Ecological aspects of transgenic sugar beet: transfer and expression of herbicide resistance in hybrids with wild beets

Summary An increasing number of genetically engineered cultivars of several crops is being experimentally released into the environment. In future, crops with new transgenic traits will probably play an important role in agricultural practice. The long-term effect of transgenes on community ecology will depend on the distribution and establishment of transgenic plants in the wild, on the sexual transfer of their new genes to the environment and on the potential ecological impact of the transgenic trait. The starting point was the use of transgenic sugar beet lines, Beta vulgaris subspec. vulgaris var. altissima DÖLL (Helm 1957), with transgenes coding for rhizomania and herbicide (BASTA^®) resistance. The first two questions to answer were: Can the transgenes be transferred via pollen to wild beets, Beta vulgaris subspec. maritima (L.) ARCANG. or cultivated relatives such as red beet or spinach beet and are they expressed in the hybrids? Can transgenes be monitored in young Beta vulgaris-hybrids? The experimental transfer of transgenes was conducted in 1993 at a field location in northern Germany. The beets were hand-pollinated with transgenic pollen. In a non destructive biotest, the hybrid seedlings were tested for herbicide resistance. Transgenic plants showed no noxious phenotypic effects whereas control plants developed leaf necroses. All herbicide resistant hybrids within the biotest were assumed to be transgenic.     

Molecular and morpho-physiological characterization of sea,ruderal and cultivated beets

Beta vulgaris genetic resources are essential for broadening genetic base of sugar beet and developing cultivars adapted to adverse environmental conditions. Wild beets (sea beets, B. vulgaris spp. maritima and their naturalized introgressions with cultivated beets known as ruderal beets) harbor substantial genetic diversity that could be useful for beet improvement. Here, we compared molecular and morpho-physiological traits of wild beets collected on the Adriatic coast of Italy with sugar beet using eight primer-pairs amplifying 194 polymorphic fragments and four root traits (glucose and fructose content in the root tip, root elongation rate, number of the of root tips, total root length and its distribution among diameters ranges). Genetic diversity was higher in the sea beet accession, which may be due to the highly variable selection pressures that occur in heterogeneous ecological niches, compared with the ruderal and cultivated beets. Sea and sugar beet accessions showed contrasting root patterns in response to sulfate deprivation: sugar beet showed an increase of reducing sugars in the root tips and higher root elongation rate, and the sea beet accession showed an increase in root tip number, total root length and fine root length (average diameter < 0.5 mm). The ruderal beet showed intermediary responses to sea and sugar beet accessions. AFLP and morpho-physiological cluster analyzes showed sea, ruderal and cultivated beets to be genetically distinct groups. The results of this study indicate variability in response to sulfate deprivation is present in undomesticated beets that could be deployed for sugar beet improvement.     

QTL for delayed bolting after winter detected in leaf beet (Beta vulgaris L.)

Growing sugar beet (Beta vulgaris L. ssp. vulgaris) as a winter crop in cool temperate climates is expected to increase yield potential. However, this requires bolting resistance after winter. One strategy to achieve complete bolting resistance is to accumulate genes for bolting delay from various genetic resources within the B. vulgaris gene pool. To identify such genes, a QTL mapping was performed in a segregating population derived from a biennial leaf beet with delayed bolting after winter. The population was tested for bolting delay after winter in two different experiments with natural or artificial vernalization. Three QTL for bolting delay were mapped on linkage groups 3, 5 and 9 affecting bolting time by up to 19 days. These QTL could be combined with recently reported bolting QTL to develop a winter sugar beet with complete bolting resistance.     

Beet mild yellowing virus resistance derived from wild and cultivated Beta germplasm

Sugar beet (Beta vulgaris ssp. vulgaris L.) has a relatively narrow genetic base and several programmes have sought to evaluate the potential for introducing novel traits from wild and cultivated Beta germplasm into the crop. In particular, resistance to important sugar beet diseases has been identified within individual Beta accessions. We report here the successful transfer of resistance to Beet mild yellowing virus (BMYV) from garden beet, fodder beet and leaf beet accessions to progeny populations in initial crosses with sugar beet. Twelve plant populations derived from different Beta accessions were inoculated with viruliferous aphids carrying BMYV and the virus content of individual plants subsequently quantified by an ELISA test. Seven populations were significantly more resistant than a control sugar beet cultivar (P ≤ 0.05). BMYV resistance was successfully inherited in BC₁ and BC₂ generations, suggesting that resistance could potentially be introgressed from these sources into elite sugar beet lines.     

Variation in some agronomically important characters in a germplasm collection of beet (Beta vulgaris L.)

Summary A geographically representative selection of germplasm of Beta vulgaris, section Beta has been assessed for characteristics important in sugarbeet breeding, including downy mildew resistance, resistance to aphid colonisation and infection by the beet virus yellow complex. The occurrence of maintainer lines for cytoplasmic male-steriles was also investigated. Desirable qualities were found in some accessions, including nothern European wild vulgaris ssp. maritima and some old multigerm cultivars of fodder beets.     

Selection of Diploid Nematode-Resistant Sugar Beet from Monosomic Addition Lines

Monosomic sugar beet/wild beet addition lines (2n = 19) with full resistance against the beet cyst nematode have been characterized in different ways. Within the B. procumbens and B. webbiana addition lines three groups could be classified according to their isozymes pattern, growth habit, transmission rate, and resistance level. It is assumed that B. procumbens and B. webbiana each possess three different chromosomes which carry genes for nematode resistance. In the offspring of the addition lines diploid translocation types appear at very low frequencies, Isozyme pattern or growth type of the resistant plants were used for selecting diploid types in the offspring of monosomic addition lines. Effective selection could be made in progenies of susceptible sugar beets pollinated by addition lines because the pollen transmission of the alien chromosome is very low. Using these methods 7 nematode-resistant sugar beet lines could be selected. The transmission rates of the resistance gene ranged from 70.6% to 100%. Threw heterozygous progenies showed a 1:1 segregation indicating monogenic dominant inheritance of resistance. The level of resistance was as high as in the addition lines.     

Partial resistance of sugarbeet to beet cyst eelworm (Heterodera schachtii Schm.)

Summary Within Beta vulgaris and B. maritima origins some partial resistance to beet cyst eelworm was found which could be raised to a very limited extent by selection. However after the second backcross to commercial sugar beet varieties and successive selection of the inbreds this resistance was lost. It was demonstrated that in the rootsystem of resistant plants as much nematodes penetrate and develop as in susceptible ones but the ratio between males and females is different. It was therefore quite probable that this resistance is polyfactorial and merely recessive.     

The creation of tetraploid beets

Summary In recent years trial fields were laid out with tetraploid families of the sugar beet varieties SX and SY, the fodder sugar beet varieties VA and VB and the fodder beet varieties VC (high content) and VD (low content).It appeared that the tetraploid beets are morphologically distinct from the diploid commercial varieties in leaf form, leaf development, form of the beet, tendency to bolt, size of the clusters and the number of seedlings per cluster.The beets of all the plots are washed, weighed and measured for dry matter, sugar and crude protein content and seed for stecklings is sown on a separate trial field.From the results it appears that there is a variation in the above-mentioned contents so that it seems possible to improve the material by selection. This also holds for the dry matter production which agrees with that of diploid commercial varieties.The investigations are being continued.     

Overwintering of genetically modified sugar beet, Beta vulgaris L. subsp. vulgaris, as a source for dispersal of transgenic pollen

The potential impact of transgenic crops on community ecology will depend on the distribution and establishment of the new transgenic traits, on the sexual transfer of their new genes to the environment (Bartsch &; Pohl-Orf, 1996) and on the potential ecological impact of the transgenic trait. Flowering and pollen dispersal is important for outcrossing of the genetically engineered trait. For a biennial plant, like the cultivars of Beta vulgaris L., overwintering is normally necessary to become generative and to produce pollen and seeds (Abe et al., 1997), which usually does not happen with sugar beet as a field crop harvested in autumn (Longden 1989). The starting point for the project was a transgenic sugar beet, Beta vulgaris L. subsp. vulgaris (Lange et al., 1998), with rhizomania and herbicide ( Basta®, Liberty®) resistance. Cold tolerance is one of the most important factors for survival of sugar beet in Central- and North-Europe. Among other ways, spreading of transgenic traits into weed beet (Boudry et al., 1993) or wild beet can occur if genetically engineered – biennial – plants survive the winter, flower in spring and spread their pollen. Field experiments were performed with transgenic breeding lines and their hybrids, transgenic and non-transgenic hybrids with Swiss chard and three conventional beet cultivars to evaluate winter survival rates at seven different field sites. We could show that survival of sugar beet – transgenic as well as conventional ones – in Germany and at the Dutch border is possible. Survival rates were well correlated with temperature data and were unexpectedly high. Differences between sugar beet hybrids and breeding lines could be detected but not within different breeding lines or hybrids. There were no differences detectable between transgenic and non-transgenic plants. The data are crucial for the risk assessment of the release of transgenic sugar beet and are the basis for further experiments towards outcrossing and establishment.     

Incremental crop tolerance to weeds: A measure for selecting competitive ability in Australian wheats

Total reliance on herbicides for weed control is unsustainable with the spread of herbicide resistance and the environmental need to reduce pesticide use. Strongly competitive wheat crops that have high tolerance to weed pressure and therefore maintain high yields in the presence of weeds are a low-cost option for reducing dependence on herbicides. We examined the feasibility of selecting for wheat tolerance to weeds by crossing varieties differing for traits associated with competitiveness. Competitive ability and yield potential must be treated as separate traits for selection. Current measures of crop tolerance to weed competition do not separate the two traits so that selection based on these measures is often synonymous with selection for yield potential rather than pure tolerance. We propose a new measure, termed Incremental Crop Tolerance (ICT) that reflects the incremental yield difference between genotypes associated with tolerance, over and above differences in underlying yield potential.     

Dormancy, germination and ageing of Lolium multiflorum seeds following contrasting herbicide selection regimes

Lolium multiflorum Lam. is an annual weed that has developed resistance to diclofop-methyl herbicide. Knowledge of the relative fitness components of resistant and susceptible biotypes is important to predict and manage resistance. Here, we use different L. multiflorum selections obtained from a long-term experiment, in which the herbicide dose (0, 280, 560 and 1120 g a.i. ha⁻¹) and gene flow chances from Festuca rubra, naturally resistant to the herbicide, were managed. The relationship among herbicide selection history, the level of resistance achieved, and seed dormancy, germination rate and ageing is evaluated. The current resistance level of the different selections was not related to the herbicide dose of selection. The level of association between herbicide resistance and germination varied with the selection treatment and after-ripening time. There was a general positive relationship between hours to 50% germination and herbicide dose of selection, which was independent from the presence of F. rubra. Two L. multiflorum selections with highest resistance level diverged in their response to alternating temperature (i.e. seed dormancy level). While one of them displayed a high germination rate and high ageing rate, the second one displayed a low ageing rate during the evaluated period, similar to the control selection. Most arguments on how weeds evolve increasing fitness on agricultural fields are centered on dose responses and rarely on the effects of herbicide selection on other traits. Our results provide evidence that directional selection for other characteristics apart from resistance, may result from herbicide control causing adaptive phenological change.     

Introgression of phenoxy herbicide resistance from Raphanus raphanistrum into Raphanus sativus

Phenoxy herbicides such as 2,4‐dichlorophenoxy acetic acid (2,4‐D) and 4‐chloro‐2‐methylphenoxy acetic acid (MCPA) are selective herbicides used extensively in agriculture for weed control. Wild radish (Raphanus raphanistrum) is a problem weed across the globe and heavily infests crop fields in Australia. Phenoxy herbicides are used to selectively control dicot weeds, including wild radish. As a result of selection, phenoxy‐resistant wild radish populations evolved in Western Australia. In this research, introgression of phenoxy resistance from wild radish to cultivated radish (Raphanus sativus) was investigated following classical breeding procedures. F₁ progeny were generated by crossing MCPA‐resistant R. raphanistrum and MCPA‐susceptible R. sativus. F₁ hybrids were screened for MCPA resistance. The MCPA‐resistant F₁ hybrids were used to produce three generations of backcross progeny. Genetic analyses of F₁ and backcross progeny demonstrated introgression of the MCPA‐resistant trait from wild radish to cultivated radish. Implications of phenoxy resistance introgression into cultivated radish include potential development of herbicide‐tolerant radish cultivars or other members of the Brassicaceae family.     

Differential occurrence of beet cryptic viruses—A new tool for germplasm characterization and evolutionary studies in beets?

Summary Beet cryptic viruses occur differentially in cultivars of chard (only BCV 1) and red beet (only BCV 2). As landraces of these crops and wild beets can contain both of the viruses, the differential occurrence in cultivars should indicate founder effects in the course of evolution of chard and red beet. Consequently, the genetic base of those crops should be considered as narrow. On the basis of the presence or absence of the viruses in beet plants within the populations conclusions can be drawn concerning the uniformity of cultivars maintained in the collections, the characterization of chard and beet landraces and some evolutionary tendencies within the variable Beta vulgaris-complex.     

Weed tolerance in soybean: a direct selection system

Weed competition can severely reduce soybean (Glycine max (L.) Merr.) yields, particularly in organic systems. An efficient screening and breeding approach is needed to increase breeding progress for weed tolerance. This study sought to (i) establish a system for direct selection of competitive genotypes, (ii) evaluate genotypic differences in weed tolerance among six early‐maturing genotypes and (iii) assess the contribution of selected morphological traits to weed tolerance. A direct selection system providing two different levels of weed competition through all development stages of a soybean crop was developed, using mixtures of selected crop species as sown competitors. Two resulting mixtures induced intermediate (<30%) and strong (>50%) yield reduction, respectively. This selection system can be applied in screening and breeding programmes to facilitate breeding for weed tolerance. No significant difference in weed tolerance was detected between six soybean genotypes of maturity groups 000 to 00. Morphological traits that might influence competitive ability, for example light absorption, leaf area and lateral shoots, were assessed, and their potential for indirect selection for weed tolerance is discussed.     

Nematode Resistance Derived from Wild Beet and its Meiotic Stability in Sugar Beet

Three different karyotypes of sugar beet with resistance against the beet cyst nematode (Heterodera schachtii) have been investigated. These comprised monosomic addition lines (2n = 19) with one complete chromosome from B. patellarris or B. procumbens, one line with a chromosomal fragment added to the normal sugar beet chromosome complement (2n =18 + fragment) and one diploid line (2n = 18). The fragment originated from a B. procumbens chromosome since during meiosis it formed a univalem. It carries the gene for nematode resistance. Meiotic disturbances like univalems. laggards, anaphase I bridges, fragments and micronuclei were observed in all resistant genotypes. These may result in an exclusion of the chromosome fragment carrying the resistance from the rest of the genome. In the diploid resistant line, a chromosome with a translocation could be distinguished from the other B. vulgaris chromosomes. Meiotic irregularities also appeared in diploid resistant types and are one main reason for low transmission of the resistance. Tin-relationship between meiotic stability and the transmission rate of the resistance gene is discussed.     

Weed Control in Sugar Beet using Genetically Modified Herbicide-tolerant Varieties – A Review of the Economics for Cultivation in Europe

Genetically modified sugar beet varieties have been developed to be tolerant to glyphosate and glufosinate. To date, research regarding other active ingredients did not result in additional herbicide‐tolerant varieties and the approval of glufosinate‐tolerant varieties for market access has been withdrawn by the applicant. Therefore, only glyphosate‐tolerant varieties could be introduced for cultivation in the short run. Results concerning efficacy and cost of weed control using these varieties and the complementary herbicides were extensively reported in various contributions, mostly on a national level. Based on these results, the economics of weed control for sugar beet production in Europe were reviewed and aspects of integrated control, risk management, and issues of sustainable development of crop production are discussed. Efficient weed control is possible in almost any field situation with glyphosate at about 2 kg a.i. ha^?1, compared with conventional herbicides at 6 kg ha^?1 or higher, depending on weed infestation level. Cost savings for weed control with glyphosate would amount to an average of €150 ha^?1, without any great deviation across different sites and states. A technology fee of about €40 ha^?1 is assumed. The high selectivity of glyphosate may result in a 1–3 % higher yield performance of the crop. All assumptions being considered, total cost savings of €180 × 10⁶ year^?1 were calculated for the area of 1.7 × 10⁶ ha in the main EU sugar beet‐growing countries. A risk management by implementing a monitoring programme is compulsory, and systems of identity preservation or quality assurance are needed in order to enable the production of conventional and genetically modified sugar beet in coexistence. To date, costs are unknown for these measures. Because of the favourable ecotoxicological behaviour of glyphosate and the possibility of threshold‐based weed control, this new technology could provide an excellent option towards sustainable development of the crop. However, political reasons and the lack of acceptance of genetically modified varieties by the consumer have prevented the market entry of GMHT sugar beet to date, so that conventional herbicides will continuously be used in the future.     

Comparison of Sugar Beet Cropping Systems with Dead and Living Mulch using a Glyphosate-resistant Hybrid

The aim of this study was to evaluate new options for sugar beet mulch systems, using a glyphosate‐resistant hybrid. In four field trials – conducted in 2002 and 2003 at two sites in a major sugar beet‐growing region in the central Germany – three different mulch systems (straw, non‐winterhardy and winterhardy cover crop) and an alternative seedbed preparation method (rotary band tillage) have been tested in comparison with a control treatment (plough, no mulch, broadcast seedbed preparation) representing the common German practice. In all systems, a set of eight different weed control programmes, including two reference treatments, one with selective conventional herbicides and five exclusively using glyphosate, has been evaluated for efficacy. It could be shown that the integration of winterhardy cover crops into sugar beet mulch systems reduced the risk of nitrogen loss by leaching. The changes in the nitrogen dynamics neither influence the yield nor the technical quality of the sugar beet. The field emergence of sugar beet decreased while using a winterhardy cover crop, but was not altered by the seedbed preparation method. Compared with the conventional broadcast seedbed preparation, the rotary band tillage did reduce the weed density. The lowest weed density was observed in the straw mulch system. It was not possible to control the remaining plants of the winterhardy cover crops completely with selective herbicides. If glyphosate was used until the four‐leaf stage of the sugar beet, a regulation of the winterhardy cover crop was achievable. With glyphosate it was also feasible to control older weeds together with the newly emerging ones with post‐emergence applications only. For most of the mulch systems tested, the sugar beet was very sensitive to weed competition between the four‐ and 10‐leaf stage. If the weeds within the sugar beet rows were controlled during this sensitive period with an early glyphosate band application, the remaining weeds between the rows could be left uncontrolled until the 10‐leaf stage of the sugar beet without any yield loss. Only in the combination of winterhardy cover crop × rotary band seedbed preparation, the weeds and cover crop had to be controlled directly after emergence of the sugar beet.     

Molecular markers linked to rhizomania resistance in sugar beet, Beta vulgaris, from two different sources map to the same linkage group

Rhizomania, one of the most important diseases of sugar beet, is caused by beet necrotic yellow vein virus, a Furovirus vectored by the fungus Polymyxa betae Keskin. Reduction of the production losses caused by this disease can only be achieved by using tolerant cultivars. The objective of this study was the identification and mapping of random amplified polymorphic DNA (RAPD) markers linked to a rhizomania resistance gene. The RAPD markers were identified using bulked segregant analysis in a segregating population of 62 individuals derived by intercrossing plants of the resistant commercial hybrid GOLF, and the resistance locus was positioned in a molecular marker linkage map made with a different population of 50 GOLF plants. The resistance locus, Rr1, was mapped to linkage group III of our map of Beta vulgaris L. ssp. vulgaris, which consisted of 76 RAPDs, 20 restriction fragment length polymorphisms (RFLPs), three sequence characterized amplified regions (SCARs) and one sequence tagged site (STS). In total, 101 molecular markers were mapped over 14 linkage groups which spanned 688.4 cM with an average interval length of 8.0 cM. In the combined map, Rr1 proved to be flanked by the RAPD loci RA411₁₈₀₀ and AS7₁₁₀₀ at 9.5 and 18.5cM, respectively. Moreover, in our I₂ population, we found that a set of markers shown by Barzen et al. (1997) to be linked to the ‘Holly’ type resistance gene was also linked to the ‘GOLF’-type resistance gene. These results appeared to indicate that the rhizomania resistance gene present in the GOLF hybrid could be the same gene underlying resistance in ‘Holly’-based resistant genotypes. Two other explanations could be applied: first, that two different alleles at the same locus could have been selected; second, that two different genes at two different but clustered loci underwent the selection process.     

Evaluation of genetic diversity and root traits of sea beet accessions of the Adriatic Sea coast

Thirty-nine sea beet [Beta vulgaris L. ssp. maritima (L.) Arcang.] accessions of the Adriatic coast were screened genetically and for their adaptive morpho-functional root traits in order to identify new sources of abiotic resistances for sugar beet breeding programs. Genetic diversity was evaluated with 21 microsatellites markers that identified 44 polymorphic alleles. Sea beets grouped into two main clusters: the West and the East Adriatic coast groups, with the latter showing higher genetic diversity. Among sea beet accessions with desirable root traits, four accessions have proved to be interesting for sugar beet [B. vulgaris (L.) ssp. vulgaris] breeding aimed to improve tolerance to nutritional stresses. Lastovo (ID 29) and Zut (ID 34) accessions were characterized by the highest values of RER, TRL, FRL and RSA still maintaining a high value of RTD, while Grado (ID 21) an Portic (ID 23) accessions were characterized by the highest RTD, but with low values of RER, TRL, FRL and RSA parameters.     

A DNA Probe for Rapid Screening of Sugar Beet (Beta vulgaris L.) Carrying Extra Chromosomes from Wild Beets of the Procumbentes Section

Genes from wild species of the Procumbentes section can be transferred to sugar beet chromosomes via translocations. Since large translocations, including for example a gene for nematode resistance, generally result in meiotic disturbances, there is a need to select new diploid resistant beets from progenies of monosomic wild beet addition lines. A dispersed repetitive DNA probe, which is closely correlated with the resistance gene and hybridizes exclusively with wild beet DNA, appears to be highly superior to selection based on isozyme markers. Characteristic ‘fingerprints’ on the available monosomic addition lines reveal the existence of at last 5 different chromosomes in the wild species each housing a gene for nematode resistance. This probe can be used advantageously to identify individuals carrying the intact or fragmented wild beet chromosomes, or even various amounts of translocated chromatin. Strategies are discussed for the identification of new translocation types using straightforward squash dot or Southern hybridization techniques in combination with the wild beet DNA probe.