We monitored pollen-mediated transgene dissemination from commercial transgenic squash CZW-3 into its wild relative Cucurbita pepo ssp. ovifera var. texana (C. texana). Transgenic squash CZW-3 expresses the neomycin phosphotransferase II (nptII) gene and the coat protein (CP) genes of Cucumber mosaic virus (CMV), Zucchini yellow mosaic virus (ZYMV), and Watermelon mosaic virus (WMV); thereby, it is resistant to these three aphid-borne viruses. The rate of NPT II and CP transgene introgression increased with overlapping flowering patterns and a high ratio of transgenic F1 hybrids (C. texana × CZW-3) to C. texana. Transgene transfer also readily occurred from transgenic F1 hybrids into C. texana over three generations in field settings where test plants grew sympatrically and viruses were not severely limiting the growth, and fruit and seed production of C. texana. In contrast, introgression of the transgenes into C. texana was not sustained under conditions of high viral disease pressure. As expected, C. texana progeny that acquired the CP transgenes exhibited resistance to CMV, ZYMV, and WMV. This is the first report on transgene dissemination from a transgenic crop that exhibits disease resistance and hybridizes with a wild plant species without loss of fertility.

Guidelines to ensure the efficient coexistence of genetically modified (GM) and conventional crops are currently being considered across the European Union. The purpose of this strategy is to describe the measures a farmer must adopt to minimize the admixture of GM and non-GM crops. Minimizing pollen / seed-mediated gene flow between GM and non-GM crops is central to successful coexistence. However no system is currently available to permit the numeric quantification of a crop’s propensity for pollen/seed-mediated gene flow. The provision of such a system could permit a background level of gene flow, specific for a particular conventional crop, to be calculated. Here we present a gene flow index model implemented using the principal arable crops in Ireland as a model dataset. The objective of this research was to establish a baseline gene flow data set for Ireland’s primary conventional crops through the provision of a simple numerical index. This Gene Flow Index (GFI) incorporates four strands of crop-mediated gene flow (crop pollen-to-crop, crop pollen-to-wild, crop seed-to-volunteer and crop seed-to-feral) into a format that permits the calculation of a crop’s gene flow potential. Responsive to regional parameters, we have applied the model to sugar beet, oilseed rape, potato, ryegrass, maize, wheat and barley. We propose that the attained indices will highlight those crops that require additional measures in order to minimize gene flow in accordance with anticipated coexistence guidelines.

Plant to plant gene flow is a route of environmental exposure for GM plants specifically since crosses with wild relatives could lead to the formation of more vigorous hybrids, which could increase the rate of introgression and the environmental impact. Here, we test the first step in the process of potential transgene introgression: whether hybrid vigor can be inherited to the next generation, which could lead to fixation of altered, i.e., elevated, quantitative traits.

The potential for a permanent elevated fitness was tested using individual autogamous progeny lineages of hybrids between the crop Lactuca sativa (Lettuce) and the wild species Lactuca serriola (Prickly Lettuce). We compared progeny from motherplants grown under either greenhouse or field conditions. The survival of young plants depended strongly on maternal environment. Furthermore, we observed that offspring reproductive fitness components were correlated with maternal fitness.

Our study demonstrates that post-zygotic genotypic sorting at the young plants stage reduces the number of genotypes non-randomly, leading to inheritance of high levels of reproductive traits in the surviving hybrid lineages, compared to the pure wild relatives.

Consequently, directional selection could lead to displacement of the pure wild relative and fixation of more vigorous genome segments originating from crops, stabilizing plant traits at elevated levels. Such information can be used to indentify segments which are less likely to introgress into wild relative populations as a target for transgene insertion.

With the rapid advancement of transgenic biotechnology, large numbers of transgenic crops have been produced and released for commercial cultivation (James, 2001), raising considerable biosafety concerns all over the world. One of the major issues is the potential ecological risk resulting from transgenes escaping into and persist-ing in the environment. The extensive on-going debate on this issue (Arriola, 1997; Wolfenbarger and Phifer, 2000; Crawley et al., 2001; Prakash, 2001; Dale et al., 2002) poses challenging questions regarding the research direc-tions that need to be taken to ensure that biotechnology outputs are responsibly deployed worldwide. Unless these biosafety issues are satisfactorily addressed, large-scale commercial release of the transgenic crops developed and further advancement of transgenic bio-technology are likely to be hampered. For an in-depth review of crop-to-wild gene flow, see Jenczewski et al. (2002) in this number of EBR. Here the more specific issue of the effectiveness of preventing gene flow from occurring will be discussed.

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.

The safety and impact on the environment of transgenic crops are important issues, and studies have shown that pollen from transgenic Bt (Bacillus thuringiensis) corn (Zea mays L.) may kill nontarget insects. To develop an algorithm for assessing the environmental effect of transgenic crops, we arranged a field experiment in Tsukuba, Japan. Pollen dispersal and deposition were measured inside and outside a cornfield throughout the flowering period. Weather conditions such as wind speed and direction were measured at the same time. Pollen dispersal peaked 1 week after the start of flowering and continued for 12 days thereafter. The variation in daily pollen dispersal was similar at all observation points. Both pollen dispersal and deposition decreased exponentially with distance from the cornfield on all days. We estimated potential pollen deposition with a quasi-mechanistic model that incorporates the effects of wind direction, wind speed, and flowering intensity. The daily potential deposition was summed over the flowering period, and then the relationship between distance from the cornfield and the integrated potential deposition was estimated. It was possible to show the effective area of the environmental risk zone posed by genetically modified pollen by combining the distance/deposition relationship with the dose/response relationship derived from a laboratory assay. The algorithm described here can be applied to various wind-pollinated plants to estimate both potential and integrated pollen deposition.

Cleveland et al. (2005, Environ. Biosafety Res.4: 197–208) offer useful suggestions for monitoring transgenes in landraces of maize, but we disagree with their statement that the scientific conclusions of our paper (Ortiz-García et al., 2005, Proc. Natl. Acad. Sci. USA102: 12338–12343) are not justified. First, contrary to their perception, our survey was not designed to evaluate transgenes in the whole State of Oaxaca, but rather to monitor a specific portion of the District of Ixtlán de Juárez where the presence of transgenes had been reported previously by Quist and Chapela (2001, Nature414: 541–543). Second, our paper described two methods for estimating frequencies of undetected transgenic seeds, while Cleveland et al. recommend a third approach that explicitly estimates effective population size. They argue that the effective population size of our seed samples is smaller than we assumed, leading to false claims about our detection accuracy. However, we employed a robust statistical approach to compensate for possible bias by using numbers of maternal plants, in addition to numbers of seeds, to provide a conservative estimate of the minimum number of independent samples. When we re-analyzed our 2004 data using effective population sizes, our conclusion that transgenic seeds were “absent or extremely rare” did not change, nor did the general range of possible frequencies of undetected transgenic seeds. Unlike Cleveland et al., we advocate using combined probability tests to analyze data across localities. Third, our critics argue that we accepted the null hypothesis that transgenes were absent. Actually, we assumed that transgenes were present in local landraces, and we used parameter estimation methods to calculate the probability of failing to detect transgenic individuals at a range of frequencies. In agreement with Cleveland et al., we reiterate that there is a clear need for additional surveys with rigorous sampling methods to provide estimates of transgene frequencies over broad geographic areas in Mexico.

What is negative about negative data? Scientists understand negative data from our training in data analysis and statistics, where we use a positive concept of negative data. Negative data are data that do not enable us to reject our null hypothesis. Such data are often difficult to publish because it is not possible to prove the null hypothesis. Every active research scientist has a large drawer where these data languish. In the area of environmental biosafety, however, some scientists have begun to use “negative data” in a second, normative way. This normative concept of negative data has socio-political connotations, where “negative” data has come to connote results that GMO proponents could use to support, and GMO opponents could use to oppose the development of GMOs. This politicization of GMO biosafety research is worthy of study in its own right, but EBR is prepared to accept any kind of “negative” or “positive” data.

Pest management can have substantial impacts on non-target species both within and outside the units being managed. Assessment of these impacts is hampered by the lack of even the most basic checklist of the species present in most systems. The maize agroecosytem is of particular interest because of the large area covered and the intensity of widely varying forms of pest management. In this study a database of lepidopteran species that occur within the maize agroecosystem in the United States was compiled. The process was initiated by developing a list of plants present in maize using published sources and the first-hand knowledge of “weed” experts. This list of plant species associated with maize was then cross-listed with lepidopteran host feeding records using published sources. Finally, phenological profiles and conservation rankings were added. Although our list is not exhaustive, we found 132 plant species in 33 families associated with maize, and 229 lepidopteran species in 21 families that feed on these plants. The database of plants and lepidopteran species can be a starting point for assessment of risk to non-target Lepidoptera in maize from chemical control, biological control, and the use of transgenic Bt maize. The lepidopteran species associated with maize were found to be significantly less imperiled, as measured by their conservation rankings, than lepidopteran species as a whole in all habitats. This finding suggests that rare or endangered lepidopteran species are unlikely to be impacted by pest management in maize. Based on the likely lack of impact of pest management in maize on individual species, future studies should focus on potential impacts on the ecological services that lepidopteran species provide.

One prominent concern about genetically modified crops is the possibility of environmental impacts from the movement of fitness-enhancing traits to wild plant populations. Decisions to deregulate Bt crops in the USA have relied strongly on arguments that these crops will not interbreed with wild relatives in the permitted growing regions. Limited attention therefore has been directed to analyses of the consequences of gene flow. To provide a transparent evaluation process for risks associated with insecticidal transgene escape, we crafted a series of questions designed to guide this aspect of the risk assessment. We then explored the current knowledge base available for answering such risk-related questions for three Bt crops (cotton, rapeseed, and rice). First, we generated a list of wild relatives of these crops. A definitive list of potential transgene recipients is not yet possible for some crops. Sufficient data are not available for some crops to eliminate certain related plant species from consideration of fertile hybrid formation, thus making lists for these crops subject to speculation. Second, we queried the HOSTS database (UK) to obtain a worldwide listing of lepidopteran species that feed on these crops and their wild relatives, and to determine the host range of the larvae. To our knowledge, this list of 502 lepidopteran species is the first such list published for these crops and wild crop relatives. Third, we used a data set maintained by the Canadian Forest Service to assess Bt toxin susceptibility for these lepidopterans. Only 3% of those species have been tested for susceptibility; and the literature suggests that generalizations about susceptibility among taxa are difficult due to the variability within families. Fourth, we consulted the literature to interpret what is known about the ability of lepidopterans to regulate plant fitness or invasiveness. We could not eliminate the possibility of ecological release due to plant resistance against lepidopterans. In fact, there is strong experimental evidence that lepidopteran herbivores do limit the distribution and/or abundances of at least some wild plant species. Neither could we eliminate the possibility that non-target lepidopterans might have important functions in the ecosystem as pollinators or alternate hosts to natural enemies of pest species. This study suggests that crucial data are lacking for the development of a credible scientific basis to confirm or deny environmental risks associated with the escape of Bt transgene constructs to wild relatives. Given the absence of information on the identity, level of susceptibility, and ecological roles of lepidopterans exploiting specific wild relatives of Bt crops, we suggest that new efforts be directed to assessing possible consequences of lepidopteran mortality on resistant wild relatives.

When cultivating genetically modified varieties, the spontaneous gene flow between crop and wild relatives could be of concern. We analyzed spontaneous hybridization between a transgenic male-sterile line of oilseed rape (Brassica napus, 2n=38, AACC) and, as pollen donors, three European populations of wild radish (Raphanus raphanistrum, 2n=18, Rr,Rr) and a variety of cultivated radish (Raphanus sativus, 2n=18, RR). Seeds showed size and shape dimorphism that correlated to the frequency of hybrids. The offspring were scored morphologically and analyzed using DNA markers (inter-simple sequence repeats) to quantify hybrid frequencies. Seed set ranged from 0.4–1.2 seeds per pod, and 0.02–0.6 seeds per pod were confirmed as hybrids. The frequency of confirmed hybrids differed significantly among populations of R. raphanistrum. In the cross with a French population, all offspring were hybrids; in the cross with a Swiss population, 53% of the offspring were hybrids; and in the cross with a Danish population, only 2% of the offspring were found to be hybrids. The remaining offspring apparently belonged to two groups: the majority was B. napus-like plants, possibly of matromorphic origin, and a minority from the Danish cross seemed to carry fragments of the Raphanus genome. In the cross with a cultivated R. sativus, all offspring were found to be hybrids. This is the first report on spontaneous hybridization between B. napus and R. sativus. Hybrids from all cross-combinations had low pollen fertility (0–15%). If R. raphanistrum occurs where male-sterile B. napus is cultivated, large regional differences in hybridization frequencies between the species could complicate environmental risk assessment of transgenic oilseed rape.

Outcrossing of non-Bt cotton (Gossypium hirsutum (L.)) in refuges by transgenic Bt cultivars could reduce the efficacy of refuges for delaying resistance in seed-feeding pests. Based on reports that outcrossing decreased as distance from Bt cotton increased in small-scale studies, we hypothesized that increasing refuge width or distance from Bt fields would reduce outcrossing. In a large-scale study in Arizona, we quantified Bt seed in refuges of experimental and commercial fields, comparing outcrossing between in-field (narrow) and external (wide) refuges and among rows of refuges at various distances from Bt fields. Some refuges, including those in tightly controlled experimental plots, contained up to 8% adventitious Bt plants. Some, but not all, Bt plants likely resulted from Bt seed in the non-Bt seed bags. We did not detect a difference in outcrossing between in-field and external refuges. However, statistical power was low because outcrossing was low (< 0.4% of seeds) in both treatments. Higher outcrossing levels (≤ 4.6% of seeds) were observed in the studies measuring outcrossing at various distances from Bt fields, yet outcrossing did not decrease as the distance from Bt fields increased. We hypothesize that Bt plants in refuges cross-pollinated surrounding non-Bt plants, overshadowing the expected association between distance from Bt fields and outcrossing.

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.

Many varieties of transgenic rice are under development in countries where wild and weedy relatives co-occur with the crop. To evaluate possible risks associated with pollen-mediated transgene dispersal, we conducted a two-year survey in Vietnam to examine overlapping flowering periods of rice (Oryza sativa L.), weedy rice (O. sativa), and wild rice (O. rufipogon Griff.), all of which are inter-fertile. We surveyed populations in two regions of the Mekong Delta, northern and southern, and at three sites in each of three habitats per region: fresh water, saline water, and acid sulfate soil. Weedy rice frequently flowered simultaneously with neighboring cultivated rice plants. Flowering was more seasonal in wild rice and often peaked in November and December. Peak flowering times of wild rice overlapped with adjacent rice fields at all of the saline sites and half of the acid sulfate sites. The longer flowering season of wild rice ensured that crop-to-wild gene flow was possible in fresh water habitats as well. Our second objective was to determine whether wild and weedy rice populations are exposed to pests that could be targeted by future transgenes, which may then provide fitness benefits. These populations shared many pathogen and insect herbivore species with cultivated rice (leaffolder, locust, cricket, planthoppers, rice bug, stem borer, sheath blight, blast, bacterial leaf blight, and brown spot). Damage by leaffolders and locusts was the most frequently observed insect feeding damage on all three rice types. Indicator species analysis revealed that most of the insect herbivores were associated with particular habitats, demonstrating the importance of broad geographic sampling for transgenic rice risk assessment. These survey data and the strong likelihood of gene flow from cultivated rice suggest that further studies are needed to examine the effects of transgenic traits such as resistance to pests on the abundance of wild and weedy rice.

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.

Pseudomonas fluorescens is a normal inhabitant of the soil rhizosphere. The use of genetically altered strains of P. fluorescens in bioremediation has led to the need for effective monitoring of such cells released into the environment. In this study, we present data on the persistence in soil of P. fluorescens harboring gfp (green fluorescent protein) or lux (bioluminescence) genes. Comparisons were made between strains marked chromosomally and strains carrying these markers on a plasmid. Overall effects of plasmid carriage on culturability were also examined. Sterile soil microcosms were inoculated with washed cells to a final concentration of ca. 106 CFU.g–1 and placed at 5, 23, and 35–37 °C. Samples were taken periodically and examined for culturability and viability, using the substrate responsiveness assay. Our results indicated no significant loss of culturability at 5 and 23 °C for a period of over one year. In contrast, cells of P. fluorescens incubated at 35–37 °C entered the viable but nonculturable state within 7 days. All cells labeled with gfp retained fluorescence regardless of culturability, suggesting that the green fluorescent protein can be of value in monitoring the presence of cells following their release to the environment. Because fluorescence was maintained regardless of the cells’ physiological state, this protein may also be an indicator of cell viability.

The release of genetically engineered apple trees raises the question of their potential environmental impact, and the transfer via pollen of transgenes to cross-compatible cultivars of Malus domestica and Malus species is deemed to be the greatest source for environmental exposure. The hybrid TNR 31-35, a descendant of Malus sieversii var. sieversii f. niedzwetzkyana, carrying a homozygous, dominant gene responsible for red pigmentation in all plant parts, was used to assess gene flow in an apple scion repository of genetic resources. The red pigmentation provides a morphological marker that enables large-scale evaluation of cross-fertilization under natural conditions. In two consecutive years, 60 and then 56 apple trees of 38 different Malus domestica cultivars were selected to serve as pollen-receptor trees. In these two years, 6876 and then 5513 seeds, respectively, were gathered from pollen-receptor trees located at different distances, 2–100 m from 15 pollen-dispenser trees. In total, 11 797 seedlings were examined. An average of 1.8% and 1.4%, respectively, of all seedlings obtained showed red-colored leaves. Considering both years of sampling, 69% of the seeds fertilized by TNR 31-35 were found at less than 10 m from the nearest pollen-dispenser tree. Almost 91% of all seeds fertilized by TNR 31-35 were found at less than 60 m from the nearest pollen-dispenser tree, which is equal to 30 adjacent trees along the row. In this study, pollen was dispersed at least 104 m. After phenotypical evaluation, seedlings selected as red-colored were investigated by simple sequence repeat (SSR) analysis. Each seedling was tested with at least one heteromorphic SSR-marker, which allows the verification of TNR 31-35 as the male parent. All but four seedlings showed one allele specific for the appropriate fruiting tree and the second allele specific for the pollen-dispenser TNR 31-35.

Gene constructs containing the Cauliflower mosaic virus (CaMV) 35S promoter and a sequence coding either for a green fluorescent protein (GFP) or for firefly luciferase were transfected into Chinese hamster ovary (CHO) cells. Both reporter genes were expressed to significant levels. The 35S promoter was 40 times less active than the human eF1α promoter, which is known to be one of the most potent promoters in mammalian cells. The 35S promoter must therefore be considered to be a promoter of significant potency in mammalian cells. RT-PCR analysis suggested that transcription initiation in CHO cells occurred between the TATA box and the transcription start site of the 35S promoter that function in plant cells. Further analysis by 5’RACE confirmed that transcription was initiated in CHO cells at different sites located essentially between the TATA box and the plant transcription start site, showing that 35S promoter activity in animal cells is due to the presence of promoter elements that are functional in mammalian cells, but that are not those used in plants. The data reported here raise the possibility that genes controlled by the 35S promoter, which is commonly used in transgenic plants, have the potential for expression in animal cells.

This study deals with hybrids between Aegilops geniculata and bread wheat, Triticum aestivum L., detected in two Ae. geniculata populations in the “Meseta Central”, Spain’s central plateau where wheat is a major crop. Morphological traits and pentaploid chromosome numbers were used to identify hybrids in 2004 and 2005. The frequency of hybridization under natural conditions was calculated for one of these populations. Six hybrids were detected from a sample of 3158 seeds from this population. The hybrids were mainly sterile but seed set can be found. This study extends the range of documented hybridization to include central Spain. The potential risks associated with natural hybridization in the context of transgenic wheat cultivation are discussed.

Transgenes from transgenic oilseed rape, Brassica napus (AACC genome), can introgress into populations of wild B. rapa (AA genome), but little is known about the long-term persistence of transgenes from different transformation events. For example, transgenes that are located on the crop’s C chromosomes may be lost during the process of introgression. We investigated the genetic behavior of transgenes in backcross generations of wild B. rapa after nine GFP (green fluorescent protein)-Bt (Bacillus thuringiensis) B. napus lines, named GT lines, were hybridized with three wild B. rapa accessions, respectively. Each backcross generation involved crosses between hemizygous GT plants and non-GT B. rapa pollen recipients. In some cases, sample sizes were too small to allow the detection of major deviations from Mendelian segregation ratios, but the segregation of GT:non-GT was consistent with an expected ratio of 1:1 in all crosses in the BC1 generation. Starting with the BC2 generation, significantly different genetic behavior of the transgenes was observed among the nine GT B. napus lines. In some lines, the segregation of GT:non-GT showed a ratio of 1:1 in the BC2, BC3, and BC4 generations. However, in other GT B. napus lines the segregation ratio of GT:non-GT significantly deviated from 1:1 in the BC2 and BC3 generations, which had fewer transgenic progeny than expected, but not in the BC4 generation. Most importantly, in two GT B. napus lines the segregation of GT:non-GT did not fit into a ratio of 1:1 in the BC2, BC3 or BC4 generations due to a deficiency of transgenic progeny. For these lines, a strong reduction of transgene introgression was observed in all three B. rapa accessions. These findings imply that the genomic location of transgenes in B. napus may affect the long-term persistence of transgenes in B. rapa after hybridization has occurred.