Monitoring the Spread of Recombinant DNA from Field Plots with Transgenic Sugar Beet Plants by PCR and Natural Transformation of Pseudomonas Stutzeri

Previous studies had shown that recombinant DNA can be detected for several months in soil after the deposition of litter from transgenic (tg) plants. Here we show by PCR monitoring of field releases of tg sugar beet plants that during the growth of the plants the soil close to the plants and also plant material contains recombinant DNA, in the form of extracellular molecules. Surprisingly, the monitoring also revealed the presence of tg DNA in many field plots (30–70%) in which tg plants were never grown. These studies and the further monitoring during other tg sugar beet release experiments by PCR and a novel bioassay (measuring the transforming potential of recombinant DNA for Pseudomonas stutzeri) indicated that recombinant DNA was only detectable in the surface soil of field plots and their vicinity where flowering of the tg beet plants was allowed. Recombinant DNA was found in soil at a distance of 50 m from pollen-producing plants surrounded by a strip with hemp plants as a containment regime. It is concluded that recombinant DNA is deposited in soil during the growth of tg sugar beets and that a major mechanism of recombinant DNA spread in the environment is the dispersal of pollen which allows recombinant DNA to persist in the field plot for at least a year.     

Studying and managing the risk of cross-fertilization between transgenic crops and wild relatives

Drawing on field studies of pollen dispersal, we identify features of the hybridization process that need quantification. Our emphasis is on standardized measures, as opposed to the idiosyncratic and often anecdotal methods with which gene flow or out-crossing data are currently reported. In addition to proposing specific maximum likelihood approaches, we summarize some results to date from small-scale field trials that bear on the risks anticipated for large-scale commercialization. We conclude that absolute containment of recombinant pollen or genes is unlikely if physical isolation is the only containment strategy. Because we conclude that the escape of transgenic pollen is inevitable, we argue that the focus of risk analysis should be shifted towards the 'invasiveness' of transgenic plants and 'mitigation' of their impact on natural, as well as agricultural systems.     

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.     

Pollen as a vehicle for the escape of engineered genes?

Recent studies of pollen exchange between neighboring populations of plants have shown that interpopulation gene flow can proceed over much greater distances and at higher rates than hitherto believed. This means that the escape of engineered genes from crop plants to their wild relatives is not only possible, but also likely. The development of containment strategies, such as extra modifications for increased self-fertilization and decreased pollen longevity in engineered crop plants, will be necessary to safeguard against such escape.     

Expression of green fluorescent protein in pollen of oilseed rape (Brassica napus L.) and its utility for assessing pollen movement in the field

Transgene movement via pollen is an important component of gene flow from transgenic plants. Here, we present proof-of-concept studies that demonstrate the monitoring of short distant movement of pollen expressing a genetically encoded fluorescent tag in oilseed rape (Brassica napus L. cv. Westar). Transgenic oilseed rape plants were produced using Agrobacterium-mediated transformation method with the pBINDC1 construct containing a green fluorescent protein (GFP) variant, mGFP5-ER, under the control of the pollen-specific LAT59 promoter from tomato. Transgenic pollen was differentiated from non-transgenic pollen in vivo by a unique spectral signature, and was shown to be an effective tool to monitor pollen movement in the greenhouse and field. GFP-tagged pollen also served as a practical marker to determine the zygosity of plants. In a greenhouse pollen flow study, more pollen was captured at closer distances from the source plant plot with consistent wind generated by a fan. Under field conditions, GFP transgenic pollen grains were detected up to a distance of 15 m, the farthest distance from source plants assayed. GFP-tagged pollen was easily distinguishable from non-transgenic pollen using an epifluorescence microscope.     

Does insect netting affect the containment of airborne pollen from (GM-) plants in greenhouses?

Greenhouses are a well-accepted containment strategy to grow and study genetically modified plants (GM) before release into the environment. Various containment levels are requested by national regulations to minimize GM pollen escape. We tested the amount of pollen escaping from a standard greenhouse, which can be used for EU containment classes 1 and 2. More specifically, we investigated the hypothesis whether pollen escape could be minimized by insect-proof netting in front of the roof windows, since the turbulent airflow around the mesh wiring could avoid pollen from escaping. We studied the pollen flow out of greenhouses with and without insect netting of two non-transgenic crops, Ryegrass (Loliummultiflorum) and Corn (Zea Mays). Pollen flow was assessed with Rotorod(?) pollen samplers positioned inside and outside the greenhouse' roof windows. A significant proportion of airborne pollen inside the greenhouse leaves through roof windows. Moreover, the lighter pollen of Lolium escaped more readily than the heavier pollen of Maize. In contrast to our expectations, we did not identify any reduction in pollen flow with insect netting in front of open windows, even under induced airflow conditions. We conclude that insect netting, often present by default in greenhouses, is not effective in preventing pollen escape from greenhouses of wind-pollinated plants for containment classes 1 or 2. Further research would be needed to investigate whether other alternative strategies, including biotic ones, are more effective. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10453-011-9237-8) contains supplementary material, which is available to authorized users.     

Analysis of gene inheritance and expression in hybrids between transgenic sugar beet and wild beets

Reciprocal gene exchange between cultivated sugar beet and wild beets in seed production areas is probably the reason for the occurence of weed beets in sugar beet production fields. Therefore, when releasing transgenic sugar beet plants into the environment, gene transfer to wild beets ( Beta vulgaris ssp. maritima ) has to be considered. In this study the transfer of BNYVV- (beet necrotic yellow vein virus) resistance and herbicide-tolerance genes from two transgenic sugar beet lines that were released in field experiments in 1993 and 1994 in Germany to different wild beet accessions was investigated. In order to evaluate the consequences of outcrossing, manual pollinations of emasculated wild beet plants with homozygous transgenic sugar beet plants were performed. In the resulting hybrids the transgenes were stably inherited according to Mendelian law. Gene expression in leaves and roots of the hybrids was in the same range as in the original transgenic sugar beet plants. Moreover, it was found that in one of the wild beet accessions, transfer and expression of the BNYVV resistance gene did considerably increase the level of virus resistance.     

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.     

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.     

Marker Based Estimation of Gene Flow in Tropical Rice Fields and Its Ecological Consequences

Pollen mediated gene flow is the most widely debated biosafety issue in case of genetically modified crop plants and is a primary determining factor for permitting their field release, particularly major food crops like rice. The alleged consequences of gene escape into wild/weedy relatives that coexist together with cultivated forms in rice fields in several countries is perceived to be a major concern. In an effort to estimate the gene flow in rice in a tropical environment, rice varieties stacked with three bacterial blight (BB) resistance alleles and a cytoplasmic male sterile (CMS) line were used as donors and receiver of pollen respectively and the pollen flow was tracked with molecular markers that are closely linked to BB resistance genes. The study could detect gene flow up to 20?m distance with environmental factors like temperature and humidity influencing it. The gene flow observed at longer distances than the 10?m distance suggested earlier advocate caution and suggests that all the relevant biosafety issues need to be addressed prior to release of GM rice in tropical countries where sympatric association of wild relatives with cultivated rice with frequent crop-weedy gene flow both ways is quite common.     

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.     

Risk of alfalfa transgene dissemination and scale-dependent effects

Pollen can function as a vehicle to disseminate introduced, genetically engineered genes throughout a plant population or into a related species. The measurement of the risk of inadvertent dispersal of transgenes must include the assessment of accidental dispersion of pollen. Factors to be considered include the rate of pollen spread, the maximal dispersion distance of pollen, and the spatial dynamics of pollen movement within seed production fields; none of which are known for alfalfa (Medicago sativa L.), an insect-pollinated crop species. Using a rare, naturally occurring molecular marker, alfalfa pollen movement was tracked from seed and hay production fields. Results indicated that leafcutter bees (Megachile spp.) used in commercial seed production show a directional, non-random bias when pollinating within fields, primarily resulting in the movement of pollen directly towards and away from the bee domicile. Within-field pollen movement was detected only over distances of 4 m or less. Dispersal of pollen from alfalfa hay and seed production fields occurs at distances up to 1000 m. By examining widely dispersed, individual escaped alfalfa plants and their progeny using RAPD markers, gene movement among escaped alfalfa plants has been confirmed for distances up to 230 m. The outcrossing frequency for large fields was nearly 10-times greater than that of research-sized plots. A minimum isolation distance of 1557 m may be required to prevent gene flow in alfalfa. Data suggest that complete containment of transgenes within alfalfa seed or hay production fields would be highly unlikely using current production practices. Received: 20 March 1999 / Accepted: 11 November 1999     

Using seed purity data to estimate an average pollen mediated gene flow from crops to wild relatives

Gene flow from crops to wild related species has been recently under focus in risk-assessment studies of the ecological consequences of growing transgenic crops. However, experimental studies addressing this question are usually temporally or spatially limited. Indirect population-structure approaches can provide more global estimates of gene flow, but their assumptions appear inappropriate in an agricultural context. In an attempt to help the committees providing advice on the release of transgenic crops, we present a new method to estimate the quantity of genes migrating from crops to populations of related wild plants by way of pollen dispersal. This method provides an average estimate at a landscape level. Its originality is based on the measure of the inverse gene flow, i.e. gene flow from the wild plants to the crop. Such gene flow results in an observed level of impurities from wild plants in crop seeds. This level of impurity is usually known by the seed producers and, in any case, its measure is easier than a direct screen of wild populations because crop seeds are abundant and their genetic profile is known. By assuming that wild and cultivated plants have a similar individual pollen dispersal function, we infer the level of pollen-mediated gene flow from a crop to the surrounding wild populations from this observed level of impurity. We present an example for sugar beet data. Results suggest that under conditions of seed production in France (isolation distance of 1,000 m) wild beets produce high numbers of seeds fathered by cultivated plants. Received: 5 February 2001 / Accepted: 26 March 2001     

GFP‐tagged pollen to monitor pollen flow of transgenic plants

In this study, the pollen‐active LAT59 promoter from tomato was used to express a green fluorescent protein (GFP) encoding gene in Nicotiana tabacum (tobacco) pollen. This promoter is preferentially expressed in anthers and pollen. Pollen in transgenic plants segregated in a 1 : 1 Mendelian ratio, and the plants were polymerase chain reaction (PCR)‐positive. GFP‐tagged pollen was developed as a tool for tracking the movement of transgenic plant pollen in the environment. Specifically, it should be a useful tool for characterizing the spatial distribution patterns of transgenic pollen, to determine pollination mechanisms, to monitor the effects on nontarget organisms, and to monitor gene flow in field conditions.     

Crossing the divide: gene flow produces intergeneric hybrid in feral transgenic creeping bentgrass population

Gene flow is the most frequently expressed public concern related to the deregulation of transgenic events ( Snow 2002 ; Ellstrand 2003 ). However, assessing the potential for transgene escape is complex because it depends on the opportunities for unintended gene flow, and establishment and persistence of the transgene in the environment ( Warwick et al. 2008 ). Creeping bentgrass (Agrostis stolonifera L.), a turfgrass species widely used on golf courses, has been genetically engineered to be resistant to glyphosate, a nonselective herbicide. Outcrossing species, such as creeping bentgrass (CB), which have several compatible species, have greater chances for gene escape and spontaneous hybridization (i.e. natural, unassisted sexual reproduction between taxa in the field), which challenges transgene containment. Several authors have emphasized the need for evidence of spontaneous hybridization to infer the potential for gene flow ( Armstrong et al. 2005 ). Here we report that a transgenic intergeneric hybrid has been produced as result of spontaneous hybridization of a feral‐regulated transgenic pollen receptor (CB) and a nontransgenic pollen donor (rabbitfoot grass, RF, Polypogon monspeliensis (L.) Desf.). We identified an off‐type transgenic seedling and confirmed it to be CB × RF intergeneric hybrid. This first report of a transgenic intergeneric hybrid produced in situ with a regulated transgenic event demonstrates the importance of considering all possible avenues for transgene spread at the landscape level before planting a regulated transgenic crop in the field. Spontaneous hybridization adds a level of complexity to transgene monitoring, containment, mitigation and remediation programmes.     

A male sterility-associated mitochondrial protein in wild beets causes pollen disruption in transgenic plants

In higher plants, male reproductive (pollen) development is known to be disrupted in a class of mitochondrial mutants termed cytoplasmic male sterility (CMS) mutants. Despite the increase in knowledge regarding CMS-encoding genes and their expression, definitive evidence that CMS-associated proteins actually cause pollen disruption is not yet available in most cases. Here we compare the translation products of mitochondria between the normal fertile cytoplasm and the male-sterile I-12CMS(3) cytoplasm derived from wild beets. The results show a unique 12 kDa polypeptide that is present in the I-12CMS(3) mitochondria but is not detectable among the translation products of normal mitochondria. We also found that a mitochondrial open reading frame (named orf129 ) was uniquely transcribed in I-12CMS(3) and is large enough to encode the novel 12 kDa polypeptide. Antibodies against a GST–ORF129 fusion protein were raised to establish that this 12 kDa polypeptide is the product of orf129. ORF129 was shown to accumulate in flower mitochondria as well as in root and leaf mitochondria. As for the CMS-associated protein (PCF protein) in petunia, ORF129 is primarily present in the matrix and is loosely associated with the inner mitochondrial membrane. The orf129 sequence was fused to a mitochondrial targeting pre-sequence, placed under the control of the Arabidopsis apetala3 promoter, and introduced into the tobacco nuclear genome. Transgenic expression of ORF129 resulted in male sterility, which provides clear supporting evidence that ORF129 is responsible for the male-sterile phenotype in sugar beet with wild beet cytoplasm.     

Enhanced drought resistance in fructan-producing sugar beet

Fructans are soluble polymers of fructose that are produced by approximately 15 % of the flowering plant species. Production of bacterial fructans in tobacco has been shown previously to lead to improved biomass production under polyethylene glycol-mediated drought stress. Here, we used the same SacB gene from Bacillus subtilis to produce bacterial fructans in sugar beet (Beta vulgaris L.). The transgenic sugar beets accumulated fructans to low levels (max. 0.5 % of dry weight) in both roots and shoots. Two independent transgenic lines of fructan-producing sugar beets showed significantly better growth under drought stress than untransformed beets. Drought stressed fructan-producing plants attained higher total dry weights (+25–35 %) than wildtype sugar beet, due to higher biomass production of leaves (+30–33 %), storage roots (+16–33 %) and fibrous roots (+37–60 %). Under well-watered conditions, no significant differences were observed between the transgenic and wildtype beets. In conclusion, the introduction of fructan biosynthesis in transgenic plants is a promising approach to improve crop productivity under drought stress.     

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.     

Environmentally friendly approaches to genetic engineering

Summary Several environmental problems related to plant genetic engineering may prohibit advancement of this technology and prevent realization of its full potential. One such common concern is the demonstrated escape of foreign genes through pollen dispersal from transgenic crop plants to their weedy relatives, creating super weeds or causing gene pollution among other crops or toxicity of transgenic pollen to nontarget insects. The high rates of gene flow from crops to wild relatives (as high as 38% in sunflower and 50% in strawberries) are certainly a serious concern. Maternal inheritance of the herbicide resistance gene via chloroplast genetic engineering has been shown to be a practical solution to these problems. Another common concern is the suboptimal production of Bacillus thuringiensis (Bt) insecticidal protein or reliance on a single (or similar) B.t. protein in commercial transgenic crops, resulting in B.t. resistance among target pests. Clearly, different insecticidal proteins should be produced in lethal quantities to decrease the development of resistance. Such hyperexpression of a novel B.t. protein in chloroplasts has resulted in 100% mortality of insects that are up to 40 000-fold resistant to other B.t. proteins. Yet another concern is the presence of antibiotic resistance genes in transgenic plants that could inactivate oral doses of the antibiotic or be transferred to pathogenic microbes in the GI tract or in soil, rendering them resistant to treatment with such antibiotics. Cotransformation and elimination of antibiotic resistant genes from transgenic plants using transposable elements via breeding are promising new approaches. Genetic engineering efforts have also addressed yet another concern, i.e., the accumulation and persistence of plastics in our environment by production of biodegradable plastics. Recent approaches and accomplishments in addressing these environmental concerns via chloroplast genetic engineering are discussed in this review.