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- Materials and methods
Given the increasing likelihood of genetically modified crops being grown on a commercial scale, there is an urgent need for more knowledge of the possibilities of gene exchange between crops and their wild relatives (Ellstrand & Hoffman 1990; Rogers & Parkes 1995; Hancock, Grumet & Hokanson 1996). The greatest concern is the escape of transgenes from the crop to the wild and their subsequent introgression into the genome of wild relatives (Raybould & Gray 1993; Dale 1994; Darmency 1994; Gliddon 1994; Kareiva, Morris & Jacobi 1994; Linder & Schmitt 1994; Raybould 1999). A distinction must be made between crops cultivated for their seeds, such as cereals, sunflower Helianthus annuus L., rapeseed Brassica napus L., etc., and crops cultivated for their vegetative parts that are not supposed to flower in the fields, for example sugar beet Beta vulgaris L. and chicory Cichorium intybus L. Most seed crops will easily exchange genes in the presence of close wild relatives (Ellstrand, Prentice & Hancock 1999). Vegetable crops pose less of a risk, but are cultivated from seed, which has to be produced, thus creating the possibility of gene exchange in seed-production areas (Bartsch et al. 1999), albeit on a smaller scale.
In our study species, the sugar beet Beta vulgaris ssp. vulgaris (biennial, self-incompatible and largely wind pollinated), there is a Mendelian gene of which the dominant B or bolting allele cancels vernalization requirement (Munerati 1931; Boudry et al. 1994; Abe, Guan & Shimamoto 1997). Instead of remaining vegetative when sown in late spring, plants possessing this allele are able to produce seeds before the roots are harvested. The B allele is present in wild (ruderal) populations near the main sugar beet seed-production regions in south-west France (Boudry et al. 1993) and recurrently contaminates the commercial sugar beet seed at a low frequency, giving a few bolting plants per hectare in the sugar beet fields. These plants can eventually lead to weed beets.
We studied the introgression of wild genes and in particular the B allele into the transgenic crop (creating an agronomic problem), rather than the introgression of transgenes into the wild populations (which may create an ecological problem outside the agro-ecosystem). Nevertheless, the presence of flowering beets in sugar beet fields near the coast may favour gene flow from the crop to the wild beets in their natural habitat, i.e. the sea beets, Beta vulgaris ssp. maritima. Beets form a crop–wild complex in which genetic information is freely exchangeable between the different parts of the complex (Van Raamsdonk 1995). Several authors have identified the potential for cultivated beet to hybridize with their wild relatives, together with the ability of the crop to produce weedy forms (Keeler 1989; Kapteijns 1993; Bartsch et al. 1996a; Van Raamsdonk & Van Der Maesen 1996; Bartsch & Schmidt 1997; Van Raamsdonk & Schouten 1997; Bartsch & Ellstrand 1999).
Weed beets have arisen as a consequence of poor agricultural practice (Williamson 1993). Since the mechanization of sugar beet cultivation in the 1950s, farmers have not eliminated bolters from their fields so thoroughly. As a consequence, numerous fields have been invaded by weedy forms of beet carrying the B allele (e.g. in northern France but also in other European countries; Hornsey & Arnold 1979; Longden 1993). Because beet has a long-lived seed bank (Desprez 1980) and can shed thousands of seeds (Bartsch et al. 1996a), weeds reappear each spring. They are easily controlled by selective herbicides in crops other than sugar beet, but because they belong to the same species as cropped beet they are insensitive to the selective herbicides used for sugar beet cultures. Traditionally, the only successful remedy has been the systematic manual removal of bolters, which is currently regarded as good agricultural practice for beet growers (Brants & Hermann 1998). Unfortunately, if weed beets are numerous, complete removal by hand is impractical.
Occasionally the beets sown by the farmer also bolt and flower. There are two types of ‘sown bolters’. First, there are the F1 crop–wild hybrids resulting from sugar beets that were pollinated by wild beets containing the B allele in the seed-production area. These hybrids are probably the original source of the weed beets (Boudry et al. 1993; Desplanque et al. 1999). The second type of bolter can occur if individuals in the crop, generally selected for a strong bolting resistance, receive sufficient cold for vernalization. The studies by Boudry et al. (1993) and Desplanque et al. (1999) excluded the possibility that weed beets originated from this type.
With the advancement of gene technology there is now a new, very attractive, opportunity to tackle the weed beet problem (together with the other weeds; for Chenopodium album L. in particular see Freckleton & Watkinson 1998; Watkinson et al. 2000): the use of sugar beets possessing a transgene for tolerance to a non-selective herbicide (for a first study on the impact on biodiversity see Elmegaard & Bruus Pedersen 2001). All plants without that tolerance gene, even the very close relatives, could be completely eliminated by the herbicide. This raises the question of the likelihood that the tolerance gene might escape to the weeds, creating a similar weed problem, this time with transgenic weed beets insensitive to the herbicide (for a discussion about ‘super-weeds’ see Kling 1998). The B allele and the tolerance gene would have to combine in a single individual, being able to survive and reproduce under herbicide treatment. Unsurprisingly, hybrids between wild beets and transgenic sugar beets are easily obtained (Bartsch & Pohl-Orf 1996; Dietz-Pfeilstetter & Kirchner 1998). It should be noted that there is no principal difference between conventional herbicide or transgenic tolerance, so all scenarios considered also apply to conventional tolerance, should it occur.
The most important variables with direct consequences for gene flow are:
the parent containing the transgene (the male-sterile seed bearer or the hermaphrodite pollinator);
in the case of the male-sterile seed bearer, the genome containing the transgene (the nuclear genome or one of the cytoplasmic genomes);
in the case of the pollinator plant, its ploidy level.
Until recently, most European sugar beets were triploid as a result of diploid male-steriles pollinated by tetraploid pollinator plants. The pollen of tetraploids is known to be less competitive compared with the pollen of diploids (Hecker 1988), and triploids may have problems with gamete production (Hecker & McClintock 1988).
In this study we quantified the present weed beet problem in northern France by estimating population dynamic parameters. We examined to what extent gene flow is possible between the different forms of beet in this area. Together with the available information about the situation in the French seed-production region, we wanted to investigate how the three variables listed above play a role in limiting gene flow. The final aim was to find the best scenario for the introduction of herbicide tolerance: the one which minimizes the probability or the speed of the appearance of transgenic weed beets.
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- Materials and methods
The weed beet densities found in the study area were extremely variable. In a large part of the fields used for sugar beet there was no weed beet problem, but more or less severely infested fields were not difficult to find, for example in field 17 in the year 2000 one part of the field was so saturated by weed beets that no roots could be harvested. Hence there is a real agronomic problem.
The infestation by weed beets starts with the seeds shed by crop–wild hybrid bolters in the sowing line within a field. These seeds stay around the mother plant unless dispersed by mechanical tillage. No zoochory is reported and there is no particular adaptation for attractiveness to animals or dispersion by wind. Our seed bank study makes clear that considerable densities of viable seeds can survive to the next sugar beet culture (usually 3 years after the previous one), for example field 14 after 4 years. Outside the agro-ecosystem, including recently abandoned fields, we have never found any wild (‘ruderal’) beets in northern France, although overwintering of beet is sometimes possible (Pohl-Orf et al. 1999). Beets are known to be poor competitors in undisturbed sites (Bartsch et al. 1996b; Fredshavn & Poulsen 1996) and there is therefore no real risk of invasion of the surrounding vegetation in the root crop region.
We examined bolters in the sowing line in 10 fields and noted that each field was contaminated by crop–wild hybrids (Table 1), although below the acceptable maximum value of 100 ha−1. This makes clear that the arrival of hybrids in the fields is a recurring phenomenon, which is in agreement with a study based on molecular polymorphism (Desplanque et al. 1999). In that study the large diversity of the weed beets compared with their short history could be explained by recurrent crop–wild hybridizations during seed production in spite of the precautions taken for more than 20 years in the seed-production region.
EVIDENCE FOR GENE FLOW IN THE CROP–WILD COMPLEX
Gene exchange between wild plants and the crop is possible in both the seed-production area and in the sugar beet fields, and is visualized in Fig. 2. The normal seed-production process and transport to the north is indicated by 1; all other figures indicate unwanted gene flow possibilities.
Figure 2. A schematic presentation of the possibilities of gene flow by seeds and pollen in the sowing seed-production area (left) and in the sugar-production area (right). The seed bearers are male-sterile, the pollinator plants are hermaphrodite; all other plants can be both. The pollinator plants can be tetrapoid (4N) or diploid (2N), leading to triploid (3N) or diploid (2N) varieties, respectively; all other plants are usually diploid.
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Gene flow in the seed-production area
Gene flow from wild plants to crop plants cannot be measured in the sugar beet seed-production area, but is manifested by the presence of diploid bolters in a triploid crop as well as by the weeds that are genetically intermediate between the ordinarily used sugar beet varieties and the wild (ruderal) plants in the area surrounding the seed-production fields (Desplanque et al. 1999). In Fig. 2 this crucial form of gene flow is indicated by 2. Gene flow from crop to wild in these areas may happen in two ways: by seeds lost during the harvest (arrow 4 in Fig. 2) or through pollination of wild plants by cultivated pollen (arrow 3).
Modern sugar beet varieties (since the 1960s) have a characteristic mitochondrial genotype, Svulg, associated with male sterility (Senda, Onodera & Mikami 1998), which is only maternally transmitted. This genotype has never been found in Mediterranean coastal populations nor in the Atlantic and western Channel populations (Cuguen et al. 1994). Desplanque (1999) found the Svulg cytoplasm in five out of 86 plants, from five different populations out of 23, within or near the area where seed production is allowed. This argues seed flow (arrow 4 in Fig. 2): the five plants are probably maternal descendants from seeds lost during the harvest.
Pollen flow from crop (all forms of Beta vulgaris ssp. vulgaris: sugar beet, fodder beet, red table beet and Swiss chard) to wild (Beta vulgaris ssp. maritima) in this area will result in cultivated characters in the progeny harvested on wild plants in the neighbourhood of the seed-production fields, and/or in triploid progeny in the case of tetraploid pollinator plants. Desplanque (1999) found evidence that 1–2% of all plants had progeny with a cultivated-like morphology, but mostly (in 10 populations out of 64) the plants concerned were pale green with broad leaves and very thick veins but no special thick root. This suggests a hybrid with Swiss chard rather than with sugar beet. Swiss chard is frequently grown in kitchen gardens and sometimes bolts and flowers before being removed. In two populations progeny were found possessing the characteristics of red table beet and in two other populations they possessed sugar beet characteristics. Desplanque et al. (1996) did not find any triploids after examination of 81 seeds harvested on different wild populations in the seed-production area. The evidence for pollen flow (arrow 3 in Fig. 2) is therefore weak, but cannot be excluded.
Gene flow in the sugarbeet fields
Notwithstanding the biennial character of the sugar beet crop, sexual reproduction can obviously happen in the sugar beet fields. Pollen and seeds can be produced by beets both inside and outside the sowing line (variety bolters and F1 hybrids vs. weed beets, respectively). The seed-bearer plants used to produce the hybrid variety are fully male-sterile. We showed, however, that this is not so for variety bolters or F1 hybrids: at least some of them can release pollen, as can be concluded from the cross results (Table 5). This can be explained by the restoration of male fertility by nuclear restorer alleles transmitted by the pollinator line or the wild (ruderal) beets, respectively. Male sterility is sometimes described in risk assessment as a way to reduce gene flow (Raybould 1999) but this is not a valid argument for safety in transgenic beets.
In the first stage of contamination of a field by weed beets (i.e. a clean field with only a few bolters per hectare), pollination could be limiting due to self-incompatibility and wind pollination. However, it is notable that we observed intense activity by Diptera (mainly syrphids) with 10–30 individuals per flowering plant, thereby confirming older reports (Archimovitch 1949; Free et al. 1975). A partial insect pollination could therefore exist (Darmency et al. 1998) and should be studied more carefully.
The offspring of the F1 hybrids will evolve to weed beets (arrow 5 in Fig. 2). Other gene flow possibilities are the cross-pollination of weed beets in neighbouring fields and the variety bolters or the F1 hybrids (arrows 6) and the gene flow by pollen or seed from weed beets to neighbouring wild coastal populations (arrows 7).
THE RISK OF THE SPREAD OF A TRANSGENE FOR HERBICIDE TOLERANCE
The weed beet problem as it exists in several parts of Europe may be combated successfully by the introduction of sugar beets carrying a transgene for non-selective herbicide tolerance. This would allow destruction of all the weeds including those belonging to the same species (Dhalluin et al. 1992). However, our results suggest that such a transgene could combine with the B allele for flowering without vernalization, thus leading to transgenic weed beets, which would mean that the success was only temporary. We discuss the following scenarios.
The transgene is incorporated in the nuclear genome of the (diploid) male-sterile seed bearers.
The transgene is incorporated in the nuclear genome of tetraploid pollinators.
As 2 but with diploid pollinators.
The transgene is incorporated in one of the cytoplasmic genomes of the seed bearers.
Scenario 1: transgenic male-sterile seed bearers
The advantage of having the transgene in the male-sterile seed bearers is that it will never be transferred by pollen to the wild populations in the seed-production region because of the absence of transgenic pollen in the seed-production process. The only direct way of transgene escape is by seed loss during or after the harvest (arrow 4 in Fig. 2). However, with the same precautions as currently used, there will be a low level of contamination of the seed by wild pollen carrying the B allele. The resulting F1 bolters in the sugar beet fields have to be systematically eliminated, otherwise transgenic weed beets will be the immediate result (arrow 5). Their removal must take place before flowering in order to prevent the arrival of the transgene in neighbouring fields with weed beets in a non-transgenic crop (arrow 6). The total eradication of bolters by hand includes the removal of the variety bolters, which, although they do not contain the B allele, may also transmit the transgene to weeds in other fields (arrow 6).
Scenario 2: transgenic tetraploid pollinator plants
Using this option, each contamination of the seed by the B-carrying wild pollen will lead to non-tolerant hybrids that are destroyed by the herbicide after germination. There is therefore no direct risk of the installation of new transgenic weed beet populations, at least when the herbicide treatment covers 100% of the field. There is the possibility, however, that the wild populations in the seed-production region would receive the transgene (arrow 3 in Fig. 2), thus generating plants that combine B and the transgene. Their pollen would be capable of contaminating the seed and thus producing herbicide-resistant hybrid bolters in the sugar beet fields (arrow 2).
When compared with scenario 1, it is clear that such a circuitous contamination of the seed, if any, will take considerable time. In the first place, the diploid pollen of the tetraploid pollinator plants appears to have a low chance of pollinating wild plants. The diploid pollen of a tetraploid beet is less readily released by the anthers (Scott & Longden 1970), and Hecker (1988) showed that when a diploid beet has an equal chance of being pollinated by haploid and diploid pollen, 89% of the effective pollinations are made by haploid pollen. Consistently, we found no triploids in the seeds sampled on wild plants in the seed-production area (Desplanque et al. 1996), in spite of the presence of high quantities of diploid pollen for several decades. Once such triploid hybrids are formed, their fitness may be low due to their triploidy, as we observed for the variety bolters. Hecker & McClintock (1988) for example, mentioned a weak germination ability (3%) for the pollen from triploid beets compared with pollen from diploid or tetraploid beets (about 40%). Seeds of the triploid varieties are sometimes described as non-viable (Longden 1993) but this is in contradiction to our findings (Tables 3 and 5).
Seeds may escape to the wild, also leading to triploids in the next generation, but, although still carrying the Svulg mitotype, they may evolve during later generations into diploid wild plants indistinguishable from normal individuals. On the other hand, introgressed ruderal plants exhibiting a strong domestication syndrome (i.e. large rosette and biennial character) will mainly escape from our investigation, as they are easy to spot by the seed producers who continuously try to destroy all ruderal beets they can find. An unwanted consequence of this behaviour could be the selection of the B allele, as annuality (together with a short life cycle) allows the ruderal plants to produce offspring before destruction.
Scenario 3: transgenic diploid pollinator plants
The crucial difference with the previous scenario is the easy passage of the transgene to the wild beets in the seed-production area. The massive amount of pollen, together with the absence of the ploidy handicap, which would also enhance the pollination of weeds in neighbouring fields by variety bolters in the crop, makes this an unattractive option.
Scenario 4: cytoplasmic herbicide tolerance
The incorporation of the transgene in one of the cytoplasmic genomes (e.g. in the chloroplast; Daniell et al. 1998) excludes all forms of exchange of the transgene by pollen, because these organelles are strictly maternally inherited in beet (Corriveau & Coleman 1988). This scenario closes a potential escape route for transgenes into the environment (Gray & Raybould 1998; Daniell 1999). The possibility of contamination by the wild B-carrying pollen, however, is not influenced. As a consequence, weed beet infestations will occur in each case of inattention to bolters, as in scenario 1. The passage to other fields or to wild sea beets at the Channel and North Sea coasts via pollen, on the other hand, is excluded, although still possible through seed transport.
Two mutually exclusive scenarios (1 and 2) both have their advantages and disadvantages, which means that a preference is not easy to justify. In the first scenario (incorporation of the transgene in the male-sterile seed bearers) there is little risk of the surrounding wild populations receiving the transgene during seed production. Transgenic weed beet populations, however, could very probably arise in a part of the sugar beet fields. It is unrealistic to think that all farmers will remove all the bolting beets from their fields. Finally, the transgene may arrive in natural sea beet populations in those areas where sugar beet is cultivated near the coast (Fig. 1).
In the second scenario (the incorporation of the transgene into the pollinator plants), the disadvantages are different. Large quantities of transgenic pollen will be generated in an area where wild plants of the same species are growing. We know that the diploid pollen of the actual tetraploid pollinator plants will probably have a low chance of pollinating the wild diploid plants, but the increasing tendency to use diploid sugar beet varieties suggests that scenario 3 (transgenic diploid pollinator plants), which certainly is the worst of all four, will be more likely. A second problem, for the seed producers, is that all pollinations by other varieties without tolerance for the same herbicide will result in plants dying after herbicide treatment of the sugar beet fields, whereas at present such unwanted pollinations are hardly visible and do not have real economic consequences.
Scenario 4 (a cytoplasmic inheritance of the transgene), has few advantages when compared with scenario 1 and suffers from the same drawback, the probable installation of transgenic weed beet populations in some of the fields.
The use of herbicide-tolerant transgenic sugar beet varieties will only be economically successful if transgenic weed beet populations do not arise within a few decades, which otherwise would bring us rapidly back to the current situation in which no herbicide is available for weed beet control. We have shown that the eventual formation of transgenic weed beets is not only possible, but even probable, whatever the scenario adopted. However, the speed with which this happens, and the scale whereon, are both of decisive economic importance. We recommend the incorporation of the transgene into the tetraploid pollinator line (scenario 2) as being the best strategy in this respect.