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.     

Seed bank dynamics govern persistence of Brassica hybrids in crop and natural habitats

Background and Aims Gene flow from crops to their wild relatives has the potential to alter population growth rates and demography of hybrid populations, especially when a new crop has been genetically modified (GM). This study introduces a comprehensive approach to assess this potential for altered population fitness, and uses a combination of demographic data in two habitat types and mathematical (matrix) models that include crop rotations and outcrossing between parental species.Methods Full life-cycle demographic rates, including seed bank survival, of non-GM Brassica rapa × B. napus F₁ hybrids and their parent species were estimated from experiments in both agricultural and semi-natural habitats. Altered fitness potential was modelled using periodic matrices including crop rotations and outcrossing between parent species.Key Results The demographic vital rates (i.e. for major stage transitions) of the hybrid population were intermediate between or lower than both parental species. The population growth rate (λ) of hybrids indicated decreases in both habitat types, and in a semi-natural habitat hybrids became extinct at two sites. Elasticity analyses indicated that seed bank survival was the greatest contributor to λ. In agricultural habitats, hybrid populations were projected to decline, but with persistence times up to 20 years. The seed bank survival rate was the main driver determining persistence. It was found that λ of the hybrids was largely determined by parental seed bank survival and subsequent replenishment of the hybrid population through outcrossing of B. rapa with B. napus.Conclusions Hybrid persistence was found to be highly dependent on the seed bank, suggesting that targeting hybrid seed survival could be an important management option in controlling hybrid persistence. For local risk mitigation, an increased focus on the wild parent is suggested. Management actions, such as control of B. rapa, could indirectly reduce hybrid populations by blocking hybrid replenishment.     

Chlorophyll breakdown in oilseed rape

Chlorophyll catabolism accompanying leaf senescence is one of the most spectacular natural phenomena. Despite this fact, the metabolism of chlorophyll has been largely neglegted until recently. Oilseed rape has been used extensively as a model plant for the recent elucidating of structures of chlorophyll catabolites and for investigation of the enzymic reactions of the chlorophyll breakdown pathway. The key reaction which causes loss of green color is catalyzed in a two-step reaction by pheophorbide a oxygenase and red chlorophyll catabolite reductase. In this Minireview, we summarize the actual knowledge about catabolites and enzymes of chlorophyll catabolism in oilseed rape and discuss the significance of this pathway in respect to chlorophyll degradation during Brassica napus seed development. This revised version was published online in June 2006 with corrections to the Cover Date.     

Hover flies are efficient pollinators of oilseed rape

Understanding the consequences of declining diversity and abundance of pollinators for crops and floral biodiversity is a major challenge for current conservation ecology. However, most studies on this issue focus on bees, while other invertebrate taxa are largely ignored. We investigated the pollination efficiency of the globally abundant hover fly Episyrphus balteatus on the common crop, oilseed rape (Brassica napus). The study was conducted over a period of 2 consecutive years by means of enclosure experiments at an agricultural site located in Central Hesse (Germany). E. balteatus significantly increased both seed set and yield. This effect was very constant in the 2 years, despite considerable interannual differences in total seed numbers and seed mass. It highlights the important role of hover flies as pollinators of arable crops under varying environmental conditions. In contrast to bees, the effect of E. balteatus was lower at high pollinator densities than at low pollinator densities. This suggests adverse effects of density-dependent factors on pollination efficiency at high densities. Thus, models ignoring the modulating effect of biotic interactions by generally assuming a simple positive relationship between pollinator density and pollination efficiency might not apply to a vital component of the pollinator community.     

Profiling of phenylpropanoids in transgenic low-sinapine oilseed rape (Brassica napus)

A dsRNAi approach silencing a key enzyme of sinapate ester biosynthesis (UDP-glucose:sinapate glucosyltransferase, encoded by the UGT84A9 gene) in oilseed rape (Brassica napus) seeds was performed to reduce the anti-nutritive properties of the seeds by lowering the content of the major seed component sinapine (sinapoylcholine) and various minor sinapate esters. The transgenic seeds have been produced so far to the T6 generation and revealed a steady suppression of sinapate ester accumulation. HPLC analysis of the wild-type and transgenic seeds revealed, as in the previous generations, marked alterations of the sinapate ester pattern of the transformed seeds. Besides strong reduction of the amount of the known sinapate esters, HPLC analysis revealed unexpectedly the appearance of several minor hitherto unknown rapeseed constituents. These compounds were isolated and identified by mass spectrometric and NMR spectroscopic analyses. Structures of 11 components were elucidated to be 4-O-glucosides of syringate, caffeyl alcohol and its 7,8-dihydro derivative as well as of sinapate and sinapine, along with sinapoylated kaempferol glycosides, a hexoside of a cyclic spermidine alkaloid and a sinapine derivative with an ether-bridge to a C₆-C₃-unit. These results indicate a strong impact of the transgenic approach on the metabolic network of phenylpropanoids in B. napus seeds. Silencing of UGT84A9 gene expression disrupt the metabolic flow through sinapoylglucose and alters the amounts and nature of the phenylpropanoid endproducts.     

Characterization of the interactions between architecture and source-sink relationships in winter oilseed rape (Brassica napus) using the GreenLab model

Background and Aims

This study aimed to characterize the interaction between architecture and source–sink relationships in winter oilseed rape (WOSR): do the costs of ramification compromise the source–sink ratio during seed filling? The GreenLab model is a good candidate to address this question because it has been already used to describe interactions between source–sink relationships and architecture for other species. However, its adaptation to WOSR is a challenge because of the complexity of its developmental scheme, especially during the reproductive phase.

Methods

Equations were added in GreenLab to compute expansion delays for ramification, flowering of each axis and photosynthesis of pods including the energetic cost of oil synthesis. Experimental field data were used to estimate morphological parameters while source–sink parameters of the model were estimated by adjustment of model outputs to the data. Ecophysiological outputs were used to assess the sources/sink relationships during the whole growth cycle.

Key Results

First results indicated that, at the plant scale, the model correctly simulates the dynamics of organ growth. However, at the organ scale, errors were observed that could be explained either by secondary growth that was not incorporated or by uncertainties in morphological parameters (durations of expansion and life). Ecophysiological outputs highlighted the dramatic negative impact of ramification on the source–sink ratio, as well as the decrease in this ratio during seed filling despite pod envelope photosynthesis that allowed significant biomass production to be maintained.

Conclusions

This work is a promising first step in the construction of a structure–function model for a plant as complex as WOSR. Once tested for other environments and/or genotypes, the model can be used for studies on WOSR architectural plasticity.     

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.     

An integrated approach to oilseed rape cultivar selection using phenotypic stability

Summary Various methods of evaluating phenotypic stability have been proposed; however, no single method can adequately describe cultivar performance. The objectives of this study were to integrate a number of methods of evaluating stability and to use this approach for cultivar selection. These objectives were considered in the context of the broad-based oilseed rape cultivar (Brassica napus spp. oleifera) evaluation system currently used in western Canada. Regression analysis was used to assess cultivar response to environments. Cluster analysis was used to assemble cultivars into groups with similar regression coefficients (b _i ) and mean yield. Three parametric stability parameters, years within locations mean square (MS; Y/L), Shukla's stability variance ( _i ² ), and Francis and Kannenberg's coefficient of variability (CV _i ), were compared to determine which method would be most suitable for selection of oilseed rape cultivars from within clustered groups. Yield data from three cultivars and six breeding lines that had been tested for 2 years at 26 locations in the Western Canola Cooperative Test A were used for all calculations. The cluster analysis was successful in identifying commercially acceptable breeding lines. The parameter MS _i Y/L was considered to be more appropriate than either CV _i or _i ² , because it measured only the unpredictable portion of the genotype x environment interaction and was independent of the other cultivars in the test. The use of cluster analysis to group entries with similar b _i values and mean yields, followed by selection for stability within groups, was advocated.Contribution No. 846 of the Plant Science Department, University of Manitoba     

Identification of differentially expressed sequences in pre-embryogenic tissue of oilseed rape by suppression subtractive hybridisation (SSH)

A subtracted cDNA library comprising of 576 clones were constructed to isolate differentially expressed sequences in the pre-embryogenic tissue (PEC) of winter oilseed repe. After differential screening, only 16 clones were identified as potential positives. Eventually, only three clones: BNPE DG3, BNPE AE4 and BNPE EG1 were confirmed by Northern analysis as upregulated in␣PEC of the oilseed rape embryogenic culture. This is the first study to report Ae4, Dg3 and Eg1 sequences as preferentially expressed in the oilseed␣rape PEC and associated with the induction of somatic embryogenesis in B.napus secondary embryogenesis. Ae4 encodes a putative proline-rich protein, Dg3 encodes a lipid transfer-like protein and Eg1 encodes a napin, member of the BNMNAP subfamily.     

Opportunities for gene transfer from transgenic oilseed rape (Brassica napus) to related species

Before novel transgenic plant genotypes are grown outside containment facilities and evaluated under field conditions, it is necessary to complete a risk assessment to consider the possible consequences of that release. An important aspect of risk assessment is to consider the likelihood and consequences of the transgene being transferred by cross-pollination to related species, including other crops, weeds and ruderal populations. The purpose of this report is to review the literature to assess the ease with whichBrassica napus can hybridize with related species. The evidence for hybridization is considered at three levels: a) by open pollination, b) by hand pollination and c) by the use ofin vitro ovule and embryo rescue techniques; and also examines the fertility and vigour of the F₁, F₂ and backcross generations. Four species are reported to hybridize withB. napus by open pollination:B. rapa andB. juncea using fully fertile parents; andB. adpressa andR. raphanistrum using a male-sterileB. napus parent. Seventeen species are reported to form hybrids (including the four species above) withB. napus when pollination is carried out manually. At least 12 of these species were unable to form F₂ progeny, and eight were unable to produce progeny when the F₁ was backcrossed to one of the parental species. Many factors will influence the success of hybridization under field conditions, including: distance between the parents, synchrony of flowering, method of pollen spread, specific parental genotypes used, direction of the cross and the environmental conditions. Even where there is a possibility of hybridization betweenB. napus and a related species growing in the vicinity of a release, poor vigour and high sterility in the hybrids will generally mean that hybrids and their progeny will not survive in either an agricultural or natural habitat.     

Genetic manipulation in cultivars of oilseed rape (Brassica napus) using Agrobacterium

Summary The response of oilseed rape cultivars to infection with Agrobacterium tumefaciens and A. rhizogenes and the possibility of regenerating genetically transformed oilseed rape plants were examined. The frequency at which Agrobacterium induced galls or hairy-roots on in vitro cultured plants ranged from 10% to 70%, depending on the cultivar. From galls induced by the tumorigenic strain T37, known to be strongly shoot inducing on tobacco, roots developed frequently. Occasionally, shoots formed and some of these produced tumour cell specific nopaline. Attempts to grow the transformed shoots into plants have so far been unsuccessful. Whole plants transformed with Ri-T-DNA, however, were regenerated. These had crinkled leaves and abundant, frequently branching roots that showed reduced geotropism, similar to previously isolated Ri T-DNA transformed tobacco and potato plants. The transformed oilseed rape plants flowered, but failed to form seeds.     

Effects of a Ficoll-agarose system on early division and development of mesophyll protoplasts of winter oilseed rape (Brassica napus L.)

A method is described for regenerating callus from mesophyll protoplasts of a winter variety of Brassica napus. The method combines the use of Ficoll in an initial liquid medium, enhancing early protoplast division and cell colony formation, with a transfer to an agarose system after 10 days culture to give rapid microcalli formation. Further transfers resulted in callus regeneration and the initiation of organogenesis.     

Frequency and distance of pollen dispersal from transgenic oilseed rape (Brassica napus)

The objective of this study was to evaluate pollen dispersal inBrassica napus (oilseed rape). The selectable marker, used to follow pollen movement, was a dominant transgene (bar) conferring resistance to the herbicide glufosinate-ammonium. Transgenic and non-transgenic plants of the cultivar Westar were planted in a 1.1 ha field trial, with the transgenic plants in a 9 m diameter circle at the centre, surrounded by non-transgenic plants to a distance of at least 47 m in all directions. A 1 m circle of non-transgenic plants was sown in the centre of the transgenic area to allow estimation of the level of pollen dispersal when plants were in close contact. Honeybee hives were placed at the trial site to optimize the opportunity for cross-pollination. During the flowering period, regular observations were made of the number of plants flowering and the number and type of insects present in 60 1 m² areas. These areas were located uniformly around the plot at distances of 1, 3, 6, 12, 24, 36 and 47 m from the edge of the 9 m circle of transgenic plants. Seed samples were harvested from each of the 7 distances so that approximately 20% of the circumference of the plot was sampled at each distance. The centre non-transgenic circle was also sampled. Plants were grown from the seed samples and sprayed with glufosinate to estimate the frequency of pollen dispersal at each distance. In order to screen enough samples to detect low frequency cross-pollination events, seed samples were tested in the greenhouse and on a larger scale in the field. Results were confirmed by testing progeny for glufosinate resistance and by Southern blot analysis. The estimated percentage of pollen dispersal in the non-transgenic centre circle was 4.8%. The frequency was estimated to be 1.5% at a distance of 1 m and 0.4% at 3 m. The frequency decreased sharply to 0.02% at 12 m and was only 0.00033% at 47 m. No obvious directional effects were detected that could be ascribed to wind or insect activity.     

Nitrogen and fungicide applications against Erysiphe cruciferarum affect quality components of oilseed rape

Oilseed rape (Brassica napus L.) is one of the most important oilseed crops in temperate climates. Erysiphe cruciferarum is an important disease of oilseed rape and causes crop loss in warmer areas of Europe. The research investigated the effect of nitrogen fertilizer and fungicidal treatment against powdery mildew infection caused by E. cruciferarum of oilseed rape on seed components, including protein, oil, oleic acid, linolenic acid and undesirable substances such as sinapic acid esters (SAE) and glucosinolates (GSL), using near infrared spectroscopy (NIRS). Five susceptible oilseed rape varieties were employed in this research using four treatment groups: no nitrogen fertilization and no fungicidal treatment (N₀–F₀); no nitrogen fertilization but fungicidal treatment (N₀–F₁); and nitrogen fertilization but no fungicidal treatment (N₁–F₀); nitrogen fertilization and fungicidal treatment (N₁–F₁). Nitrogen fertilization increased the protein, but lowered the oil content, of the seeds. Fungicidal treatments significantly increased oil contents in all varieties tested, however reduced protein levels in fertilized and non-fertilized plots. The level of linolenic acid did not change significantly in any plots of any treatment combinations; a similar result was observed in the level of oleic acid in most of the genotypes. Nitrogen fertilization increased GSL and SAE levels, whereas fungicidal treatment had no effect. Our findings demonstrated that nitrogen fertilization can markedly influence some quality parameters in oilseed rape; however, the application of fungicides reduced side effects of nitrogen fertilizer and resulted a reduction on GSL, SAE and protein contents but an increase on total oil and oleic acid contents.     

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.     

Cold pretreatment enhances microspore embryogenesis in oilseed rape (Brassica napus L.)

Stress is an essential component during embryogenesis induction in microspore culture. Cold pretreatment has been used in cereal microspore culture but very seldom attempted in Brassica microspore culture. The effect of cold pretreatment of flower buds subjected to a liquid medium on microspore embryogenesis was investigated in spring and winter Brassica napus, as well as in B. rapa and B. oleracea. Cold pretreatment significantly enhanced microspore embryogenesis (by 1–7 fold) compared to commonly used microspore culture protocol in B. napus, while it was less effective in B. rapa or even negative in B. oleracea. The appropriate duration of cold pretreatment was found to be 2–4 days, which stimulated the best microspore embryogenesis. Cold pretreatment was also able to promote embryo development including the improvement of embryo quality and acceleration of embryogenesis. When incorporating with medium refreshing, cold pretreatment could initiate the most microspore embryogenesis than any other treatment used. With further improvement cold pretreatment method may have a positive potential in Brassica breeding programmes.     

Genetic investigation of the origination of allopolyploid with virtually synthesized lines: application to the C subgenome of Brassica napus

Although there are a number of different allopolyploids in the plant kingdom, the exact ancestral parents of some allopolyploids have not been well characterized. We propose a strategy in which virtual allopolyploid lines derived from different types of parental species are used to investigate the progenitors of an allopolyploid. The genotypes of the parental lines and the natural allopolyploid were established using a set of DNA molecular markers. The genotypes of the virtual lines were then derived from those of the parental lines, and compared extensively with that of the natural allopolyploid. We applied this strategy to investigate the progenitors of the C subgenome of Brassica napus (rapeseed, AACC). A total of 39 accessions from 10 wild and 7 cultivated types of the B. oleracea cytodeme (CC), and 4 accessions of B. rapa (AA) were used to construct 156 virtual rapeseed lines. Genetic structure was compared among natural rapeseed, virtual rapeseed lines, and their parental lines by principal component analysis and analysis of ancestry. Our data showed that the C subgenome of natural rapeseed was related closely to the genome of cultivated B. oleracea and its related wild types, such as B. incana, B. bourgeaui, B. montana, B. oleracea ssp. oleracea and B. cretica. This finding indicated that these types or their progeny might be ancestral donors of the C subgenome of rapeseed. The successful application of the strategy of virtual allopolyploidy in rapeseed demonstrates that it can possibly be used to identify the progenitors of an allopolyploid species.     

Coat protein-mediated resistance to Turnip mosaic virus in oilseed rape (Brassica napus)

Oilseed rape (Brassica napus) lines transformedwith the coat protein (CP) gene of Turnip mosaic virus(TuMV) were used to determine the effectiveness of resistance to TuMV mediatedby CP RNA or coat protein. Lines with one, two, or more copies of transgeneswere produced. T₂ and T₃ lines containing the CP genewitha functional start codon synthesised coat protein and showed high, but variablelevels of resistance to TuMV (21–96% resistant plants per line). TheT₁ and T₂ progeny of all lines carrying the CP gene withamutated start codon so that RNA but not protein was expressed, were assusceptible to TuMV as controls. Thus, in these experiments we were able toinduce CP-mediated resistance, but not RNA-mediated resistance.