Genetic variation for secondary seed dormancy and seed longevity in a set of black‐seeded European winter oilseed rape cultivars

Secondary seed dormancy in oilseed rape is a phenomenon that allows seeds to survive in the soil for many years without germination. Following soil cultivation, dormant seeds may germinate in subsequent years, and they are the main reason for the occurrence of volunteer oilseed rape plants in successive crops. Inheritance of secondary dormancy may be related to seed longevity (SL) in the soil. Genetic reduction in secondary dormancy and SL could provide a mean to reduce the frequency of volunteer plants and especially the dispersal of transgenic oilseed rape. The aim of the present study was to analyse secondary dormancy, germination rate and SL of 28 black‐seeded winter oilseed rape cultivars using in vitro laboratory tests. The material was tested in field experiments at six different locations in Germany in 2008/2009. Significant effects of the genotype and the location on all traits were found. Heritability was high for secondary dormancy (0.97) and moderate for germination rate (0.70) and SL (0.71). Results indicate that a selection for low secondary dormancy would be effective.     

Degradation of oxalic acid by transgenic oilseed rape plants expressing oxalate oxidase

Summary Oxalic acid is thought to have a primary role in the pathogenicity of several plant pathogens, notably Sclerotinia selerotiorum. A gene coding for the enzyme oxalate oxidase was isolated from barley roots and introduced into oilseed rape as a means of degrading oxalic acid in vivo. This report describes the production of several transgenic plants of oilseed rape and the characterisation of these plants by Southern, Western and enzyme activity assays. Plants were shown to contain an active oxalate oxidase enzyme and were tolerant of exogenously supplied oxalic acid.     

Occasional loss of expression of phosphinothricin tolerance in sexual offspring of transgenic oilseed rape (Brassica napus L.)

The commercial and economic value of genetically modified crops is determined by a predictable, consistent and stable transmission and expression of the transgenes in successive generations. No gene inactivation is expected after selfings or crosses with non-transformed plants of homozygous transgenic oilseed rape plants if the expression of the transgene in homozygous or hemizygous nature in such plants is stable. The segregation ratios of phosphinothricin (PPT) tolerance in successive generations of selfings and mutual crosses of a few independent transgenic PPT-tolerant oilseed rape plants indicated a dominant, monogenic inheritance. In within-variety and between-variety crosses no transgene inactivation was observed. However, after selfings and backcrosses with non-transgenic oilseed rape infrequent loss of the expression of the PPT tolerance transgene was observed independent from its homozygous or hemizygous nature. Molecular analysis of PPT-susceptible plants showed that the loss of expression was due to gene inactivation and not to the absence of the transgene. Methylation and co-suppression are mechanisms that might cause reduced or even loss of expression of the transgene in later generations. The implications of this observation for seed multiplication of varieties and breeding activities with transgenic oilseed rape are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Herbicide-tolerant crops in agriculture: oilseed rape as a case study

Oilseed rape has been modified extensively by conventional breeding for the production of varieties useful for human consumption (blended vegetable oil and margarine) and industrial processes (rubber additives and high‐temperature lubricants). Because much is now known about its genetic and biochemical composition, it has been an obvious choice for genetic modification and is now at the forefront of the commercial development of genetically modified (GM) or transgenic crops. Around the world, the increase in commercial plantings of all transgenic crops has been rapid. In 1996, 1.7 million hectares were planted, but by 1998 this figure had jumped to 27.8 million ha. The area in the year 2001 is likely to be about 50 million ha. With the possible introduction of transgenic varieties into European agriculture, it is essential that the associated farming practices employed are appropriate for their growth, both from a commercial and an environmental viewpoint. Some of the first transgenic crops are those carrying agronomic traits, e.g. herbicide tolerance transgenes. However, before full commercialization occurs, important agronomic and environmental questions need to be answered. How are these new crops to be incorporated into existing cropping practices? How will this change the current herbicide use profile for a given crop? Do herbicide‐tolerant varieties enhance or impede integrated pest management schemes? What is the likely uptake of such crops in agriculture? What are the ecological implications of their introduction? Are there effective measures to control the spread of transgenes to wild relatives? This paper addresses these questions, with special emphasis on oilseed rape production in the UK, but includes examples from other crops and countries where appropriate.     

Hybridization of radish (Raphanus sativus L.) and oilseed rape (Brassica napus L.) through a flower-culture method

Summary Hybridization between radish and oilseed rape has been cumbersome, requiring elaborate embryo rescue techniques. With a modified flower culture method, we have achieved successful hybridization between radish and (transgenic) oilseed rape without the laborious and technically demandingin vitro ovule or embryo rescue techniques.The hybrid nature of the intergeneric hybrids was demonstrated using morphological traits, and DNA analyses. The described method will facilitate the generation ofRaphanobrassica hybrids useful for biosafety studies of the potential for transgenes to spread in weedyCruciferae as well as for breeding programs aimed at introducing useful radish genes, e.g. nematode resistance genes, into oilseed rape.     

Population dynamics of volunteer oilseed rape (Brassica napus L.) affected by tillage

Volunteer plants of oilseed rape (Brassica napus L.) from persistent seeds in soil can affect subsequent crops. Apart from the agricultural disadvantages, the environment and the marketing of the seeds may also be affected, particularly if plants with special ingredients or genetically modified (gm) plants are grown. In order to investigate the influence of soil cultivation and genotype on seed persistence and gene flow via volunteers, a field experiment was set up testing four tillage treatments and two cultivars in a split-plot design. The cultivars tested were near-isogenic to two gm cultivars. To simulate harvesting losses, 10 000 seeds m⁻² were broadcast on a soil in July. The subsequent tillage treatments were combinations of immediate or delayed stubble tillage by a rotary tiller, primary tillage with plough or cultivator, or zero tillage. Over the following year, the fate of the seeds was determined. Immediate stubble tillage with following cultivator or plough resulted in 586 resp. 246 seeds m⁻² in the soil seed bank. After delayed stubble tillage with following plough, 76 seeds m⁻² were found, and no soil seed bank was built up in the zero tillage treatment. Nevertheless, in the zero tillage treatment, several robust volunteer plants survived the herbicide application before the direct drilling in autumn until following spring. In the zero tillage treatment and in the cultivator treatment, 0.19 volunteers m⁻² resp. 0.06 volunteers m⁻² flowered simultaneously to ordinarily sown oilseed rape in the following crop of winter wheat and produced 73 resp. 18 seeds m⁻². Delayed stubble tillage reduced the risk of gene escape via the soil seed bank, while zero tillage resulted in the highest risk of gene escape by pollen and by production of a new generation of seeds. In terms of a labelling threshold for gm food this number of seeds would be below the threshold of 0.9% of transgenic parts in conventially bred food or feed.     

Reducing potential gene escape in time by appropriate post-harvest tillage—Evidence from field experiments with oilseed rape at 10 sites in Europe

Persistence of oilseed rapeseed in soil can result in weed problems but also reduce oil quality of following rape crops or result in unwanted gene escape which is particularly relevant in the context of genetically modified oilseed rape. In this paper data from 13 field experiments at sites in England, Austria and Germany are presented where tillage operations were tested that potentially reduce the build-up of a seed bank. In the majority of experiments seed losses were artificially simulated by broadcasting ca. 10,000 freshly ripened rapeseed m⁻² onto cereal stubbles. Oilseed rapeseedlings in autumn, the seed bank in winter–spring and yields of the following crop winter wheat were assessed as a function of tillage regime. During summer and autumn 19–70% of the seeds germinated and emerged. This part of the population was killed by following tillage operations or herbicide applications. However, 0–29%, in moist years 0–5%, of the initially broadcasted seeds developed dormancy and remained ungerminated in the soil until the following winter–spring.

Delaying incorporation of the seeds by leaving the stubble untouched for up to 4 weeks resulted in a reduced seed bank in almost every case. Also, repeated stubble tillage compared to an early single stubble tillage operation resulted in a smaller seed bank. The type of primary tillage (ploughing versus non-inversion cultivation) had no clear effect. No relation was found between the number of seedlings in autumn and the size of the seed bank the following winter–spring. Grain yield of the following crop winter wheat was not adversely affected by delayed stubble tillage.

The results indicate that stubble tillage aiming at a reduced seed bank of oilseed rape should focus on conditions avoiding induction of secondary dormancy rather than improving germination conditions. This means that, under the climatic conditions of central and western Europe, the stubble should be left untouched for several weeks after harvest before starting the usual tillage sequence with stubble tillage and ploughing or a non-inversion tillage sequence.     

Outcrossing frequencies from multiple high erucic acid oilseed rape fields to a central receptor field

Genetically modified oilseed rape is currently grown on about 23 % of the global oilseed rape acreage. In order to separate transgenic and non-transgenic oilseed rape production and to ensure co-existence of different agricultural cultivation schemes, as is specified by the European legislation, confinement measures have to be defined. Pollen-mediated gene flow is the most important means by which transgenes are dispersed between fields. In contrast to the majority of the previous investigations the objective of this study was to assess the extent of gene flow in the case of multiple pollen-donor fields. A high erucic acid rape genotype was used as biochemical marker for the quantification of outcrossing into a low erucic acid oilseed rape variety. Outcrossing data were obtained from two experimental locations. As expected, multiple pollen sources in a fragmented landscape can result in high gene transfer frequencies, thus requiring larger isolation distances than a field design with a single pollen donor source. The results of the study are transferable to homozygous transgenic oilseed rape varieties.     

Predictive study of the advantages of cleistogamy in oilseed rape in limiting unwanted gene flow

Summary The aim of this study was to evaluate the interest of beginning the selection process on a new genetic characteristic, cleistogamy, to manage gene flow in oilseed rape. The first step was to introduce this characteristic in an existing model of gene flow between oilseed rape populations in time and space, GeneSys-Rape. The second step was to evaluate the parameters of the model linked to this characteristic using field experimentations. Cleistogamous oilseed rape was shown to have an autogamy rate as high as 94% and to emit 10 times less pollen than an open-flowered oilseed rape in the same conditions. But the cleistogamous character was also shown to be unstable in the genotypes tested. The third step was to evaluate the interest of cleistogamy using simulations comparing several genotypes with or without cleistogamy in two different cropping systems. These simulations showed that an oilseed rape both dwarf and cleistogamous was interesting to limit gene escape and that a 99%-autogamous oilseed rape was interesting to limit both gene escape from and harvest contamination of the 99%-autogamous oilseed rape.     

抗除草剂基因在黄瓜杂种纯度快速鉴定上的应用研究

黄瓜抗除草剂转基因T0植株经除草剂抗性筛选,连续2代自交获得T1和T2种子;分别对T1植株进行bar基因PCR检测和T2植株Southern杂交检测,证明bar基因已整合到黄瓜染色体上;以非转基因黄瓜为对照,摸索出田间抗性鉴定和室内种子抗性鉴定的除草剂临界浓度;从田间和室内筛选除草剂抗性纯合性转基因株系3个;以表现较好的2个抗性纯合转基因株系为父本,与非转基因母本杂交,获得F1种子,并在室内进行了杂交种纯度鉴定,鉴定效率达100％。建立了一套在种子发芽阶段或2片真叶期进行黄瓜杂交种纯度鉴定的新技术。     

Seed germination in weedy Brassica campestris and its hybrids with B. napus: Implications for risk assessment of transgenic oilseed rape

We studied the germination behaviour of the following types of seeds: weedy Brassica campestris, oilseed rape (Brassica napus), B. campestris (♀) × B. napus (♂), B. napus (♀) × B. campestris (♂) and, finally, seeds harvested on B. napus (♀) × B. campestris (♂) hybrids in open pollination with B. campestris and B. napus. The seeds were germinated in Petri dishes, using three different consecutive treatments and assaying the viability of non-germinated seeds with tetrazolium staining. B. campestris seeds varied in the treatment they required in order to germinate and many seeds were dormant, in contrast to B. napus seeds, which lacked dormancy. B. campestris (♀) × B. napus (♂) and B. napus (♀) × B. campestris (♂) seeds both resembled B. napus being non-dormant whereas seeds harvested on B. napus (♀) × B. campestris (♂) hybrids were more B. campestris-like in germination behaviour. We discuss implications for risk of transgene spread from oilseed rape to weedy B. campestris. This revised version was published online in July 2006 with corrections to the Cover Date.     

Outcrossing frequencies and distribution of transgenic oilseed rape (Brassica napus L.) in the nearest neighbourhood

The outcrossing of transgenic oilseed rape in the neighbourhood is of major concern with regard to the actual EU labelling threshold of 0.9% for transgenic contamination in food and feed and the regulations for the co-existence of cultivation of genetically modified crops, conventional and organic farming. In a two-year field trial, the outcrossing frequencies and distribution from plots with different ratios of transgenic plants (100%, 1.0% and 0.1%) containing the pat-gene for resistance towards the broad-range herbicide glufosinate-ammonium were determined in surrounding acceptor plots within a distance of 3–11 m. Randomly distributed outcrossing with isolated pollination events became apparent, and an average gene flow of 0.28%, 0.01% and 0.0065% was detected for 100%, 1.0% and 0.1% transgenic donor plots, respectively. Significant effects on the distribution of outcrossing were found for distance, but not for the prevailing wind direction. The random distribution in combination with the behaviour of honey-bees and bumble-bees gives strong evidence that insects play an important role for short distance gene dispersal. A curve fit assuming an exponential decline was performed with the experimental outcrossing data as a function of distance and was applied to the actual EU labelling threshold. The contamination limit of 0.9% in food and feed could be kept without cultivation distances to the transgenic source. The impact of effective detection limits of analytical methods on the establishment of specific rules for the co-existence is discussed.     

Genetic relationships among resynthesized,semi-resynthesized and natural <Emphasis Type="Italic">Brassica napus</Emphasis> L. genotypes

The allopolyploidization event that created cultivated oilseed rape Brassica napus L, followed by intense breeding, reduced its genetic diversity. Resynthesized (RS) B. napus L. obtained by interspecific hybridization between genotypes of B. rapa L. and B. oleracea L. can be a valuable source for broadening genetic diversity in cultivated oilseed rape. In this study, we determined the extent of DNA polymorphism among natural accessions of oilseed rape, resynthesized B. napus, their parental species and double-low quality semi-RS lines carrying the Rfo gene. Using 10 selected primer combinations, 522 polymorphic AFLP markers were scored in the complete set of 100 Brassica sp. To detect relationships between these genotypes, a cluster analysis was performed using the Jaccard’s distance. Resynthesized allopolyploids clustered directly between their diploid parents. Cultivated accessions of oilseed rape created a compact group away from resynthesized allopolyploids as well as semi-RS lines. The natural oilseed rape group, which consists of 49 cultivars and breeding lines of oilseed rape, is characterized by lower genetic diversity than the group of 33 accessions of resynthesized oilseed rape, and the analysis showed that the double-low quality semi-RS lines represent a specific genetic variation of B. napus. The de novo resynthesized B. napus lines and the semi-RS lines of double-low quality generated from them, provide a significant opportunity for enrichment the gene pool of oilseed rape.     

转反义TRX s基因对小麦萌发过程中储藏蛋白降解的影响

以转反义TRX s基因豫麦18和对照（豫麦18）为试材，测定了小麦种子萌发过程中胚乳内thiocalsin、苹果酸脱氢酶、谷丙转氨酶的活性以及游离氨基酸含量的变化。结果表明，反义TRX s基因的导入能够增加对thiocalsin和苹果酸脱氢酶的抑制，降低其活性，使储存蛋白更难于被降解，谷丙转氨酶增高的速度减慢，种子氨基酸代谢减弱。说明蛋白质代谢缓慢是转反义TRX s基因小麦抗穗发芽的一个主要原因。     

Leaf glucosinolate profiles and their relationship to pest and disease resistance in oilseed rape

Summary Glucosinolates are sulphur-containing glycosides which occur within vegetative and reproductive tissue of oilseed rape. Following tissue damage, glucosinolates undergo hydrolysis catalysed by the enzyme myrosinase to produce a complex array of products which include volatile isothiocyanates and several compounds with goitrogenic activity. Many of these products have been implicated in the interaction betweenBrassica and their pests and pathogens and some may have a role in defence mechanisms. Low glucosinolate (00) oilseed rape cultivars have been shown to possess similar concentrations of leaf glucosinolates as high glucosinolate (0) oilseed rape cultivars. Likewise, despite considerable speculation to the contrary, 00 cultivars have been shown not to be more susceptible to pests and pathogens than 0 cultivars. The potential to enhance pest and disease resistance of oilseed rape by manipulating the leaf glucosinolate profile without reducing seed quality is discussed.     

Influence of leaf and silique photosynthesis on seeds yield and seeds oil quality of oilseed rape (Brassica napus L.)

With the purpose of enhancing oil production, the present work was carried out to elucidate relationships between photosynthesis of leaves, siliques and seeds yield and seeds oil accumulation of oilseed rape. Field trials, in which two repeated experiments was carried out during 2012–2014 growth season, a rape hybrids the “Qin You No.7” (Brassica napus L.) variety was taken into account. The results showed that, on rape plant the photosynthetic capacity and chlorophyll a, b, carotenoid contents of leaf were significantly higher than that of silique shell. Oil content of 94.7% was achieved in young seeds (sampled at 25th day after flowering ending stage of the tested rape plant) versus to mature seeds, saturated fatty acids percent was higher whereas oleic acid percent was lower of oil extracted from young seeds. During flowering period of the rape plants tested, area and dry weight of leaves attained maximum, treatments of removing leaves induced reduction in seeds number per silique, siliques number, seeds yield per plant and seeds oil content, these indexes were respectively decreased by 73.6%, 43.4%, 83.4% and 10.5% in maximum, and seeds oil composition was not significantly influenced; during seeds growing period of the tested plants, surface area and dry weight of siliques attained maximum, under shading siliques treatment, the 1000-seed weight, seeds yield per plant and seeds oil content were respectively reduced by 57.5%, 61.4% and 44.7% in maximum, and seeds oil oleic acid (C18:1) and linolenic acid (C18:3) percent was decreased, linolic acid (C18:2) and erucidic acid (C22:1) percent was increased. So for oilseed rape plant during flowering period, surface area and photosynthesis of leaves dramatically influenced siliques number, seeds number and seeds yield; while in seeds growth period, surface area and photosynthesis of siliques greatly influenced 1000-seed yield, seeds yield, seeds oil content and oil composition; oil accumulation in rape seeds initiated early since seeds commencing growth, seed mature degree influenced oil composition of seeds.     

Estimation of additive and epistatic gene effects of doubled haploid lines of winter oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis> L.)

In this paper 60 doubled haploid lines of oilseed rape (Brassica napus L.) were studied. Genetic parameters as additive and epistasis effects were estimated for nine quantitative traits. The results indicate the importance of both additive and epistasis gene effects of number of branches per plant, number of siliques per plant, number of seeds per silique and silique length in this study.     

Biochemical side effects of the herbicide FINALE<Superscript>®</Superscript> on <Emphasis Type="Italic">bar</Emphasis> gene-containing transgenic pineapple plantlets

Pineapple is one of the most important tropical fruits and therefore intensive genetic improvement programs are being carried out in many countries, including Cuba. Our research team has previously introduced the bar gene, along with chitinase and AP24 genes, into the pineapple genome. Herein, we report on the biochemical side effects of the herbicide FINALE^® on these transgenic plantlets during hardening. Levels of aldehydes and chlorophylls, and peroxidase activity were recorded. The transformed clone studied here, not sprayed with FINALE^®, showed the following side effects because of transgenesis only. Levels of malondialdehyde, other aldehydes, chlorophyll b, and total chlorophyll pigments decreased. The most remarkable biochemical differences between transgenic and non-transgenic plantlets after application of FINALE^® follow. Levels of malondialdehyde and other aldehydes in transgenic material were not decreased by FINALE^®, perhaps because these levels were already low as a result of transformation. FINALE^® increased peroxidase activity in transgenic plantlets but such increase was higher in non-transgenic material. The herbicide increased contents of chlorophyll pigments (a, b, total) in transformed plantlets. However, as expected, non-transgenic plantlets decreased levels of chlorophylls (a, b, total) after application of FINALE^®. The genetic transformation of pineapple with the bar gene not only conferred resistance to the herbicide FINALE^®, but also promoted other biochemical changes.     

Interspecific hybridization of Brassica napus and B. juncea through ovary,ovule and embryo culture

Summary Employing in vitro culture of ovaries, ovules and embryos, interspecific hybrids have been obtained amongst two important oilseed crops, Brassica napus x B. juncea and their reciprocal. The test-tube hybrid plants have been transferred to the field, and reared to maturity. The F₁ seeds obtained from the hybrid ovaries showed normal germination, and the hybrid plants exhibited a range of variation of characters.     

Status and perspectives of breeding for enhanced yield and quality of oilseed crops for Europe

Vegetable oils are a high-value agricultural commodity for use in refined edible oil products and as renewable industrial or fuel oils, and as the world population increases demand for high-quality seed oils continues to grow. Worldwide the oilseed market is dominated by soybean (Glycine max), followed by oilseed rape/canola (Brassica napus). In Europe the major oilseed crop is oilseed rape (B. napus), followed some way behind by sunflower (Helianthus annuus) and other minor crops like linseed (Linum usitatissimum) or camelina (Camelina sativa). The seed oil of these crops is characterized by a specific quality, i.e. fatty acid composition and other fat-soluble compounds: Camelina and linseed oils are characterised by high contents of linolenic acid (C18:3); in sunflower very high-oleic (up to 90% C18:1) types exist in addition to classical high-linoleic (C18:2) oilseeds; in B. napus a broad diversity of oil-types is available in addition to the modern 00 (canola) type, e.g. high-erucic acid rapeseed or high-oleic and low-linolenic cultivars. Moreover, vegetable oils contain valuable minor compounds such as tocopherols (vitamin E). Increases of such contents by breeding have lead to value-added edible oils. After oil extraction, oilseed meals—such as rapeseed extraction meal—contain a high-quality protein that can be used as a valuable animal feed. However, in comparison to soybean the meal from oilseed rape also contains relatively high amounts of anti-nutritive fibre compounds, phenolic acids, phytate and glucosinolates. Breeding efforts with respect to meal quality are therefore aimed at reduction of anti-nutritive components, while increasing the oil content, quality and yield also remains a major aim in oilseed rape breeding. This review article provides a general overview of the status of oilseed production in Europe and uses examples from winter oilseed rape to illustrate key breeding aims for sustainable and high-yielding production of high-quality vegetable oil. Emphasis is placed on analytical tools for high-throughput selection of overall seed quality.