Gene Flow,Risk Assessment and the Environmental Release of Transgenic Plants

The release of genetically modified plants is governed by regulations that aim to provide an assessment of potential impact on the environment. One of the most important components of this risk assessment is an evaluation of the probability of gene flow. In this review, we provide an overview of the current literature on gene flow from transgenic plants, providing a framework of issues for those considering the release of a transgenic plant into the environment. For some plants gene flow from transgenic crops is well documented, and this information is discussed in detail in this review. Mechanisms of gene flow vary from plant species to plant species and range from the possibility of asexual propagation, short- or long-distance pollen dispersal mediated by insects or wind and seed dispersal. Volunteer populations of transgenic plants may occur where seed is inadvertently spread during harvest or commercial distribution. If there are wild populations related to the transgenic crop then hybridization and eventually introgression in the wild may occur, as it has for herbicide resistant transgenic oilseed rape (Brassica napus). Tools to measure the amount of gene flow, experimental data measuring the distance of pollen dispersal, and experiments measuring hybridization and seed survivability are discussed in this review. The various methods that have been proposed to prevent gene flow from genetically modified plants are also described. The current “transgenic traits” in the major crops confer resistance to herbicides and certain insects. Such traits could confer a selective advantage (an increase in fitness) in wild plant populations in some circumstances, were gene flow to occur. However, there is ample evidence that gene flow from crops to related wild species occurred before the development of transgenic crops and this should be taken into account in the risk assessment process.     

Tracing back seed and pollen flow within the crop-wild Beta vulgaris complex: genetic distinctiveness vs. hot spots of hybridization over a regional scale

Hybrids between transgenic crops and wild relatives have been documented successfully in a wide range of cultivated species, having implications on conservation and biosafety management. Nonetheless, the magnitude and frequency of hybridization in the wild is still an open question, in particular when considering several populations at the landscape level. The Beta vulgaris complex provides an excellent biological model to tackle this issue. Weed beets contaminating sugar beet fields are expected to act as a relay between wild populations and crops and from crops-to-crops. In one major European sugar beet production area, nine wild populations and 12 weed populations were genetically characterized using cytoplasmic markers specific to the cultivated lines and nuclear microsatellite loci. A tremendous overall genetic differentiation between neighbouring wild and weed populations was depicted. However, genetic admixture analyses at the individual level revealed clear evidence for gene flow between wild and weed populations. In particular, one wild population displayed a high magnitude of nuclear genetic admixture, reinforced by direct seed flow as evidenced by cytoplasmic markers. Altogether, weed beets were shown to act as relay for gene flow between crops to wild populations and crops to crops by pollen and seeds at a landscape level.     

Gene flow from transgenic to nontransgenic soybean plants in the Cerrado region of Brazil

Evaluation of transgenic crops under field conditions is a fundamental step for the production of genetically engineered varieties. In order to determine if there is pollen dispersal from transgenic to nontransgenic soybean plants, a field release experiment was conducted in the Cerrado region of Brazil. Nontransgenic plants were cultivated in plots surrounding Roundup Ready transgenic plants carrying the cp4 epsps gene, which confers herbicide tolerance against glyphosate herbicide, and pollen dispersal was evaluated by checking for the dominant gene. The percentage of cross-pollination was calculated as a fraction of herbicide-tolerant and -nontolerant plants. The greatest amount of transgenic pollen dispersion was observed in the first row, located at one meter from the central (transgenic) plot, with a 0.52% average frequency. The frequency of pollen dispersion decreased to 0.12% in row 2, reaching 0% when the plants were up to 10 m distance from the central plot. Under these conditions pollen flow was higher for a short distance. This fact suggests that the management necessary to avoid cross-pollination from transgenic to nontransgenic plants in the seed production fields should be similar to the procedures currently utilized to produce commercial seeds.     

Evidence for gene flow via seed dispersal from crop to wild relatives in Beta vulgaris (Chenopodiaceae): consequences for the release of genetically modified crop species with weedy lineages

Gene flow and introgression from cultivated to wild plant populations have important evolutionary and ecological consequences and require detailed investigations for risk assessments of transgene escape into natural ecosystems. Sugar beets (Beta vulgaris ssp. vulgaris) are of particular concern because: (i) they are cross-compatible with their wild relatives (the sea beet, B. vulgaris ssp. maritima); (ii) crop-to-wild gene flow is likely to occur via weedy lineages resulting from hybridization events and locally infesting fields. Using a chloroplastic marker and a set of nuclear microsatellite loci, the occurrence of crop-to-wild gene flow was investigated in the French sugar beet production area within a 'contact-zone' in between coastal wild populations and sugar beet fields. The results did not reveal large pollen dispersal from weed to wild beets. However, several pieces of evidence clearly show an escape of weedy lineages from fields via seed flow. Since most studies involving the assessment of transgene escape from crops to wild outcrossing relatives generally focused only on pollen dispersal, this last result was unexpected: it points out the key role of a long-lived seed bank and highlights support for transgene escape via man-mediated long-distance dispersal events.     

Pollen and seed dispersal inferred from seedling genotypes: the Bayesian revolution has passed here too

Understanding precisely how plants disperse their seeds and pollen in their neighbourhood is a central question for both ecologists and evolutionary biologists because seed and pollen dispersal governs both the rate of spread of an expanding population and gene flow within and among populations. The concept of a 'dispersal kernel' has become extremely popular in dispersal ecology as a tool that summarizes how dispersal distributes individuals and genes in space and at a given scale. In this issue of Molecular Ecology, the study by Moran & Clark (2011) (M&C in the following) shows how genotypic and spatial data of established seedlings can be analysed in a Bayesian framework to estimate jointly the pollen and seed dispersal kernels and finally derive a parentage analysis from a full-probability approach. This approach applied to red oak shows important dispersal of seeds (138 m on average) and pollen (178 m on average). For seeds, this estimate contrasts with previous results from inverse modelling on seed trap data (9.3 m). This research gathers several methodological advances made in recent years in two research communities and could become a cornerstone for dispersal ecology.     

Fine-scale geographical structure of genetic diversity in inland wild beet populations

Introgression arising from crop-to-wild gene flow provides novel sources of genetic variation in plant species complexes. Hybridization within the Beta vulgaris species complex is of immediate concern; crop lineages ( B .  vulgaris ssp. vulgaris ) hybridize easily with their wild relatives ( B .  vulgaris ssp. maritima ) thereby threatening wild beet gene diversity with genetic swamping. Hybridization 'hotspots' occur in European seed production areas because inland ruderal wild beets occur and reproduce in sympatry with cultivated beets. We studied gene flow occurring between seed-producing cultivars and ruderal wild B .  vulgaris in southwestern France to determine whether feral beets, arising from unharvested cultivated seed, represent an opportunity for crop-to-wild gene flow. We surveyed 42 inland ruderal beet populations located near seed production fields for nucleo-cytoplasmic variation and used a cytoplasmic marker diagnostic of cultivated lines. Occurrence of cultivated-type cytoplasm within ruderal populations clearly reflected events of crop seed escape. However, we found no genetic signatures of nuclear cultivated gene introgression, which suggests past introgression of cultivated cytoplasm into a wild nuclear background through seed escape rather than recent direct pollen flow. Overall, patterns of genetic structure suggested that inland ruderal wild beet populations act as a metapopulation, with founding events involving a few sib groups, followed by low rates of seed or pollen gene flow after populations are established. Altogether, our results indicate that a long-lived seed bank plays a key role in maintaining cultivated-type cytoplasm in the wild and highlight the need for careful management of seed production areas where wild and cultivated relatives co-occur.     

Modelling and estimating pollen movement in oilseed rape (Brassica napus) at the landscape scale using genetic markers

Understanding patterns of pollen movement at the landscape scale is important for establishing management rules following the release of genetically modified (GM) crops. We use here a mating model adapted to cultivated species to estimate dispersal kernels from the genotypes of the progenies of male-sterile plants positioned at different sampling sites within a 10 x 10-km oilseed rape production area. Half of the pollen clouds sampled by the male-sterile plants originated from uncharacterized pollen sources that could consist of both large volunteer and feral populations, and fields within and outside the study area. The geometric dispersal kernel was the most appropriate to predict pollen movement in the study area. It predicted a much larger proportion of long-distance pollination than previously fitted dispersal kernels. This best-fitting mating model underestimated the level of differentiation among pollen clouds but could predict its spatial structure. The estimation method was validated on simulated genotypic data, and proved to provide good estimates of both the shape of the dispersal kernel and the rate and composition of pollen issued from uncharacterized pollen sources. The best dispersal kernel fitted here, the geometric kernel, should now be integrated into models that aim at predicting gene flow at the landscape level, in particular between GM and non-GM crops.     

Pollen dispersal in sugar beet production fields

Pollen-mediated gene flow has important implications for biodiversity conservation and for breeders and farmers’ activities. In sugar beet production fields, a few sugar beet bolters can produce pollen as well as be fertilized by wild and weed beet. Since the crop, the wild beets, and the weed beets are the same species and intercross freely, the question of pollen flow is an important issue to determine the potential dispersal of transgenes from field to field and to wild habitats. We report here an experiment to describe pollen dispersal from a small herbicide-resistant sugar beet source towards male sterile target plants located along radiating lines up to 1,200 m away. Individual dispersal functions were inferred from statistical analyses and compared. Pollen limitation, as expected in root-production fields, was confirmed at all the distances from the pollen source. The number of resistant seeds produced by bait plants best fitted a fat-tailed probability distribution curve of pollen grains (power–law) dependent on the distance from the pollen source. A literature survey confirmed that power–law function could fit in most cases. The b coefficient was lower than 2. The number of fertilized flowers by background (herbicide-susceptible) pollen grains was uniform across the whole field. Airborne pollen had a fertilization impact equivalent to that of one adjacent bolter. The individual dispersal function from different pollen sources can be integrated to provide the pollen cloud composition for a given target plant, thus allowing modeling of gene flow in a field, inter-fields in a small region, and also in seed-production area. Long-distance pollen flow is not negligible and could play an important role in rapid transgene dispersal from crop to wild and weed beets in the landscape. The removing of any bolting, herbicide-resistant sugar beet should be compulsory to prevent the occurrence of herbicide-resistant weed beet, thus preventing gene flow to wild populations and preserving the sustainable utility of the resistant varieties. Whether such a goal is attainable remains an open question and certainly would be worth a large scale experimental study.     

Long distance pollen-mediated gene flow at a landscape level: the weed beet as a case study

Gene flow is a crucial parameter that can affect the organization of genetic diversity in plant species. It has important implications in terms of conservation of genetic resources and of gene exchanges between crop to wild relatives and within crop species complex. In the Beta vulgaris complex, hybridization between crop and wild beets in seed production areas is well documented and the role of the ensuing hybrids, weed beets, as bridges towards wild forms in sugar beet production areas have been shown. Indeed, in contrast to cultivated beets that are bi-annual, weed beets can bolt, flower and reproduce in the same crop season. Nonetheless, the extent of pollen gene dispersal through weedy lineages remains unknown. In this study, the focus is directed towards weed-to-weed gene flow, and we report the results of a pollen-dispersal analysis within an agricultural landscape composed of five sugar beet fields with different levels of infestation by weed beets. Our results, based on paternity analysis of 3240 progenies from 135 maternal plants using 10 microsatellite loci, clearly demonstrate that even if weedy plants are mostly pollinated by individuals from the same field, some mating events occur between weed beets situated several kilometres apart (up to 9.6 km), with rates of interfield-detected paternities ranging from 11.3% to 17.5%. Moreover, we show that pollen flow appears to be more restricted when individuals are aggregated as most mating events occurred only for short-distance classes. The best-fit dispersal curves were fat-tailed geometric functions for populations exhibiting low densities of weed beets and thin-tailed Weibull function for fields with weed beet high densities. Thus, weed beet populations characterized by low density with geographically isolated individuals may be difficult to detect but are likely to act as pollen traps for pollen emitted by close and remote fields. Hence, it appears evident that interfield pollen-mediated gene flow between weed beets is almost unavoidable and could contribute to the diffusion of (trans)genes in the agricultural landscape.     

Consequences of recurrent gene flow from crops to wild relatives

Concern about gene flow from crops to wild relatives has become widespread with the increasing cultivation of transgenic crops. Possible consequences of such gene flow include genetic assimilation, wherein crop genes replace wild ones, and demographic swamping, wherein hybrids are less fertile than their wild parents, and wild populations shrink. Using mathematical models of a wild population recurrently receiving pollen from a genetically fixed crop, we find that the conditions for genetic assimilation are not stringent, and progress towards replacement can be fast, even for disfavoured crop genes. Demographic swamping and genetic drift relax the conditions for genetic assimilation and speed progress towards replacement. Genetic assimilation can involve thresholds and hysteresis, such that a small increase in immigration can lead to fixation of a disfavoured crop gene that had been maintained at a moderate frequency, even if the increase in immigration is cancelled before the gene fixes. Demographic swamping can give rise to 'migrational meltdown', such that a small increase in immigration can lead to not only fixation of a disfavoured crop gene but also drastic shrinkage of the wild population. These findings suggest that the spread of crop genes in wild populations should be monitored more closely.     

Establishment of transgenic herbicide-resistant creeping bentgrass (Agrostis stolonifera L.) in nonagronomic habitats

Concerns about genetically modified (GM) crops include transgene flow to compatible wild species and unintended ecological consequences of potential transgene introgression. However, there has been little empirical documentation of establishment and distribution of transgenic plants in wild populations. We present herein the first evidence for escape of transgenes into wild plant populations within the USA; glyphosate-resistant creeping bentgrass (Agrostis stolonifera L.) plants expressing CP4 EPSPS transgenes were found outside of cultivation area in central Oregon. Resident populations of three compatible Agrostis species were sampled in nonagronomic habitats outside the Oregon Department of Agriculture control area designated for test production of glyphosate-resistant creeping bentgrass. CP4 EPSPS protein and the corresponding transgene were found in nine A. stolonifera plants screened from 20,400 samples (0.04 +/- 0.01% SE). CP4 EPSPS-positive plants were located predominantly in mesic habitats downwind and up to 3.8 km beyond the control area perimeter; two plants were found within the USDA Crooked River National Grassland. Spatial distribution and parentage of transgenic plants (as confirmed by analyses of nuclear ITS and chloroplast matK gene trees) suggest that establishment resulted from both pollen-mediated intraspecific hybridizations and from crop seed dispersal. These results demonstrate that transgene flow from short-term production can result in establishment of transgenic plants at multi-kilometre distances from GM source fields or plants. Selective pressure from direct application or drift of glyphosate herbicide could enhance introgression of CP4 EPSPS transgenes and additional establishment. Obligatory outcrossing and vegetative spread could further contribute to persistence of CP4 EPSPS transgenes in wild Agrostis populations, both in the presence or absence of herbicide selection.     

Increased pre-dispersal seed predation in sunflower crop-wild hybrids

The fitness of crop-wild hybrids can influence gene flow between crop and wild populations. Seed predation levels in crop-wild hybrid plants can be an important factor in determining plant fitness, especially in large-seeded crops such as sunflower. To determine patterns of pre-dispersal seed predation, seeds were collected from wild sunflowers (Helianthus annuus L.) and wild×crop F₁ hybrids at three experimental field sites in eastern Kansas. Seed heads were dissected and each seed was counted and scored for categories of seed damage by lepidopteran and coleopteran larvae. Hybrid seed heads showed significantly higher levels of insect-damaged seeds. The average hybrid plant had 36.5% of its seeds (or 45.1 seeds per plant) eaten by insect larvae while the average wild plant lost only 1.8% (or 95 seeds) to seed predators. Hybrid populations had higher levels of total insect damage even when date of flowering, flower head diameter, and the number of open heads within the study site were accounted for. These results suggest that the reduced fecundity of F₁ crop-wild sunflower hybrids demonstrated in other studies may be augmented by the increased seed predation in hybrid flower heads. Fecundity estimates of crop-wild hybrid and wild plants that disregard differential seed predation levels may not accurately reflect the actual relative contributions of hybrid and wild plants to future generations. Received: 21 December 1998 / Accepted: 8 July 1999     

Engineered selective plant male sterility through pollen‐specific expression of the EcoRI restriction endonuclease

Unintended gene flow from transgenic plants via pollen, seed and vegetative propagation is a regulatory concern because of potential admixture in food and crop systems, as well as hybridization and introgression to wild and weedy relatives. Bioconfinement of transgenic pollen would help address some of these concerns and enable transgenic plant production for several crops where gene flow is an issue. Here, we demonstrate the expression of the restriction endonuclease EcoRI under the control of the tomato pollen‐specific LAT52 promoter is an effective method for generating selective male sterility in Nicotiana tabacum (tobacco). Of nine transgenic events recovered, four events had very high bioconfinement with tightly controlled EcoRI expression in pollen and negligible‐to‐no expression other plant tissues. Transgenic plants had normal morphology wherein vegetative growth and reproductivity were similar to nontransgenic controls. In glasshouse experiments, transgenic lines were hand‐crossed to both male‐sterile and emasculated nontransgenic tobacco varieties. Progeny analysis of 16 000–40 000 seeds per transgenic line demonstrated five lines approached (>99.7%) or attained 100% bioconfinement for one or more generations. Bioconfinement was again demonstrated at or near 100% under field conditions where four transgenic lines were grown in close proximity to male‐sterile tobacco, and 900–2100 seeds per male‐sterile line were analysed for transgenes. Based upon these results, we conclude EcoRI‐driven selective male sterility holds practical potential as a safe and reliable transgene bioconfinement strategy. Given the mechanism of male sterility, this method could be applicable to any plant species.     

Population genetic structure of Japanese wild soybean (Glycine soja) based on microsatellite variation

The research objectives were to determine aspects of the population dynamics relevant to effective monitoring of gene flow in the soybean crop complex in Japan. Using 20 microsatellite primers, 616 individuals from 77 wild soybean (Glycine soja) populations were analysed. All samples were of small seed size (< 0.03 g), were directly collected in the field and came from all parts of Japan where wild soybeans grow, except Hokkaido. Japanese wild soybean showed significant reduction in observed heterozygosity, low outcrossing rate (mean 3.4%) and strong genetic differentiation among populations. However, the individual assignment test revealed evidence of rare long-distance seed dispersal (> 10 km) events among populations, and spatial autocorrelation analysis revealed that populations within a radius of 100 km showed a close genetic relationship to one another. When analysis of graphical ordination was applied to compare the microsatellite variation of wild soybean with that of 53 widely grown Japanese varieties of cultivated soybean (Glycine max), the primary factor of genetic differentiation was based on differences between wild and cultivated soybeans and the secondary factor was geographical differentiation of wild soybean populations. Admixture analysis revealed that 6.8% of individuals appear to show introgression from cultivated soybeans. These results indicated that population genetic structure of Japanese wild soybean is (i) strongly affected by the founder effect due to seed dispersal and inbreeding strategy, (ii) generally well differentiated from cultivated soybean, but (iii) introgression from cultivated soybean occurs. The implications of the results for the release of transgenic soybeans where wild soybeans grow are discussed.     

Historical and contemporary gene dispersal in wild carrot (Daucus carota ssp. carota) populations

Background and Aims

Wild carrot is the ancestor of cultivated carrot and is the most important gene pool for carrot breeding. Transgenic carrot may be released into the environment in the future. The aim of the present study was to determine how far a gene can disperse in wild carrot populations, facilitating risk assessment and management of transgene introgression from cultivated to wild carrots and helping to design sampling strategies for germplasm collections.

Methods

Wild carrots were sampled from Meijendel and Alkmaar in The Netherlands and genotyped with 12 microsatellite markers. Spatial autocorrelation analyses were used to detect spatial genetic structures (SGSs). Historical gene dispersal estimates were based on an isolation by distance model. Mating system and contemporary pollen dispersal were estimated using 437 offspring of 20 mothers with different spatial distances and a correlated paternity analysis in the Meijendel population.

Key Results

Significant SGSs are found in both populations and they are not significantly different from each other. Combined SGS analysis indicated significant positive genetic correlations up to 27 m. Historical gene dispersal σ_g and neighbourhood size N_b were estimated to be 4–12 m [95 % confidence interval (CI): 3–25] and 42–73 plants (95 % CI: 28–322) in Meijendel and 10–31 m (95 % CI: 7–∞) and 57–198 plants (95 % CI: 28–∞) in Alkmaar with longer gene dispersal in lower density populations. Contemporary pollen dispersal follows a fat-tailed exponential-power distribution, implying pollen of wild carrots could be dispersed by insects over long distance. The estimated outcrossing rate was 96 %.

Conclusions

SGSs in wild carrots may be the result of high outcrossing, restricted seed dispersal and long-distance pollen dispersal. High outcrossing and long-distance pollen dispersal suggest high frequency of transgene flow might occur from cultivated to wild carrots and that they could easily spread within and between populations.     

Impact of gene flow from cultivated beet on genetic diversity of wild sea beet populations

Gene flow and introgression from cultivated plants may have important consequences for the conservation of wild plant populations. Cultivated beets (sugar beet, red beet and Swiss chard: Beta vulgaris ssp. vulgaris) are of particular concern because they are cross-compatible with the wild taxon, sea beet (B.vs. ssp. maritima). Cultivated beet seed production areas are sometimes adjacent to sea beet populations; the numbers of flowering individuals in the former typically outnumber those in the populations of the latter. In such situations, gene flow from cultivated beets has the potential to alter the genetic composition of the nearby wild populations. In this study we measured isozyme allele frequencies of 11 polymorphic loci in 26 accessions of cultivated beet, in 20 sea beet accessions growing near a cultivated beet seed production region in northeastern Italy, and 19 wild beet accessions growing far from seed production areas. We found one allele that is specific to sugar beet, relative to other cultivated types, and a second that has a much higher frequency in Swiss chard and red beet than in sugar beet. Both alleles are typically rare in sea beet populations that are distant from seed production areas, but both are common in those that are near the Italian cultivated beet seed production region, supporting the contention that gene flow from the crop to the wild species can be substantial when both grow in proximity. Interestingly, the introgressed populations have higher genetic diversity than those that are isolated from the crop. The crop-to-wild gene flow rates are unknown, as are the fitness consequences of such alleles in the wild. Thus, we are unable to assess the long-term impact of such introgression. However, it is clear that gene flow from a crop to a wild taxon does not necessarily result in a decrease in the genetic diversity of the native plant.     

Gene flow assessment in transgenic plants

In most of the important crops in the world, gene flow between cultivars and between wild and weedy relatives has always taken place. Factors influencing this gene flow, such as the mating system, mode of pollination, mode of seed dispersal and the particular characteristics of the habitat where the crops grow, are difficult to evaluate and in consequence, the quantification of gene flow is not easy. Transgene flow from engineered crops to other cultivars or to their wild and weedy relatives is one of the major concerns in relation to the ecological risks associated with the commercial release of transgenic plants. With transgenic crops it is important to quantify this gene flow and to try to establish strategies to control or minimise it, taking into account the possible ecological effect of the newly introduced genes, whether advantageous or disadvantageous. The use of transgenic plants has proven to be an effective tool to quantify the gene flow to other cultivars of the same species or to wild and weedy relatives in all crops analysed. Here we review the major studies in this area, and conclude that the potential risk of gene flow has to be assessed case by case and caution is necessary when making general conclusions.     

Founder effect in crop-plant evolution

Seed-crop plants apparently originated from a limited number of mutants in which seed dispersal was changed from that found in nondomesticated populations. Seed nonshattering in cultivated plants may be controlled by a single gene or a small number of genes. Allopolyploid crop plants were derived from a limited number of interspecific hybridizations followed by chromosome doubling. The consequence of this founder effect is a narrow genetic variability in the crop population compared to its wild progenitor. Natural hybridization between the two is prevented by various isolating mechanisms, and gene flow, if it exists, is apparently more effective in the direction from the cultivated to the wild populations. Founder effect in crop-plant evolution indicates the value and the breeding potential of the genetic variability remaining in its wild relatives.     

Seed size variation and predation of seeds produced by wild and crop-wild sunflowers

The movement of pollen between crop and wild sunflowers (both Helianthus annuus) has led to concerns about the possible introduction of crop transgenes into wild populations. The persistence of crop traits in wild populations will depend in part on the relative fitness of crop-wild hybrid vs. wild plants. Using seeds from two large experimental field plots, we found that seeds produced by crop-wild plants were twice the size of wild seeds and differed in coloration. Head diameter, date of flowering, identity of mother plant, and levels of predispersal predation explained some variation in mean seed size. We hypothesized that postdispersal vertebrate seed predation would be affected by seed size, with hybrid seeds preferentially eaten. In each of three field trials, significantly more hybrid seeds were eaten (62% of hybrid seed; 42% of wild seed). Within the category of wild seeds, larger seeds were preferentially eaten; however among hybrid seeds, predation was not significantly related to seed size. In this study, differential predation thus reduces hybrid fitness and would presumably slow the spread of transgenes into wild populations.