This is the report of the first open field release of genetically modified microorganisms (GMMs) in Italy. It covers ten years of monitoring, and follows in-field GMM dynamics from strain release to disappearance below detection limits, as well as assessment of impact on resident microorganisms. The bacteria released belong to the nitrogen fixing legume endosymbiont Rhizobium leguminosarum bv. viciae, and were engineered with non-agronomically-proficient traits, in order to assess their behavior and fate without GMM-specific positive feedback from the plant. A DNA cassette containing mercury resistance and ß-galactosidase genes was introduced in either plasmid-borne or chromosomally integrated versions, in order to test the resulting strain stability. A synthetic promoter was used to drive the lacZ gene, conferring high catabolic activity to the GMM. Two different wild-type Rhizobium backgrounds were tested, comparing a non-indigenous vs. an indigenous, highly competitive strain. The latter had much greater persistence, since it was able to survive and establish at technically detectable levels for over four years after release. Selection factors, such as reiterated presence of the plant host, or lactose substrate supply, enhanced long-term survival to different extents. The lactose treatment showed that even a single trophic supplementation can surpass the benefits of symbiotic interaction for a period of several years. Concerning impact, the GMMs did not alter substantially the other soil community general microbiota. However, there were some significant differences in microbiota as a consequence of the Rhizobium inoculation. This effect was observed with either the WT or GMM, and was more evident in the release of the indigenous Rhizobium. Moreover, as the indigenous GMM had its parental, dominant wild-type in the same soil, it was possible to evaluate to what extent the GMM version could result in parent displacement (“self-impact”), and how much the two rhizobia would additively contribute to nodulation.

Horizontal gene transfer (HGT) refers to the acquisition of foreign genes by organisms. The occurrence of HGT among bacteria in the environment is assumed to have implications in the risk assessment of genetically modified bacteria which are released into the environment. First, introduced genetic sequences from a genetically modified bacterium could be transferred to indigenous micro-organisms and alter their genome and subsequently their ecological niche. Second, the genetically modified bacterium released into the environment might capture mobile genetic elements (MGE) from indigenous micro-organisms which could extend its ecological potential. Thus, for a risk assessment it is important to understand the extent of HGT and genome plasticity of bacteria in the environment. This review summarizes the present state of knowledge on HGT between bacteria as a crucial mechanism contributing to bacterial adaptability and diversity. In view of the use of GM crops and microbes in agricultural settings, in this mini-review we focus particularly on the presence and role of MGE in soil and plant-associated bacteria and the factors affecting gene transfer.

Based on international findings, Irish co-existence guidelines for the cultivation of GM potato stipulate that an isolation distance of 20 m is required to minimize the spread of transgenic pollen in accordance with required labeling thresholds. As potato tolerant to Phytophthora infestans is the most applicable GM crop from an Irish context, we tested the efficacy of this isolation distance under Irish environmental conditions using the conventional variety Désirée as a pollen donor and the male-sterile variety British Queen as a pollen receptor. Gene flow was determined by scoring for berry presence on receptor plants and confirmed using a microsatellite marker system designed to assess paternity in F1 seedlings. 99.1% of seedlings recovered were identified as having Désirée paternity. Whereas 19.9% (140/708) of total berries formed on receptor plants occurred at a distance of 21 m from the pollen source, only 4 of these berries bore viable true potato seed (TPS), from which 23 TPS germinated. TPS-bearing berry formation was negatively correlated with distance from the pollen source, and although overall distribution of berries and seeds was non-random across the plot, no significant correlation was evident with respect to wind direction. Microsatellite markers were also used to confirm that the foraging beetle Meligethes aeneus is a vector for the transmission of potato pollen, but a more detailed statistical analysis of this dataset was limited by inclement weather during the trial. To conclude, we recommend that a two-tiered system be established in regard to establishing isolation distances for the experimental trial and commercial cultivation of GM potato in Ireland, and that responsible crop management be adopted to minimize the establishment of TPS-derived volunteers, which we have noted will emerge through a rotation as a result of pollen-mediated gene flow.

Many crop species are able to hybridize with related weedy or wild relatives, which could lead to transfer of cultivar genes, and among them transgenes, into wild populations. It is not clear, however, whether the hybrids and their descendants are able to survive and reproduce in natural habitats, as inherited cultivar traits may be maladaptive under such conditions. To test this, we produced hybrid (F1) seeds by controlled crosses between wild and cultivated carrots (Daucus carota ssp. carota and ssp. sativa, respectively) and sowed them into three Danish grasslands of different age, in parallel with seeds of wild carrots. Replicate plots were sown in fall and spring. Survival and flowering of the emerging plants were monitored for the following three years. Both hybrid and wild carrots survived and flowered in highest frequency at a recently disturbed site, and much less at two older sites. Hybrids emerged in higher proportions than wild carrots in the first year and survived to similar or slightly lower frequencies at the end of the experiment. Hybrids flowered as frequently or slightly less frequently than wild plants, and developed fewer and smaller umbels. Despite a somewhat lower reproductive potential compared to wild carrots, first generation hybrids between cultivated and wild carrots are likely to survive and produce offspring in natural grasslands in Denmark. This, together with other studies, suggests that cultivar genes may transfer relatively easily into wild carrot populations.

Effects of tobacco genetically modified to express the protease inhibitor bovine spleen trypsin inhibitor (BSTI) were examined in laboratory assays against three earthworm and one collembolan species. BSTI is a serine protease inhibitor that can bind to the digestive trypsins of insects feeding on modified plants, resulting in reduced growth and survival. Protease inhibitors are active against a broad range of insects, so may have a large impact on non-target organisms. Survival and fecundity of the collembolan Folsomia candida were unaffected by consumption of artificial diet containing BSTI-expressing tobacco leaf or powdered freeze-dried BSTI-expressing tobacco leaf that was added to soil. Similarly, mortality and growth of earthworms Aporrectodea caliginosa and Lumbricus rubellus did not differ significantly between soil augmented with BSTI-expressing tobacco leaves or unmodified control leaves. The redworm Eisenia fetida gained less weight when provided with BSTI-expressing leaves in one assay, but when the experiment was repeated, there was no significant difference between treatments. BSTI-expressing tobacco and unmodified control leaves decomposed at the same rate, indicating that the inhibitor had no effect on the overall function of the decomposer community of micro-flora and fauna in soil.

This review addresses the possible ecological effects of transgenic plants on micro-organisms in the field, hence, in the phytosphere and in the soil matrix. The important steps involved in the interaction between plant DNA and bacteria and the factors that influence the horizontal gene transfer (HGT) process will be discussed. HGT is a process in which two partners are involved, even if indirectly. In the first section, aspects concerning bacteria, such as their physico-chemical, biological and genetic characteristics, are described. Parameters affecting transgenic DNA fate in the environment are described in the second section. Subsequently, terrestrial habitats are evaluated in terms of their capacity to favor horizontal gene transfer. Finally, we focused on several studies in order to evaluate possible perturbations of soil bacterial community composition due to cultivation of transgenic plants in the field.

Concerns have been raised in Europe about the efficiency, sustainability, and environmental impact of the first genetically modified crops. The committees and regulators in charge of approving procedures have encouraged a field trial approach for safety assessment studies under current agronomic conditions. We describe the gene flow from sugar beet (Beta vulgaris L.) in a multi-year and multi-crop monitoring study on farmers' fields at two locations that has been carried out since 1995. We analyzed two sugar beet lines that have been genetically transformed for herbicide resistance. One sugar beet has resistance to glufosinate and the other to glyphosate. Large differences among lines, years and locations were observed. These differences provided a broad range of situations to estimate the risks. Sugar beet bolters produced the majority (86%) of the herbicide-resistant seeds harvested in the field. Direct pollen flow from sugar beet bolters to weed beets that were growing within the same field as well as in a neighboring field that was left fallow accounted for only 0.4% of the resistant seeds released over the years and locations. Descendants of the hybrids between the sugar beet and the weed beet produced the remaining 13.6% of resistant seeds. Herbicide-resistant seeds from the progeny of the weed beet were recorded up to 112 m away from the closest transgenic pollen donor. Indications were observed of non-randomness of the weed beet producing resistant progeny. We also analyzed pollen flow to male-sterile bait plants located within and outside of the sugar beet field. Herbicide-resistant pollen flow was recorded up to 277 m, and fitted with an inverse power regression. Using sugar beet varieties with no, or very low, sensitivity to bolting and destroying bolters are two necessary measures that could delay gene flow.

Development of transgenic triticale as a platform for novel bio-industrial products is predicated on an environmental biosafety assessment that quantifies the potential risks associated with its release. Pollen-mediated gene flow to related species and conventional triticale varieties is one pathway for transgene movement. A tier 1 quantification of triticale hybridization was conducted by emasculating and hand pollinating flowers under greenhouse conditions. Approximately 2000 manual pollinations were conducted for each cross and its reciprocal between two triticale genotypes: a modern triticale cultivar (AC Alta) and primary triticale (89TT108), and common wheat, durum wheat and rye. The frequency of outcrossing, hybrid seed appearance and weight, and F1 emergence and fertility were recorded. Outcrossing, F1 emergence and fertility rates were high from crosses between triticale genotypes. Outcrossing in inter-specific crosses was influenced by the species, and the genotype and gender of the triticale parent. In crosses to common and durum wheat where triticale was the male parent, outcrossing was ≥73.0% and ≥69.5%, respectively, but ≤23.9% and ≤3.0% when triticale was the female parent. Overall, outcrossing with rye was lower than with common and durum wheat. F1 hybrid emergence was greater when triticale was the female parent. With the exception of a single seed, all wheat-triticale F1 hybrid seeds were non-viable when triticale was the male parent in the cross. Only seven durum wheat-triticale F1 hybrids emerged from 163 seeds sown, and all were produced with triticale 89TT108 as female parent. With rye, 8 F1 hybrids emerged from 38 seeds sown, and all were produced from crosses to AC Alta; five with AC Alta as the female parent and three as the male. Interspecific F1 hybrids were self-sterile, with the exception of those produced in crosses between common wheat and triticale where triticale was the female parent. Tier 2 hybridization quantification will be conducted under field conditions.

The introduction of genetically modified organisms (GMOs) has called for an improved understanding of the fate of DNA in various environments, because extracellular DNA may also be important for transferring genetic information between individuals and species. Accumulating nucleotide sequence data suggest that acquisition of foreign DNA by horizontal gene transfer (HGT) is of considerable importance in bacterial evolution. The uptake of extracellular DNA by natural transformation is one of several ways bacteria can acquire new genetic information given sufficient size, concentration and integrity of the DNA. We review studies on the release, breakdown and persistence of bacterial and plant DNA in soil, sediment and water, with a focus on the accessibility of the extracellular nucleic acids as substrate for competent bacteria. DNA fragments often persist over time in many environments, thereby facilitating their detection and characterization. Nevertheless, the long-term physical persistence of DNA fragments of limited size observed by PCR and Southern hybridization often contrasts with the short-term availability of extracellular DNA to competent bacteria studied in microcosms. The main factors leading to breakdown of extracellular DNA are presented. There is a need for improved methods for accurately determining the degradation routes and the persistence, integrity and potential for horizontal transfer of DNA released from various organisms throughout their lifecycles.

RecA-mediated recombination requires regions of homology between donor and recipient DNA for successful integration. This paper investigates the effect of the relationship between the length of gene-sized inserts (434, 733, 2228 and 2400 bp) and flanking sequence homology (100 – ca. 11 000 bp) on transformation frequency in Acinetobacterbaylyi strain BD413. Both insert size and size of the homologous region were varied, which improves on previous studies that kept insert size constant and varied only the homologous flank size. Transfer frequency of a non-homologous single small gene for gentamicin resistance (aac(3)I; 773 bp) was increased 18-fold when flanking homology was changed from about 2000 bp to 8000 bp, but was reduced 234-fold when two genes were inserted (nptII-gfp; 2400 bp) between similar homologous regions. To investigate the effect of smaller regions of flanking homology (100 – 2000 bp), a partial nptII-gfp deletion (434 bp) was restored. This confirmed that a minimum of 500 bp on each flank was required for transformation to be affected by flanking homology. The data obtained allowed development of a multiple regression equation to predict transformation frequency from homology, insert size and total fragment size for gene insertions. We also show that the ratio of flanking homology to insert size and not the total size of donor DNA is the most important variable determining transformation frequency. The equation developed was consistent with results previously reported by others, and so will be useful when using A. baylyi as a model for gene transfer by transformation in the laboratory, environment and for biosafety.

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.

Four GM plant species (Arabidopsis thaliana, Brassica napus, Nicotiana benthamiana and N. tabacum), each expressing the gene encoding the jellyfish green fluorescent protein (GFP) regulated by the cauliflower mosaic virus (CaMV) 35S RNA promoter, were assessed for the extent of transgene silencing and viral genome integration following infection by CaMV. The first three species are systemic hosts of CaMV, while N. tabacum is only a local host for a few strains of CaMV. A generalized systemic silencing of the GFP transgene was not observed in a total of 100 plants of each species infected with CaMV, although some localized loss of GFP was observed in CaMV-infected N. benthamiana leaves, and some loss of fluorescence was observed in older leaves of uninfected as well as infected plants. Progeny seedlings obtained from the above infected plants also did not exhibit transgene silencing showing that virus infection did not affect the stability of the transgene. These progeny plants also did not show signs of virus infection, indicating that the presence of the CaMV 35S RNA promoter sequences in the plant genome did not potentiate seed transmission of the virus. Integration of infective CaMV into the CaMV 35S RNA promoter could not be detected in 944 samples taken from leaves of the above infected plant species or in 2912 samples taken from progeny seedlings. Based on a detection limit of one copy per 4000 haploid genomes, we conclude that if integration of virus does occur into the CaMV 35S RNA promoter, then it occurs at such a low frequency as to be insignificant.

The European honey bee (Apis mellifera) is important both for pollination and for honey production. Pollen is the major protein source for bees, which exposes them directly to changes in pollen quality e.g. through genetic engineering. In order to create a worst case scenario regarding pea lectin (PSL) expressed transgenically in oilseed rape anthers and pollen, the maximum amount of dried pollen that could be mixed in an artificial diet without negatively affecting larval performance (1.5% w/w) was fed to bee larvae. Pollen from two transgenic plant lines expressing PSL up to 1.2% of total soluble protein and pollen from one non-transgenic line was added to the same diet and used as a pollen control. When these three pollen diets and the control diet (without added pollen) were compared, no negative effect from the pollen of the transgenic plants could be detected on larval mortality, weight, or development time. An increased weight and a reduced developmental time were recorded for larvae on all diets containing pollen when compared to the diet without pollen.

The likelihood of gene transfer from transgenic plants to bacteria is dependent on gene number and the presence of homologous sequences. The large number of transgene copies in transplastomic (transgenes contained in the chloroplast genome) plant cells as well as the prokaryotic origin of the transgene, may thus significantly increase the likelihood of gene transfer to bacteria that colonize plant tissues. In order to assess the probability of such transfer, the length of homologous DNA sequences required between the transgene and the genome of the bacterial host was assessed. In addition, the probability that bacteria, which co-infect diseased plants, are transformable and have sequences similar to the flanking regions of the transgene was evaluated. Using Acinetobacter baylyi strain BD143 and transplastomic tobacco plants harboring the aadA gene (streptomycin and spectinomycin resistance), we found that sequences identical to the flanking regions containing as few as 55 nucleotides were sufficient for recombination to occur. Consequently, a collection of bacterial isolates able to colonize tobacco plant tissue infected by Ralstonia solanacearum strain K60 was obtained, screened for DNA sequence similarity with the chloroplastic genes accD and rbcL flanking the transgene, and tested for their ability to uptake extracellular DNA (broad host-range pBBR1MCS plasmids) by natural or electro-transformation. Results showed that among the 288 bacterial isolates tested, 8% presented DNA sequence similarity with one or both chloroplastic regions flanking the transgene. Two isolates, identified as Pseudomonas sp. and Acinetobacter sp., were able to integrate exogenous plasmid DNA by electro-transformation and natural transformation, respectively. Our data suggest that transplastomic plant DNA recipients might be present in soil bacterial communities.

Transformation of plant-associated bacteria by plant DNA has never been demonstrated in agricultural fields. In total 552 bacterial isolates from stems of Ralstonia solanacearum-infected and healthy tomato plants and from stems and leaves of healthy potato plants were tested for natural genetic competence using plasmid pSKTG DNA and homologous DNA extracts. Control strain Acinetobacterbaylyi ADP1 was transformable with both DNA extracts. No transformable isolates were observed after treatment with plasmid pSKTG DNA. Two isolates, P34, identified as Pseudomonas trivialis and A19, identified as Pseudomonas fragi, were selected on the basis of the consistently higher Rp-resistant CFU numbers after treatment with DNA from Rp-resistant cells than with that from wild-type cells. P34 showed 2.1-fold and A19 1.5-fold higher Rp-resistant CFU numbers after treatment with DNA from homologous Rp-resistant cells versus that from wild-type cells. It is concluded that bacteria capable of in vitro capture and integration of exogenous DNA into their genomes are relatively rare in culturable bacterial communities associated with tomato and potato plants, or that conditions conducive to transformation were not met in transformation assays.


Natural transformation is assumed to be the most likely mechanism by which DNA from transgenic plants could be horizontally transferred to bacteria. In order to determine the occurrence of naturally transformable bacteria amongst bulk and rhizosphere soil bacteria, different transformation strategies were employed using either plasmid DNA (IncQ plasmids pSM1890 and pSM1885, conferring GFP, Smr, Gmr and GFP, Smr, Tcr, respectively) or genomic DNA from rhizosphere isolates, which were chromosomally tagged with mini-Tn5~(GFP, Tcr, as transforming DNA. Transformation assays were done in microtiter plates (262 isolates and pSM1890 or pSM1885), on filters (i) with rhizosphere bacterial community mixed with pSM1890 or pSM1885, (ii) with 24 rhizosphere or soil bacterial isolates mixed with genomic DNA of the corresponding mini-Tn5-tagged strains, and in the rhizosphere of tobacco plants inoculated with rifampicin-resistant bacterial isolates and genomic DNA of the corresponding mini-Tn5-tagged strains added. One transformant colony was obtained when Brevundimonas vesicularis was transformed with genomic DNA of the corresponding mini-Tn5-tagged strain. Attempts to reproduce this result were unsuccessful. With this single exception, transformants were neither detected in the collection of isolates nor in the rhizosphere bacterial community. Acinetobacter baylyi BD413 used as a positive control showed drastically reduced transformation frequencies with plasmid pSM1890 as transforming DNA when mixed with the rhizosphere pellet. All transformants were characterized by BOX-PCR fingerprints, and three different BOX patterns were revealed. Sequencing the 16S rRNA gene showed that all transformants could be assigned to Acinetobacter sp. Since transformants were only observed in the positive control, the introduced BD413 either underwent genomic rearrangements, or competence of the Acinetobacter population present in the rhizosphere was stimulated by the introduction of BD413. The various transformation assays performed indicate that the proportion of rhizosphere or bulk soil bacteria which are naturally transformable is negligibly low.

To provide estimates of horizontal gene transfer from transgenic crops to indigenous soil bacteria, transformation frequencies were obtained for naturally transformable Acinetobacter baylyi BD413 using a chromosomally integrated plant transgene. The transgene comprised sequences for two phenotypic markers: kanamycin resistance (npt II) and green fluorescent protein (gfp), expressed from their own bacterial promoters. Recipient bacteria carried a copy of these two genes, with deletions in their 3'-termini abolishing the marker activity, these genes were integrated into a 16S rRNA gene in the bacterial chromosomal genome or carried on a broad host range plasmid. Successful recombination between the plant transgene and the bacterial genome resulted in restoration of the markers, allowing detection through antibiotic selection and fluorescence. Transformation parameters of increasing complexity, without any enrichment steps, were used to approach the field conditions, while still obtaining measurable transformation frequencies. In pure culture filter experiments, transformation was detected using ground, chopped and whole leaves, as well as whole sterile seedlings, and ground roots. In sterile soil microcosms, transformation was detected using pure plant DNA (3.6 × 10-8 transformants per recipient) and ground leaves (2.5 × 10-11). Transformation was also detected for the first time in non-sterile soil using pure plant DNA (5.5 × 10-11). Since the same constructs were used throughout, these data allow predictions of even more complex environmental systems where measurable frequencies are not easily obtainable.