Scale effect on rice pollen‐mediated gene flow: implications in assessing transgene flow from genetically engineered plants

Transgene flow from genetically engineered (GE) crops to non‐GE varieties and wild relatives via pollen‐mediated gene flow (PMGF) may create food and environmental biosafety concerns. Assessing the level of PMGF from GE crops is required before commercialization. Whether the level of PMGF estimated at relatively small scales can sufficiently represent the actual scenario at large production scales remains unresolved. Here, we estimated average PMGF frequencies from three insect‐resistant GE rice lines to their non‐GE counterparts at four scales ranging from 9 to 576 m², having the number of GE to non‐GE plants constantly at the ratio of 8:1. Based on nearly 1.3 million examined seedlings from non‐GE rice plots, very low frequencies (<0.1%) of transgene flow were detected. The highest frequencies were found in plots at the smallest scales. Scale had a significantly negative effect on the frequency of PMGF in rice, with decreased gene flow at increased scale. An extended PMGF model could well represent the experimental data. Field experiments at relatively small scales combined with mathematical modelling could provide useful prediction on the level of rice transgene flow at large production scales. This is probably applicable for other crop species with wind‐ and self‐pollination.     

Modelling pollen‐mediated gene flow in rice: risk assessment and management of transgene escape

Fast development and commercialization of genetically modified plants have aroused concerns of transgene escape and its environmental consequences. A model that can effectively predict pollen‐mediated gene flow (PMGF) is essential for assessing and managing risks from transgene escape. A pollen‐trap method was used to measure the wind‐borne pollen dispersal in cultivated rice and common wild rice, and effects of relative humidity, temperature and wind speed on pollen dispersal were estimated. A PMGF model was constructed based on the pollen dispersal pattern in rice, taking outcrossing rates of recipients and cross‐compatibility between rice and its wild relatives into consideration. Published rice gene flow data were used to validate the model. Pollen density decreased in a simple exponential pattern with distances to the rice field. High relative humidity reduced pollen dispersal distances. Model simulation showed an increased PMGF frequency with the increase of pollen source size (the area of a rice field), but this effect levelled off with a large pollen‐source size. Cross‐compatibility is essential when modelling PMGF from rice to its wild relatives. The model fits the data well, including PMGF from rice to its wild relatives. Therefore, it can be used to predict PMGF in rice under diverse conditions (e.g. different outcrossing rates and cross‐compatibilities), facilitating the determination of isolation distances to minimize transgene escape. The PMGF model may be extended to other wind‐pollinated plant species such as wheat and barley.     

转基因逃逸及其环境生物安全评价研究进展——抗虫水稻案例分析

转基因技术研发为提高我国水稻产量和减少劳动力投入提供了巨大机遇。我国对转基因水稻研发进行了大量的投入,目前已培育了具有不同新性状的转基因水稻品系,许多品系已进入生物安全评价阶段。风险评价对转基因水稻的安全生产至关重要,是其商品化生产之前必须解决的问题,其中包括转基因逃逸及其潜在环境影响。对水稻抗虫转基因逃逸及其潜在环境风险的评价包括3个重要环节：（1）通过田间试验和模型模拟检测转基因漂移到非转基因栽培稻及其野生近缘种的频率;（2）检测转基因在栽培稻和野生近缘种后代中的表达;（3）确定转基因对野生近缘种群体适合度和进化潜力的影响。大量研究表明,在近距离的空间范围内栽培稻品种之间的基因漂移频率很低（〉0.1%）,但栽培稻与其野生近缘种的基因漂移频率变异很大。进一步研究还表明,Bt抗虫转基因在栽培稻与普通野生稻后代中均能正常表达,但在其不同生长阶段,表达量有很大变异。在有较高水平的害虫虫压下,含有抗虫转基因的栽培稻及野生近缘种杂交后代与不含转基因的对照相比,抗虫性显著提高且适合度利益明显;但是,在虫害发生水平较低时,含有抗虫转基因的群体与不含抗虫转基因的群体相比没有显著的适合度优势。综上,转基因逃逸到非转基因水稻的频率极低,并且可以通过空间隔离阻断其逃逸。虽然抗虫转基因向杂草稻以及与栽培稻距离较近的野生稻群体的逃逸无法避免,但是野生稻和杂草稻群体周围环境中的总体虫压较低,所以基因漂移带来的环境影响应十分有限。     

Gene flow from genetically modified rice to its wild relatives: Assessing potential ecological consequences

Pollen-mediated gene flow is the major pathway for transgene escape from GM rice to its wild relatives. Transgene escape to wild Oryza species having AA-genome will occur if GM rice is released to environments with these wild Oryza species. Transgenes may persist to and spread in wild populations after gene flow, resulting unwanted ecological consequences. For assessing the potential consequences caused by transgene escape, it is important to understand the actual gene flow frequencies from GM rice to wild relatives, transgene expression and inheritance in the wild relatives, as well as fitness changes that brought to wild relatives by the transgenes. This article reviews studies on transgene escape from rice to its wild relatives via gene flow and its ecological consequences. A framework for assessing potential ecological consequences caused by transgene escape from GM rice to its wild relatives is discussed based on studies of gene flow and fitness changes.     

Low crop plant population densities promote pollen-mediated gene flow in spring wheat (Triticum aestivum L.)

Transgenic wheat is currently being field tested with the intent of eventual commercialization. The development of wheat genotypes with novel traits has raised concerns regarding the presence of volunteer wheat populations and the role they may play in facilitating transgene movement. Here, we report the results of a field experiment that investigated the potential of spring wheat plant population density and crop height to minimize gene flow from a herbicide-resistant (HR) volunteer population to a non-HR crop. Pollen-mediated gene flow (PMGF) between the HR volunteer wheat population and four conventional spring wheat genotypes varying in height was assessed over a range of plant population densities. Natural hybridization events between the two cultivars were detected by phenotypically scoring plants in F₁ populations followed by verification with Mendelian segregation ratios in the F_1:2 families. PMGF was strongly associated with crop yield components, but showed no association with flowering synchrony. Maximum observed PMGF was always less than 0.6%, regardless of crop height and density. The frequency of PMGF in spring wheat decreased exponentially with increasing plant population density, but showed no dependence on either crop genotype or height. However, increasing plant densities beyond the recommended planting rate of 300 cropped wheat plants m⁻² provided no obvious benefit to reducing PMGF. Nevertheless, our results demonstrate a critical plant density of 175–200 cropped wheat plants m⁻² below which PMGF frequencies rise exponentially with decreasing plant density. These results will be useful in the development of mechanistic models and best management practices that collectively facilitate the coexistence of transgenic and nontransgenic wheat crops.     

Investigating Pollen and Gene Flow of WYMV-Resistant Transgenic Wheat N12-1 Using a Dwarf Male-Sterile Line as the Pollen Receptor

Pollen-mediated gene flow (PMGF) is the main mode of transgene flow in flowering plants. The study of pollen and gene flow of transgenic wheat can help to establish the corresponding strategy for preventing transgene escape and contamination between compatible genotypes in wheat. To investigate the pollen dispersal and gene flow frequency in various directions and distances around the pollen source and detect the association between frequency of transgene flow and pollen density from transgenic wheat, a concentric circle design was adopted to conduct a field experiment using transgenic wheat with resistance to wheat yellow mosaic virus (WYMV) as the pollen donor and dwarf male-sterile wheat as the pollen receptor. The results showed that the pollen and gene flow of transgenic wheat varied significantly among the different compass sectors. A higher pollen density and gene flow frequency was observed in the downwind SW and W sectors, with average frequencies of transgene flow of 26.37 and 23.69% respectively. The pollen and gene flow of transgenic wheat declined dramatically with increasing distance from its source. Most of the pollen grains concentrated within 5 m and only a few pollen grains were detected beyond 30 m. The percentage of transgene flow was the highest where adjacent to the pollen source, with an average of 48.24% for all eight compass directions at 0 m distance. Transgene flow was reduced to 50% and 95% between 1.61 to 3.15 m, and 10.71 to 20.93 m, respectively. Our results suggest that climate conditions, especially wind direction, may significantly affect pollen dispersal and gene flow of wheat. The isolation-by-distance model is one of the most effective methods for achieving stringent transgene confinement in wheat. The frequency of transgene flow is directly correlated with the relative density of GM pollen grains in air currents, and pollen competition may be a major factor influencing transgene flow.     

Transgene escape: what potential for crop–wild hybridization?

To date, regional surveys assessing the risk of transgene escape from GM crops have focused on records of spontaneous hybridization to infer the likelihood of crop transgene escape. However, reliable observations of spontaneous hybridization are lacking for most floras, particularly outside Europe. Here, we argue that evidence of interspecific reproductive compatibility derived from experimental crosses is an important component of risk assessment, and a useful first step especially where data from field observations are unavailable. We used this approach to assess the potential for transgene escape via hybridization for 123 widely grown temperate crops and their indigenous and naturalized relatives present in the New Zealand flora. We found that 66 crops (54%) are reproductively compatible with at least one other indigenous or naturalized species in the flora. Limited reproductive compatibility with wild relatives was evident for a further 12 crops (10%). Twenty-five crops (20%) were found to be reproductively isolated from all their wild relatives in New Zealand. For the remaining 20 crops (16%), insufficient information was available to determine levels of reproductive compatibility with wild relatives. Our approach may be useful in other regions where spontaneous crop-wild hybridization has yet to be well documented.     

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.     

Evaluating genetic containment strategies for transgenic plants

One of the primary concerns about genetically engineered crop plants is that they will hybridize with wild relatives, permitting the transgene to escape into the environment. The likelihood that a transgene will spread in the environment depends on its potential fitness impact. The fitness conferred by various transgenes to crop and/or wild-type hybrids has been evaluated in several species. Different strategies have been developed for reducing the probability and impact of gene flow, including physical separation from wild relatives and genetic engineering. Mathematical models and empirical experimental evidence suggest that genetic approaches have the potential to effectively prevent transgenes from incorporating into wild relatives and becoming established in wild populations that are not reproductively isolated from genetically engineered crops.     

Current knowledge of gene flow in plants: implications for transgene flow

Plant evolutionary biologists' view of gene flow and hybridization has undergone a revolution. Twenty-five years ago, both were considered rare and largely inconsequential. Now gene flow and hybridization are known to be idiosyncratic, varying with the specific populations involved. Gene flow typically occurs at evolutionarily significant rates and at significant distances. Spontaneous hybridization occasionally has important applied consequences, such as stimulating the evolution of more aggressive invasives and increasing the extinction risk for rare species. The same problems have occurred for spontaneous hybridization between crops and their wild relatives. These new data have implications for transgenic crops: (i) for most crops, gene flow can act to introduce engineered genes into wild populations; (ii) depending on the specific engineered gene(s) and populations involved, gene flow may have the same negative impacts as those observed for traditionally improved crops; (iii) gene flow's idiosyncratic nature may frustrate management and monitoring attempts; and (iv) intercrop transgene flow, although rarely discussed, is equally worthy of study.     

Establishment of a rice transgene flow model for predicting maximum distances of gene flow in southern China

We aimed to establish a rice gene flow model based on (i) the Gaussian plume model, (ii) data from a three-location x 3-yr field experiment on transgene flow to common rice cultivars (Oryza sativa), male sterile (ms) lines (O. sativa) and common wild rice (Oryza rufipogon), and (iii) 32-yr historical meteorological data collected from 38 meteorological stations in southern China during the rice flowering period. The concept of the gene flow coefficient (GFC) is proposed; that is, the ratio of the transgene flow frequency (G%) obtained from field experiments to the aggregated pollen dispersal frequency (P%) calculated based on the pollen dispersal model. The maximum distances of gene flow (MDGF) to traditional rice cultivars, ms lines, and common wild rice at a threshold value of either 1.0 or 0.1% were determined. The MDGF and its spatial distribution in southern China show that the gene flow pattern is significantly affected by the monsoon climate, the topography, and the outcrossing ability of recipients. We believe that the information provided in this study will be useful for the risk assessment of transgenic rice in other rice-growing regions.     

Gene flow from cultivated rice (Oryza sativa) to its weedy and wild relatives

BACKGROUND AND AIMS: Transgene escape through gene flow from genetically modified (GM) crops to their wild relative species may potentially cause environmental biosafety problems. The aim of this study was to assess the extent of gene flow between cultivated rice and two of its close relatives under field conditions. METHODS: Experiments were conducted at two sites in Korea and China to determine gene flow from cultivated rice (Oryza sativa L.) to weedy rice (O. sativa f. spontanea) and common wild rice (O. rufipogon Griff.), respectively, under special field conditions mimicking the natural occurrence of the wild relatives in Asia. Herbicide resistance (bar) and SSR molecular finger printing were used as markers to accurately determine gene flow frequencies from cultivated rice varieties to their wild relatives. KEY RESULTS: Gene flow frequency from cultivated rice was detected as between approx. 0.011 and 0.046 % to weedy rice and between approx. 1.21 and 2.19 % to wild rice under the field conditions. CONCLUSIONS: Gene flow occurs with a noticeable frequency from cultivated rice to its weedy and wild relatives, and this might cause potential ecological consequences. It is recommended that isolation zones should be established with sufficient distances between GM rice varieties and wild rice populations to avoid potential outcrosses. Also, GM rice should not be released when it has inserted genes that can significantly enhance the ecological fitness of weedy rice in regions where weedy rice is already abundant and causing great problems.     

Surveying of pollen-mediated crop-to-crop gene flow from a wheat field trial as a biosafety measure

Outcrosses from genetically modified (GM) to conventional crops by pollen-mediated gene flow (PMGF) are a concern when growing GM crops close to non-GM fields. This also applies to the experimental releases of GM plants in field trials. Therefore, biosafety measures such as isolation distances and surveying of PMGF are required by the regulatory authorities in Switzerland. For two and three years, respectively, we monitored crop-to-crop PMGF from GM wheat field trials in two locations in Switzerland. The pollen donors were two GM spring wheat lines with enhanced fungal resistance and a herbicide tolerance as a selection marker. Seeds from the experimental plots were sampled to test the detection method for outcrosses. Two outcrosses were found adjacent to a transgenic plot within the experimental area. For the survey of PMGF, pollen receptor plots of the conventional wheat variety Frisal used for transformation were planted in the border crop and around the experimental field up to a distance of 200 m. Although the environmental conditions were favorable and the donor and receptor plots flowered at the same time, only three outcrosses were found in approximately 185,000 tested seedlings from seeds collected outside the experimental area. All three hybrids were found in the border crop surrounding the experimental area, but none outside the field. We conclude that a pollen barrier (border crop) and an additional isolation distance of 5 m is a sufficient measure to reduce PMGF from a GM wheat field trial to cleistogamous varieties in commercial fields below a level that can be detected.     

Transgene flow in Mexican maize revisited: Socio‐biological analysis across two contrasting farmer communities and seed management systems

The flow of transgenes into landraces and wild relatives is an important biosafety concern. The case of transgene flow into local maize varieties in Mexico (the center of origin of maize) has been intensively debated over the past 15 years, including legal, political, and environmental disputes fanned by the existence of a significant scientific controversy over the methods used for the detection of transgenes. The use of diverse approaches and a lack of harmonized methods specific to the detection and monitoring of transgenes in landraces have generated both positive and negative results regarding contamination of Mexican maize with genetically modified material over the years. In this paper, we revisit the case of transgene contamination in Mexican maize and present a novel research approach based on socio‐biological analysis of contrasting communities and seed management systems. Two communities were used to investigate how different social and biological factors can affect transgene flow and impact transgene spread in Mexico. Our results show the presence of transgenes in one community and thus support the position that transgenes are highly likely to be present in Mexican maize landraces. However, our work also demonstrates that the extent and frequency with which transgenes can be found will significantly depend on the societal characteristics and seed management systems of the local communities. Therefore, we argue that future analysis of transgene presence should include social research on the seed management practices in the sampling area so that more robust and comprehensive understandings and conclusions can be drawn.     

The EPSPS gene flow from glyphosate-resistant Brassica napus to untransgene B. napus and wild relative species Orychophragmus violaceus

Gene flow from transgenic plants to compatible wild relatives is one of the major impediments to the development of the culture of genetically engineered crop plants. In this work, the flow of EPSPS (conferring resistance to glyphosate) gene of transgene Brassica napus toward the untransgene B. napus and wild relative species Orychophragmus violaceus in an open field (1 ha) was studied. The data related to only the 2004 and 2005 autumn season on one location of southwest of China. Pollen dispersal and fertilization of the target plants were favored and a detailed analysis of the hybrid offspring was performed. In field, the data studied show that the gene flow frequency was 0.16% between GM and non-GM B. napus at a distance of 1 m from the transgenic donor area. The crosspollination frequency was 0.05% between GM and non-GM B. napus at a distance of 5 m from the transgenic donor area. At a distance of 10 m, no crosspollination was observed. According to the results of this study, B. napus transgene flow was low. However, the wild relative species O. violaceus could not be fertilized by the transgenic pollen of B. napus, no matter what the distance was.     

Lessons we can learn from ecological biosafety research

The last decade has seen an increasing number of biosafety related publications focusing on transgenic organisms. Recent extensive field studies suggest that harmful laboratory effects on non-target organisms rarely occur in the environment. Moreover, biosafety studies typically show no difference in hybridisation between genetically modified plants (GMPs) or non-GMPs and related wild species. Since risk is a product of both exposure and hazard, biosafety research should clearly not only target gene flow exposure but specifically concentrate on expected hazards emerging from successful transgene flow to wild relatives of GMPs. Generally, transgenic plants behave in an ecologically similar manner to non-GMPs if the modified trait confers a neutral advantage under environmental or experimental conditions. However, GMPs perform better than non-GMPs if the new phenotype is challenged by conditions ecologically advantageous for the modified trait. Since biosafety research is a laborious process it will have to concentrate resources on thoughtful, thorough experiments, and target ecologically 'riskier' organisms. So far, we have no evidence that the use of GMPs contradicts sustainable agriculture and nature conservation per se.     

Bioclimatic model of tree radial growth: application to the French Mediterranean Aleppo pine forests

The potential effects of global changes on forests are of increasing concern. Dendrochronology, which deals with long-term records of tree growth under natural environmental conditions, can be used to evaluate the impact of climatic change on forest productivity. However, assessment of climatic change impacts must be supported by accurate and reliable models of the relationships between climate and tree growth. In this study, a bioclimatic model is used to explore the relationships between tree radial growth and bioclimatic variables closely related to the biological functioning of a tree. This model is at an intermediate level of complexity between purely empirical and process-based models. The method is illustrated with data for 21 Aleppo pine (Pinus halepensis Mill.) stands grown under a Mediterranean climate in south-east France. The results show that Aleppo pine growth is mainly controlled by soil water availability during the growing season. The bioclimatic variable which best expresses the observed inter-annual tree growth variations is the actual evapotranspiration (AET). Four parameters were adjusted to simulate dendrochronological data: the soil water capacity, the wilting point, the minimum temperature for photosynthesis, and the end of the growing season. The bioclimatic model gives better results than the standard response function and provides better insight into the functional processes involved in tree growth. The convincing results obtained by the bioclimatic model as well as the limited numbers of parameters it requires demonstrate the feasibility of using it to explore future climatic change impacts on Aleppo pine forests.     

Identification of a hybridization window that facilitates sizeable reductions of pollen-mediated gene flow in spring wheat

Transgenic wheat (Triticum aestivum L.) with improved agronomic traits is currently being field-tested. Gene flow in space is well-documented, but isolation in time has not received comparable attention. Here, we report the results of a field experiment that investigated reductions in intraspecific gene flow associated with temporal isolation of flowering between T. aestivum conspecifics. Pollen-mediated gene flow (PMGF) between an imazamox-resistant (IR) volunteer wheat population and a non-IR spring wheat crop was assessed over a range of volunteer emergence timings and plant population densities that collectively promoted flowering asynchrony. Natural hybridization events between the two populations were detected by phenotypically scoring plants in F₁ populations followed by verification with Mendelian segregation ratios in the F_1:2 lines. Based on the examination of >545,000 seedlings, we identified a hybridization window in spring wheat approximately 125 growing degree-days (GDD) in length. We found a sizeable reduction (two- to four-fold) in gene flow frequencies when flowering occurred outside of this window. The hybridization window identified in this research also will serve to temporally isolate neighboring wheat crops. However, strict control of volunteer populations or spatial isolation of neighbouring crops emerging within a 125 GDD hybridization window will be necessary to maintain low frequencies of PMGF in spring wheat fields. The model developed herein also is likely to be applicable to other wind-pollinated species.     

A large-scale field study of transgene flow from cultivated rice (Oryza sativa) to common wild rice (O. rufipogon) and barnyard grass (Echinochloa crusgalli)

The introgression of transgenes into wild relatives or weeds through pollen-mediated gene flow is a major concern in environmental risk assessment of transgenic crops. A large-scale (1.3–1.8 ha) rice gene flow study was conducted using transgenic rice containing the bar gene as a pollen donor and Oryza rufipogon as a recipient. There was a high frequency of transgene flow (11%−18%) at 0–1 m, with a steep decline with increasing distance to a detection limit of 0.01% by 250 m. To our knowledge, this is the highest frequency and longest distance of gene flow from transgenic rice to O. rufipogon reported so far. On the basis of these data, an adequate isolation distance from both conventional and transgenic rice should be taken for in situ conservation of common wild rice. Meanwhile, there is no evidence of transgene introgression into barnyard grass, even when it has coexisted with transgenic rice containing the bar gene for five successive years. Thus, the environmental risk of gene flow to this weedy species is of little concern.