstrated. The approaches taken to realise this outcome will be discussed, as will their prospects for providing a sustainable resource for the future.

With the development of plant genetic engineering, many transformation methods can be used to transfer heterogeneous genes into plants to develop genetic crops. However, a lot of research results have shown that transgene expression remains largely unpredictable and there is great variation of expression level in different transgenic plant lines. Plant genetic engineering research is reviewed in the present paper. We analysed the reasons why low efficiency of heterogeneous gene expression has happened in transgenic plants in terms of DNA modification, localization of proteins and methods of transformation used. Some strategies for improving heterogeneous gene expression in transgenic plants are also discussed.

Over the last decade, plant virus-based vectors have been developed and successfully exploited for high-yield production of heterologous proteins in plants. However, widespread application of recombinant viruses raises concerns about possible risks to the environment. One of the primary safety issues that must be considered is the uncontrolled spread of the genetically engineered virus from experimental plants to susceptible weeds or crops. Using a movement-deficient Potato virus X (PVX)-based transient gene expression vector which harbors the β-glucuronidase (gus) gene, we established a plant viral expression system that provides containment of the recombinant virus and allows for safe and efficient protein production. By deletion of the viral 25k movement protein gene, systemic spread of the modified virus in non-transgenic Nicotiana benthamiana plants was successfully inhibited. In transgenic N. benthamiana plants expressing the 25K viral movement protein, this deficiency was complemented, thus resulting in systemic infection with the movement-deficient virus. While no differences in virus spread and accumulation were observed compared to infection caused by wild-type PVX in non-transgenic plants, the movement protein transgenic plants exhibited none of the normal symptoms of viral infection. Several biosafety aspects were investigated including the potential for recombination between the defective virus and the movement protein transgene, as well as complementation effects in non-transgenic plants doubly infected with the defective and the wild-type virus. Furthermore, the applicability of the safety system for the production of heterologous proteins was evaluated with gus as a model gene. With respect to the stability of the gus insert and the expression level of the GUS protein, there were no differences between the novel system developed and the conventional PVX-based expression system.

Regeneration of sweet potato (Ipomoea batatas cv. Lizixiang) transgenic plants with oryzacystatin-I (OCI) gene was achieved using an Agrobacterium tumefaciens-mediated method. A. tumefaciens strain LBA4404, harbouring a binary vector pBinh with neomycin phosphotransferase II and OCI genes, was used. After 3 days of subculture, Lizixiang embryogenic suspension cultures were co-cultivated with LBA4404 (OD600nm=0.5) for 4 days. Next, the infected suspension cultures were first cultured for 5 days in MS medium with 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 300 mg/l carbencillin (Carb) but without kanamycin (Kan) and then transferred to MS medium supplemented with 2 mg/l 2,4-D, 50 mg/l Kan and 300 mg/l Carb for the selection culture. Four weeks after selection, 200 Kan-resistant cell aggregates (∼1 mm in size) from the embryogenic suspension cultures were transferred to MS solid medium supplemented with 2 mg/l 2,4-D, 50 mg/l Kan and 300 mg/l Carb, and eight embryogenic calluses were obtained. After transferring to MS medium supplemented with 1 mg/l ABA, 50 mg/l Kan and 100 mg/l Carb, these embryogenic calluses formed 13 plantlets via somatic embryogenesis. PCR and PCR–Southern blot analysis indicated that seven of the 13 plantlets were transgenic.

Transgenic plants result from the isolation and insertion of genes into plants by means other than conventional breeding techniques, making it possible to isolate a gene that controls a valuable trait without also inserting thousands of other genes, as can occur with other methods. Transgenic plants potentially can provide substantial benefits to humans, but they can also pose risks to ecosystems, nontarget species and even to humans. I utilize the U.S. regulatory experience with chemical substances to provide some background for locating the strengths and weaknesses of different legal structures as well as providing an opportunity to learn from them. Learning from that case study and different legal structures utilized therein, as well as from the state of the world in which transgenic plants will be introduced and the state of the relevant sciences combined with a National Research Council Report on transgenic plants, this essay assesses the regulatory procedures that the U.S. Department of Agriculture uses to evaluate the risks from transgenic plants. These legal structures, although well suited for identifying risks before there is extensive exposure, have a number of shortcomings for reviewing the products of a new and not well-understood technology. The U.S. could take some steps toward improving its reasonable, tiered, pre-market approach for reviewing the risks from transgenic plants by following the NRC recommendations and learning from shortcomings of the regulation of chemical risks. Whether it will be or not remains to be seen.

Studies on genetically modified (GM) feedstuffs for poultry (and other livestock species) have not added any substance to public concerns in Europe about their safety for human or bird health. The compositions of maize lines engineered for insect resistance (Bt-maize) or herbicide tolerance (glyphosate) and herbicidetolerant soybean have all proved to be essentially indistinguishable from their conventional counterparts. Consequently, and not surprisingly, comparative feeding studies with broilers and layers in which conventional maize (50 to 78%) or soybeans (27%) were replaced in feeds by transgenic varieties, also have failed to show differences of any significance in production parameters. These data indicate that feeding studies with target livestock species contribute very little to the safety assessment of crops engineered for input traits that have little or no detectable effect on chemical composition. However, comparative growth studies made with broiler chicks, particularly sensitive to any change in nutrient supply or the presence of toxic elements in their feed, can be used to screen for any unintended adverse consequence of the recombinant event not detected by compositional analysis. This does, however, depend on whether the GM plant can be matched to a parental line or another suitable control and its suitability for inclusion in broiler diets. The discovery that DNA fragments from the digestive tract can be found in the tissues of animals evoked interest in the fate of ingested transgenes. Plant DNA derived from feed has been detected in the muscle, liver, spleen and kidneys of broilers and layers, although not in eggs. However, no fragments of transgenic DNA or its expressed protein have been found to date in poultry meat or eggs or in any other animal tissues examined.

To obtain information on differences between the metabolic pathways of the herbicide glufosinate (trade names: BASTA®, LIBERTY®) in non-transgenic, glufosinate-sensitive plants and in transgenic, glufosinate-resistant plants, the metabolism of 14C-labeled glufosinate and its enantiomers L- and D-glufosinate was studied using cell cultures of oilseed rape and corn. Transformation of glufosinate in both sensitive and transgenic rape cells remained at a low rate of about 3-10% in contrast to corn cells, where 20% was transformed in sensitive and 43% in transgenic cells after 14 days of incubation, the rest remaining as unchanged glufosinate. In sensitive rape and corn cells the main metabolite was 4-methylphosphinico-2-oxo-butanoic acid (PPO) with 7.3 and 16.4%, respectively, together with low amounts of 3-methylphosphinicopropionic acid (MPP), 4-methylphosphinico-2-hydroxybutanoic acid (MHB), 4-methylphosphinicobutanoic acid (MPB) and 2-methylphosphinicoacetic acid (MPA). An additional metabolite formed in transgenic cell cultures was 2-acetamido-4-methylbutanoic acid (N-acetyl-L-glufosinate, NGA), which was formed at rates of 3.2% in rape and 16.1% in corn. A further minor metabolite, not yet identified, was detected in both cell types. The liberation of 0.2% 14CO2 indicates further metabolic steps prior to a limited mineralization in plant cell cultures. L-glufosinate was transformed into the same metabolites as the glufosinate racemate. D-glufosinate was not metabolized.

In higher plants, RNA–DNA interactions can trigger de novo methylation of genomic sequences via a process that is termed RNA-directed DNA methylation (RdDM). In potato spindle tuber viroid (PSTVd)-infected tobacco plants, this process can potentially lead to methylation of all C residues at symmetrical and nonsymmetrical sites within chromosomal inserts that consist of multimers of the 359-bp-long PSTVd cDNA. Using PSTVd cDNA subfragments, we found that genomic targets with as few as 30 nt of sequence complementarity to the viroid RNA are detected and methylated. Genomic sequencing analyses of genome-integrated 30- and 60-bp-long PSTVd subfragments demonstrated that de novo cytosine methylation is not limited to the canonical CpG, CpNpG sites. Sixty-base-pair-long PSTVd cDNA constructs appeared to be densely methylated in nearly all tobacco leaf cells. With the 30-bp-long PSTVd-specific construct, the proportion of cells displaying dense transgene methylation was significantly reduced, suggesting that a minimal target size of about 30 bp is necessary for RdDM. The methylation patterns observed for two different 60-bp constructs further suggested that the sequence identity of the target may influence the methylation mechanism. Finally, a link between viroid pathogenicity and PSTVd RNA-directed methylation of host sequences is proposed.

Current control of plant parasitic nematodes often relies on highly toxic and environmentally harmful nematicides. As their use becomes increasingly restricted there is an urgent need to develop crop varieties with resistance to nematodes. The limitations surrounding conventional plant breeding ensure there is a clear opportunity for transgenic resistance to lessen current dependence on chemical control. The increasing use of molecular biology techniques in the field of plant nematology is now providing useful information for the design of novel defences to meet the new needs. Plant responses to parasitism are being investigated at the molecular level and nematode gene products that could be targets for a direct anti-nematode defence are being characterized. The potential of an anti-feedant approach to nematode control has been demonstrated. It is based on the transgenic expression of proteinase inhibitors. The rational development of this strategy involves characterization of nematode proteinase genes and optimization of inhibitors by protein engineering. Durability of the resistance can be enhanced by stacking transgenes directed at different nematode targets.

Fructans are linear or branched polymers containing a single sucrose and repeating fructose residues. An early model for fructan biosynthesis in higher plants suggested that partial synthesis of the polymer occurred in the cell cytosol. The current model suggests that synthesis requires the interaction of two separate fructosyltransferases located in the vacuole. Tobacco lines containing a chemically induced promoter, directing expression of the Bacillus amyloliquefaciens SacB gene in the present study, provided an opportunity to regulate and target fructan synthesis to the cytosol of transgenic plants. Induced expression of the gene led to rapid destruction of leaf tissue. Amino acid substitution at a highly conserved site (Arg331) in the SacB gene reduced the fructosyltransferase efficiency without reducing the invertase activity of the enzyme. Expression of the mutant gene in transgenic tobacco also resulted in leaf damage. However, the appearance of necrotic tissue was greatly delayed. The results suggest that the phenotype is due to accumulation of fructan in the cytosol. Fructan metabolism in the cytosol of potato tubers was also detrimental to development. Tuber size and starch synthesis was significantly reduced in lines containing the untargeted gene. Transgenic tobacco and potato containing the SacB gene offer an opportunity to study the metabolism of fructan and the effect of accumulation on plant cell development.