Competitiveness of transgenic oilseed rape

The competitiveness of two transgenic oilseed rape (Brassica napus ssp.napus) lines and their fertile transgenic hybrid was tested in field trials in Belgium and Denmark. The lines contained genes for male sterility, restoration of fertility and herbicide resistance. The competitiveness of the three transgenic lines was related to three non-transformed commercially-grown oilseed rape varieties: Drakkar, Topas and Line. As a reference of a more aggressive crucifer, white mustard (Sinapis alba) was also included in the experiment. The experimental design was a complete block design with two locations, monocultures and mixtures with barley (Hordeum vulgare), three plant densities, four harvest times and four blocks. The yield density relationship of the transgenic oilseed rape lines was not different from that of the non-transgenic varieties in either location. The first harvest times showed a vigorous biomass production of white mustard, which in turn produced a significant difference in the competitive ability between oilseed rape and white mustard. Later, this difference decreased, and in Belgium there was no difference at the last harvest time. Variations within populations may blur actual differences between lines and varieties, and it is argued that unless the experimental design covers a range of competitiveness for which it is possible to detect significant differences, test results reporting a lack of difference between transgenic and non-transgenic plants are of little value.     

Gene dispersal from transgenic crops

The risk of release of genetically modified oilseed rape (Brassica napus) was investigated in relation to interspecific gene flow with hoary mustard (Hirschfeldia incana). Microscopic studies showed polymorphism within the population of hoary mustard for pollen germination on oilseed rape flowers. The transgenic herbicide-resistant and a commercial cultivar of oilseed rape were not different for pollen behaviour and ovule fertilization. Pollen tube growth was slow and erratic in interspecific crosses. Fertilization efficiency of oilseed rape and hoary mustard pollen in interspecific crosses was 15% and 1.3%, respectively, of that in intraspecific crosses. This unequal efficiency in reciprocal crosses was confirmed by hybrid seed set in pods. There was no post-zygotic barrier to the development of hybrid embryos in hoary mustard pods. Up to 26 spontaneous hybrids per male sterile oilseed rape plant, and one per hoary mustard plant, were obtained in field experiments. Hybrids were identified by isozyme electrophoresis, morphology and cytology. All hybrids were triploid with 26 chromosomes, and had low fertility. They produced 0.5 seeds per plant after spontaneous backcrossing with hoary mustard. Some of these descendants were produced from unreduced gametes. Our results suggest that gene flow is likely to occur, but its actual frequency under crop growing conditions remains to be estimated.     

Maternal Inheritance of Chloroplasts between <Emphasis Type="Italic">Brassica rapa</Emphasis> and F<Subscript>1</Subscript>-hybrids Demonstrated by cpDNA Markers Specific to Oilseed Rape and <Emphasis Type="Italic">B. rapa</Emphasis>

Controlled reciprocal crosses between B. rapa and F₁-hybrids (B. napus (♀) × B. rapa), giving 20 pair-crossings, were made to reveal possible irregularities in chloroplast inheritance during production of BC₁s. Despite the close relationship of chloroplasts in B. rapa and B. napus, development of PCR-based molecular markers specific to B. rapa chloroplasts and B. napus chloroplasts was successful. Offspring from each cross were investigated and among these, we found no irregular chloroplast inheritance, since their plastid genotypes in all cases were identical to that of their mother. With a certainty of 95% our data indicate that the probability that chloroplasts are being inherited paternally is less than 0.015. In oilseed rape, pollen-mediated transgene-dispersal poses a well-known risk. Our results support development of transplastomic oilseed rape as an approach to reduce transgene dispersal.     

Unexpected Diversity of Feral Genetically Modified Oilseed Rape (Brassica napus L.) Despite a Cultivation and Import Ban in Switzerland

Despite cultivation and seed import bans of genetically modified (GM) oilseed rape (Brassica napus L.), feral GM plants were found growing along railway lines and in port areas at four sites in Switzerland in 2011 and 2012. All GM plants were identified as glyphosate-resistant GM event GT73 (Roundup Ready, Monsanto). The most affected sites were the Rhine port of Basel and the St. Johann freight railway station in Basel. To assess the distribution and intra- and interspecific outcrossing of GM oilseed rape in more detail, we monitored these two sites in 2013. Leaves and seed pods of feral oilseed rape plants, their possible hybridization partners and putative hybrid plants were sampled in monthly intervals and analysed for the presence of transgenes by real-time PCR. Using flow cytometry, we measured DNA contents of cell nuclei to confirm putative hybrids. In total, 2787 plants were sampled. The presence of GT73 oilseed rape could be confirmed at all previously documented sampling locations and was additionally detected at one new sampling location within the Rhine port. Furthermore, we found the glufosinate-resistant GM events MS8xRF3, MS8 and RF3 (all traded as InVigor, Bayer) at five sampling locations in the Rhine port. To our knowledge, this is the first time that feral MS8xRF3, MS8 or RF3 plants were detected in Europe. Real-time PCR analyses of seeds showed outcrossing of GT73 into two non-GM oilseed rape plants, but no outcrossing of transgenes into related wild species was observed. We found no hybrids between oilseed rape and related species. GM plants most frequently occurred at unloading sites for ships, indicating that ship cargo traffic is the main entry pathway for GM oilseed rape. In the future, it will be of major interest to determine the source of GM oilseed rape seeds.     

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.     

Endophytic Bacteria Induce Growth Promotion and Wilt Disease Suppression in Oilseed Rape and Tomato

To determine whether bacteria isolated from within plant tissue can have plant growth-promotion potential and provide biological control against soilborne diseases, seeds and young plants of oilseed rape (Brassica napus L. cv. Casino) and tomato (Lycopersicon lycopersicum L. cv. Dansk export) were inoculated with individual bacterial isolates or mixtures of bacteria that originated from symptomless oilseed rape, wild and cultivated. They were isolated after surface sterilization of living roots and stems. The effects of these isolates on plant growth and soilborne diseases for oilseed rape and tomato were evaluated in greenhouse experiments. We found isolates that not only significantly improved seed germination, seedling length, and plant growth of oilseed rape and tomato but also, when used for seed treatment, significantly reduced disease symptoms caused by their vascular wilt pathogens Verticillium dahliae Kleb and Fusarium oxysporum f. sp. lycopersici (Sacc.), respectively.     

Incorporation of hygromycin resistance in Brassica nigra and its transfer to B. napus through asymmetric protoplast fusion

Summary With the idea to develop a selection system for asymmetric somatic hybrids between oilseed rape (Brassica napus) and black mustard (B. nigra), the marker gene hygromycin resistance was introduced in this last species by protoplast transformation with the disarmed Agrobacterium tumefaciens strain C58 pGV 3850 HPT. The B. nigra lines used for transformation had been previously selected for resistance to two important rape pathogens (Phoma lingam, Plasmodiophora brassicae). Asymmetric somatic hybrids were obtained through fusion of X-ray irradiated (mitotically inactivated) B. nigra protoplasts from transformed lines as donor with intact protoplasts of B. napus, using the hygromycin resistance as selection marker for fusion products. The somatic hybrids hitherto obtained expressed both hygromycin phosphotransferase and nopaline synthase genes. Previous experience with other plant species had demonstrated that besides the T-DNA, other genes of the donor genome can be co-transferred. In this way, the produced hybrids constitute a valuable material for studying the possibility to transfer agronomically relevant characters — in our case, diseases resistances — through asymmetric protoplast fusion.     

Effect of a turnip rape <Emphasis Type="Italic">(Brassica rapa)</Emphasis> trap crop on stem-mining pests and their parasitoids in winter oilseed rape <Emphasis Type="Italic">(Brassica napus)</Emphasis>

Concerns about the negative effects of chemical control of oilseed rape (Brassica napus L.) pests on non-target species, human safety, and development of insecticide resistance, require alternative control strategies such as the use of trap crops and biocontrol to be developed. Psylliodes chrysocephala(L.) (Coleoptera: Chrysomelidae) (cabbage stem flea beetle) and Ceutorhynchus pallidactylus (Marsh.) (Coleoptera: Curculionidae) (cabbage stem weevil) are two major stem-mining pests of oilseed rape. This study investigated the phenology of these pests and their main parasitoids in the UK, the potential use of turnip rape (Brassica rapa L.) as a trap crop to reduce oilseed rape infestation, and the effects of insecticide treatment on pest incidence and larval parasitism. Water trap samples, plant dissections and pest larval dissections were done to determine: the incidence of adult pests and their parasitoids, the level of plant infestation by the pests and percentage larval parasitism, respectively. The turnip rape trap crop borders reduced P. chrysocephalabut not C. pallidactylus infestation of oilseed rape plots. Treatment of the trap crop with insecticide had little effect on either pest or parasitoid incidence in the oilseed rape. TersilochusmicrogasterSzép. andT. obscurator Aub. (Hymenoptera: Ichneumonidae) were the main larval parasitoids of P. chrysocephalaand C. pallidactylus, respectively. Tersilochus microgasteris reported for the first time in the UK. The implications for integrated pest management are discussed.     

Association of gene-linked SSR markers to seed glucosinolate content in oilseed rape (Brassica napus ssp. napus)

Breeding of oilseed rape (Brassica napus ssp. napus) has evoked a strong bottleneck selection towards double-low (00) seed quality with zero erucic acid and low seed glucosinolate content. The resulting reduction of genetic variability in elite 00-quality oilseed rape is particularly relevant with regard to the development of genetically diverse heterotic pools for hybrid breeding. In contrast, B. napus genotypes containing high levels of erucic acid and seed glucosinolates (++ quality) represent a comparatively genetically divergent source of germplasm. Seed glucosinolate content is a complex quantitative trait, however, meaning that the introgression of novel germplasm from this gene pool requires recurrent backcrossing to avoid linkage drag for high glucosinolate content. Molecular markers for key low-glucosinolate alleles could potentially improve the selection process. The aim of this study was to identify potentially gene-linked markers for important seed glucosinolate loci via structure-based allele-trait association studies in genetically diverse B. napus genotypes. The analyses included a set of new simple-sequence repeat (SSR) markers whose orthologs in Arabidopsis thaliana are physically closely linked to promising candidate genes for glucosinolate biosynthesis. We found evidence that four genes involved in the biosynthesis of indole, aliphatic and aromatic glucosinolates might be associated with known quantitative trait loci for total seed glucosinolate content in B. napus. Markers linked to homoeologous loci of these genes in the paleopolyploid B. napus genome were found to be associated with a significant effect on the seed glucosinolate content. This example shows the potential of Arabidopsis-Brassica comparative genome analysis for synteny-based identification of gene-linked SSR markers that can potentially be used in marker-assisted selection for an important trait in oilseed rape. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Differential expression of members of the napin storage protein gene family during embryogenesis inBrassica napus

S1 nuclease analysis and sub-family-specific oligonucleotide probes were used to characterize the expression during embryogenesis of the napin storage protein gene family ofBrassica napus (oilseed rape). The expression of one sub-class represented by the napin gene gNa peaks and declines earlier than the other members of the family. This sub-class was highly expressed representing ca. 20% of napin mRNA at 26 days after anthesis.     

Extracellular localization of napin in the embryogenic tissues of <Emphasis Type="Italic">Brassica napus</Emphasis> spp. <Emphasis Type="Italic">oleifera</Emphasis>

Napin, a storage protein, has been reported to be transcribed abundantly during the pre-embryogenic stage and associated with the induction of Brassica napus secondary embryogenesis. In this study, we studied the distribution pattern of napin in the winter oilseed rape embryogenic tissue in comparison to that of the non-embryogenic tissue using the indirect immunofluorescence localisation coupled with the ultrastructural immunogold labelling techniques. Immunolocalisation studies revealed that the extracellular matrix layer outside the outer epidermal cell wall of B. napus embryogenic tissues contained napin. This is the first study to report the extracellular localisation of napin. In addition, we have also further characterised the expression pattern of Eg1 that encodes for napin in the B. napus embryogenic tissue.     

Presence of Unintended Agrobacterium tumefaciens Cloning Vector Sequences in Genetically Modified Plants

Agrobacterium transformation was used in the production of genetically modified plants from oilseed rape (Brassica napus) and tobacco (Nicotiana tabacum). After inoculation stop with the antibiotic timentin, a subsequent one-week treatment eliminated the vector bacterium from the oilseed rape plate explant cultures. From the tobacco, however, we recorded vector-derived signals one week after potting the regenerants in the greenhouse and still 10 weeks later. Genetically modified plants produced through Agrobacterium-transformation therefore cannot be guaranteed to be completely free of unintended vector sequences after antibiotic treatment.     

Hybridisation between oilseed rape (Brassica napus) and tetraploid Brassica rapa under field conditions

Cross-compatible relatives of crop species contribute to the uncertainty regarding the potential risk of transgene escape from genetically modified varieties. The most successful crossing partner of oilseed rape (Brassica napus L.) is diploid Brassica rapa L. Variation of ploidy level among B. rapa cultivars has, until recently, been neglected in the context of gene flow and hybridisation with oilseed rape. We estimated the extent of hybridisation between autotetraploid B. rapa varieties (female) and B. napus (pollen donor) under experimental field conditions. Morphology, variation of relative DNA amount, and microsatellite markers were used to distinguish between intraspecific offspring of tetraploid B. rapa and interspecific hybrids with B. napus. Of 517 seed progenies of tetraploid B. rapa, 45 juvenile plants showed species specific morphological traits of oilseed rape. The detection of putative hybrids based on variation in relative DNA amounts was problematic due to the occurrence of aneuploidy. In total, 84 offspring showed relative DNA amounts deviating from tetraploid B. rapa, four of which were hexaploids. Of the 205 offspring analysed at three microsatellite loci, 67 had oilseed rape alleles. Based on molecular evidence a minimum hybridisation rate of 13.0% was estimated. A few mother plants accounted for the majority of hybrids. The mean pollen viability of hybrids between B. napus and tetraploid B. rapa (80.6%) was high in comparison with mean pollen viability of triploid hybrids between B. napus and diploid B. rapa. Therefore, the occurrence of tetraploid B. rapa should be taken into consideration when estimating the likelihood of gene flow from oilseed rape to close relatives at the landscape level. Tetraploid B. rapa is a common component of several seed mixtures and establishes feral populations in northwest Germany. Assuming a similar abundance of diploid and tetraploid B. rapa, gene flow from B. napus to tetraploid may be more likely than gene flow to diploid B. rapa.     

A method for quantifying the gene flow that results from a single bumblebee visit using transgenic oilseed rape,Brassica napus L. cv. Westar

Genetically modified plants containing selectable markers offer a unique opportunity for pollination biologists to investigate some of the major, but intractable questions about paternity distributions and their causes. Here, a method is reported that uses transgenic plants to enable the quantification of the outcrossed fertilizations that result from a single pollinator visit. Gene flow mediated by worker bumblebees (Bombus terrestris) was studied among plants of oilseed rape (Brassica napus L. cv. Westar) where transgenic paternity in seeds of a non-transgenic plant was manifested as herbicide resistance. Overall, 91% of the resistant seeds resulted from the first four flowers that were visited after the bumblebee left the transgenic plant, and none was found beyond the 14th successively visited flower. The possibilities for developing the method to address various questions in pollination biology are discussed.     

Heterogeneity in the distribution of genetically modified and conventional oilseed rape within fields and seed lots

The implementation of co-existence in the commercialisation of GM crops requires GM and non-GM products to be segregated in production and supply. However, maintaining segregation in oilseed rape will be made difficult by the highly persistent nature of this species. An understanding of its population dynamics is needed to predict persistence and develop potential strategies for control, while to ensure segregation is being achieved, the production of GM oilseed rape must be accompanied by the monitoring of GM levels in crop or seed populations. Heterogeneity in the spatial distribution of oilseed rape has the potential to affect both control and monitoring and, although a universal phenomenon in arable weeds and harvested seed lots, spatial heterogeneity in oilseed rape populations remains to be demonstrated and quantified. Here we investigate the distribution of crop and volunteer populations in a commercial field before and during the cultivation of the first conventional oilseed rape (winter) crop since the cultivation of a GM glufosinate-tolerant oilseed rape crop (spring) three years previously. GM presence was detected by ELISA for the PAT protein in each of three morphologically distinguishable phenotypes: autumn germinating crop-type plants (3% GM), autumn-germinating 'regrowths' (72% GM) and spring germinating 'small-type' plants (17% GM). Statistical models (Poisson log-normal and binomial logit-normal) were used to describe the spatial distribution of these populations at multiple spatial scales in the field and of GM presence in the harvested seed lot. Heterogeneity was a consistent feature in the distribution of GM and conventional oilseed rape. Large trends across the field (50 x 400 m) and seed lot (4 x 1.5 x 1.5 m) were observed in addition to small-scale heterogeneity, less than 20 m in the field and 20 cm in the seed lot. The heterogeneity was greater for the 'regrowth' and 'small' phenotypes, which were likely to be volunteers and included most of the GM plants detected, than for the largely non-GM 'crop' phenotype. The implications of the volunteer heterogeneity for field management and GM-sampling are discussed.     

Divergent responses of two cereal aphids to previous infestation of their host plant

The role of pollen odour in resource location by the pollen beetle, Meligethes aeneus (Fabricius) (Coleoptera: Nitidulidae), a pollen-feeding insect regarded as a pest of oilseed rape, Brassica napus L., (Brassicaceae) crops, was investigated in a linear track olfactometer. Both male and female beetles were attracted to the odour of whole oilseed rape flowers, indicating that these insects can locate their host plants using floral odours as cues. The attractive odour of flowers was found to emanate from all floral parts tested: the petals/sepals, the anthers, and from pollen itself. Therefore, at least part of the attractive odour of oilseed rape flowers emanates from pollen. Beetles were more attracted to floral samples containing anthers than those without anthers when these odours were directly compared in a choice-test, and this indicates that there were detectable differences between them. Anthers and pollen may therefore release distinctive odours that are quantitatively and/or qualitatively different from the odour of the rest of the flower. These experiments support the hypothesis that pollen-seeking insects use pollen odour cues to locate this food source.     

Spatial and temporal patterns of green fluorescent protein (GFP) fluorescence during leaf canopy development in transgenic oilseed rape,<Emphasis Type="Italic"> Brassica napus</Emphasis> L.

The green fluorescent protein (GFP) holds promise as a field-level transgene marker. One obstacle to the use of GFP is fluorescence variability observed within leaf canopies. In growth chamber and field experiments, GFP fluorescence in transgenic oilseed rape (Brassica napus) was shown to be variable at each leaf position over time and among different leaves on the same plant. A leaf had its highest GFP fluorescence after emergence and, subsequently, its fluorescence intensity decreased. GFP fluorescence intensity was directly correlated with the concentration of soluble protein. The concentration of the genetically linked recombinant Bacillus thuringiensis (Bt) cry1Ac endotoxin protein also was examined, and GFP fluorescence was positively correlated with Bt throughout development. The results show that GFP can be used as an accurate transgene marker but that aspects of plant developmental should be taken into account when interpreting fluorescence measurements.Communicated by M.C. Jordan     

The glucosinolate-degrading enzyme myrosinase in Brassicaceae is encoded by a gene family

A full-length cDNA clone (MB3) and three partial clones (MA1, MB1 and MB2) which encode myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1) were isolated from a Sinapis alba (white mustard) cDNA library. Nucleotide sequence analysis of these clones revealed that they are encoded by a gene family. Southern blot analysis with gene-specific probes showed that the gene family consists of a least two subfamilies (MA and MB) each with several members both in S. alba and in Brassica napus (oilseed rape). In Arabidopsis thaliana (wall cress) only three myrosinase genes seem to be present. Northern blot analysis indicated that all the myrosinase mRNA species have the same size, approximately 1.95 kb.     

Effects of 5-Aminolevulinic Acid on Oilseed Rape Seedling Growth under Herbicide Toxicity Stress

Weeds are one of the major constraints in oilseed Brassica production. Use of effective herbicides to control weeds in the fields is one of the major objectives of agronomists. To improve weed control efficacy and minimize the application costs, complex combinations of 5-aminolevulinic acid (ALA) and a new postemergence herbicide, propyl 4-(2-(4,6-dimethoxypyrimidin-2-yloxy)benzylamino)benzoate (ZJ0273), were used to investigate their combined effects in relation to seedling growth and development of oilseed rape (Brassica napus cv. ZS 758). Brassica seeds were treated with different concentrations of ZJ0273 [100 (normal dose for rape), 200, 500, and 1000 mg/L] and ALA (0.1, 1, 10, and 50 mg/L). ALA was applied as pre- and post-treatment alone and in combination with ZJ0273. We found that ZJ0273 stress imposed negative effects on rape seedling growth. Shoot fresh weight, shoot length, and root fresh weight were inhibited significantly under ZJ0273 stress, and the rate of decline increased consistently with increased ZJ0273 concentration. Root oxidizability was also inhibited significantly under ZJ0273 stress conditions, and the higher the concentration of the herbicide ZJ0273, the lower the oxidizability. Herbicide ZJ0273 treatment produced a gradual decrease in antioxidant enzymes (peroxidase, superoxide dismutase, and ascorbate peroxidase) and an increase in peroxidation substance (malondialdehyde accumulation). The increase and decrease were consistent with the ZJ0273 dosage. Our results indicated that pre- and post-treatments with a lower dosage of ALA (1 mg/L) improved rape seedling growth and root oxidizability parameters, whereas a higher concentration of ALA (50 mg/L) depressed growth. We also found that plants treated with 1 mg/L ALA produced the highest shoot fresh weights, shoot lengths, root fresh weights, and root oxidizability when the seeds were treated with different concentrations of ZJ0273. Lower dosages of ALA improved the activities of antioxidant enzymes, whereas the highest dosage of ALA increased the accumulation of peroxidation substance. These results indicate that ALA has promotive effects in the recovery of growth and development of rape seedlings under herbicide ZJ0273 toxicity stress.     

Recovery of transgenic plants by pollen-mediated transformation in <Emphasis Type="Italic">Brassica juncea</Emphasis>

The aroA-M1 encoding the mutant of 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPS) was introduced into the Brassica juncea genome by sonication-assisted, pollen-mediated transformation. The plasmid DNA and collected pollen grains were mixed in 0.3 mol/L sucrose solution and treated with mild ultrasonication. The treated pollen was then pollinated onto the oilseed stigmas after the stamens were removed artificially. Putative transgenic plants were obtained by screening germinating seeds on a medium containing glyphosate. Southern blot analysis of glyphosate-resistant plants indicated that the aroA-M1 gene had been integrated into the oilseed genome. Western blot analysis further confirmed that the EPSPS coded by aroA-M1 gene was expressed in transgenic plants. The transgenic plants exhibited increased resistance to glyphosate compared to untransformed plants. Some of those transgenic plants had considerably high resistance to glyphosate. The genetic analysis of T₁ progeny further confirmed that the inheritance of the introduced genes followed the Mendelian rules. The results indicated that foreign genes can be transferred by pollen-mediated transformation combined with mild ultrasonication.