High Erucic Acid Rapeseed Research Articles

Crambe (Crambe abyssinica Hochst): A Non-Food Oilseed Crop with Great Potential: A Review

Crambe (Crambe abyssinica Hochst) is an oilseed crop in the Brassicaceae family. Crambe’s ability to survive in diverse environmental conditions, its unique oil composition, the high oil content, suitability for the production of slip agents for plasticizers, the capacity to be easily included in common crop rotations, and its adaptability to equipment used for small grain cultivation has renewed the interest in this emerging crop. Crambe is considered one of the main sources of erucic acid, which can be up to 60% of its seed oil content. Erucic acid (C22:1) is a fatty acid with industrial importance since it is used to produce erucamide, key ingredient in the plastic industry. Inclusion of crambe into crop rotations can be beneficial because of its short life cycle, low fertility requirements, resistance to pest and diseases, and relative drought tolerance. Currently high erucic acid rapeseed (Brassica napus L.) (HEAR) is the principal source for erucic acid. However, the risk of contaminating food quality rapeseed (i.e., canola) by cross-pollination and the negative impact on climate, due to high inputs, are potential limitations to expand HEAR cultivation. Crambe has thus great potential to, at least, partially replace HEAR as a source of erucic acid, if the current knowledge-gap in agronomic management and crop improvement (seed yield and quality) can be addressed. Seed yield needs to be increased to be able to compete with HEAR. In addition, reducing glucosinolates and fiber in crambe meal may increase its inclusion in monogastrics rations. The objective of this review was to compile and summarize new and existing information on agricultural practices in crambe production and management to identify gaps in knowledge and areas for future research to increase the cultivation of crambe.

Conversion of waste materials into very long chain fatty acids by the recombinant yeast Yarrowia lipolytica.

Erucic acid (C22:1Δ13) has several industrial applications including its use as a lubricant, surfactant and biodiesel and composite material constituent. It is produced by plants belonging to the Brassicaceae family, especially by the high erucic acid rapeseed. The ability to convert oleic acid into erucic acid is facilitated by FAE1. In this study, FAD2 (encoding Δ12-desaturase) was deleted in the strain Po1d to increase oleic acid content. Subsequently, FAE1 from Thlaspi arvense was overexpressed in Yarrowia lipolytica with the Δfad2 genotype. This resulted in the YL10 strain producing very long chain fatty acids, especially erucic acid. The YL10 strain was cultivated in media containing crude glycerol and waste cooking oil as carbon substrates. The cells grown using glycerol produced microbial oil devoid of linoleic acid, which was enriched with very long chain fatty acids, mainly erucic acid (9% of the total fatty acids). When cells were grown using waste cooking oil, the highest yield of erucic acid was obtained (887mg L-1). However, external linoleic and α-linolenic were accumulated in cellular lipids when yeasts were grown in an oil medium. This study describes the possibility of conversion of waste material into erucic acid by a recombinant yeast strain.

Evaluation of the Risks of Contaminating Low Erucic Acid Rapeseed with High Erucic Rapeseed and Identification of Mitigation Strategies

High erucic acid rapeseed (HEAR) oil is under increasing demand for various industrial applications. However, many growers are concerned that if they grow the crop, they will not be able to revert to other rapeseed varieties in the future due to the risk of erucic acid (EA) contamination of the harvested seed and inability to maintain acceptable erucic acid thresholds. This review considered published literature and, using the same criteria as that used to contain transgenic crops, aimed to identify the key risks of erucic acid contamination, broadly prioritise them and identify pragmatic mitigation options. Oilseed rape has a number of traits that increase the risk of low erucic acid rapeseed (LEAR) crops being contaminated with EA from HEAR varieties. The quantity of seed produced and the potential for seed dormancy coupled with partial autogamy (self-fertilisation) facilitate the establishment and persistence of volunteer and feral populations. The large quantities of pollen produced when the crop is in flower mean there is also a high potential for cross-pollination. Self-sown volunteer plants represent the highest potential contamination risk, followed by the presence of arable weeds (e.g., wild mustard) whose seeds are also high in EA. Other risks arise from the cross-pollination of compatible wild relatives and the mixing of seed prior to sowing. It is important that both HEAR and LEAR varieties are appropriately managed since risks and their potential for mitigation arise throughout the entire LEAR crop production process. The length of rotation, type of tillage, cultivar choice, buffer zones, effective weed management and basic machinery hygiene are all factors that can reduce the risk of erucic acid contamination of LEAR crops and maintain the required thresholds.

HYHEAR 3 Roundup Ready® high erucic acid, low glucosinolate hybrid summer rape

HYHEAR 3 summer rape (Brassica napus L.) is a Roundup Ready®, high erucic acid, low glucosinolate cultivar. On average, HYHEAR 3 yielded 15.6% more seed, 4 g kg−1 less seed oil, and 22 g kg−1 less meal protein than Red River 1861 Roundup Ready®, high erucic acid, low glucosinolate summer rape. HYHEAR 3 has an erucic acid content of 50.9% in isolated field trials of high erucic acid rapeseed (HEAR) genotypes and is adapted to the southern B. napus growing regions of western Canada.

Crambe abyssinica a non-food crop with potential for the Mediterranean climate: Insights on productive performances and root growth

Within a framework of renewed interest in crambe (Crambe abyssinica Hochst ex R.E. Fries) sourcing raw materials for the bio-based industry, the adaptability and productive performances of this oil crop have been evaluated under contrasting Mediterranean environments (i.e., a fertile site in the northern part of Po valley vs. a semi-arid site of southern Sicily) during two consecutive growing seasons, aiming at its possible stable introduction in this area. The trial set in northern Italy compared three commercial varieties of crambe (Galactica, Nebula and Mario) in spring sowing, while in southern Italy only the var. Mario was tested with autumn sowing. Regardless of location and variety, thermal time for maturity was quite stable (1200–1400°C), and the crop provided satisfactory seed yields (grand mean 2.29Mghulledseedsha−1), with average oil content of ∼400gkg−1 (on dehulled seeds) and ∼52% of erucic acid. Significantly higher seed and oil yields were reached in northern than in southern Italy. Furthermore, crambe thermal use efficiency (THUE) was also higher in the north than in the south, possibly due to better environmental adaptability of the crop. The limited intraspecific variability within crambe was confirmed, with better productive performances showed by the domestic selection Mario. Promising traits were revealed in Nebula, showing greater seed weight, root length density and area, and thinner roots, although the root growth of crambe was generally modest compared with modern high erucic acid rapeseed hybrids. Available crambe varieties could be efficiently included in crop rotations across a wide range of environments within the Mediterranean basin. The short growth cycle represents an outstanding added value for this species, allowing the avoidance of prolonged drought and heat stress typical of late spring/early summer months under the Mediterranean climate. However, increased yields are needed to meet the market requests; nonetheless, the little genetic variability suggests that there is large scope for future breeding improvements, maybe exploiting advanced techniques to improve the existing genetic resources.

Molecular evidence for blocking erucic acid synthesis in rapeseed (Brassica napus L.) by a two-base-pair deletion in FAE1 (fatty acid elongase 1)

DNA sequences of fatty acid elongase 1 genes FAE1.1 (EA) and FAE1.2 (EC) were isolated and characterized for 30 commercialized low erucic acid rapeseed (LEAR) cultivars in China. Four types of independent mutation leading to low erucic acid trait were found, i.e., a single-base transition (eA1), a two-base deletion (eC2) and four-base deletion (eC4) as well as single-base transition with a four-base deletion (eA*). Three genotypes, i.e., eA1eA1eC2eC2, eA1eA1eC4eC4 and eA*eA*eC4eC4 were responsible for LEA content in storage lipids of different rapeseed cultivars. Most of the LEAR cultivars had a genotype of eA1eA1eC2eC2, which were descended from the first LEAR cultivar, Oro. Yeast expression analysis revealed that two-base-pair (AA) deletion (eC2) at the base sites of 1422–1423 in the C genome FAE1 gene resulted in the absence of the condensing enzyme and led to the failure to produce erucic acid. Coexpression of FAE1 and ketoacyl-CoA reductase (KCR) or enoyl-CoA reductase (ECR) was found in high erucic acid rapeseed (HEAR) but not in LEAR (eA1eA1eC2eC2 or eA1eA1eC4eC4). Moreover, KCR and ECR were still coordinately regulated in eA1eA1eC2eC2 or eA1eA1eC4eC4 genotypes, suggesting that the expression of two genes was tightly linked. In addition, specific detection methods were developed by high-resolution melting curve analysis in order to detect eA1 and eC4.

Erucic acid rapeseed: 1. Prospects of improvements

In the current context of boosting production of high erucic acid rapeseed, because of the wide range of its industrial applications, this literature review is designed to provide a general overview of available varieties, current knowledge of plant improvement and paths of developing research to increase competitiveness of varieties with high erucic acid content. A limited market dominated by a few companies, cropping burdens of high erucic acid rapeseed varieties among the majority “00” varieties and the still low erucic acid content in rapeseed, explains the reduced and uncompetitive varietal offers. To improve this situation, new varieties could be developed, thanks to the classical methods of selection and biotechnology.

Nutritional and Antioxidant Properties of Rapeseed (<i>Brassica Napus</i>) Cultivars with High and Low Erucic Acid Content

Since consumers pay more and more attention to healthy food, and rapeseed germplasm provides a wide choice of new cultivars, their comprehensive nutraceutical properties are now under evaluation in order to select outstanding genotypes, which may link breeding and marketing. In this study, two genotypes of high-erucic acid rapeseed and three genotypes of low-erucic acid rapeseed grown in two locations were analyzed and compared for their fatty acid composition, lutein and tocopherol profiles, total phenolic contents as well as total soluble and insoluble bound sinapic acid and antioxidant capacities. The antioxidant properties were examined as their relative DPPH• scavenging capacity, oxygen radical absorbing capacity, and ferric reducing ability of plasma. The correlation between individual chemical composition and antioxidant properties was also determined. The results indicated that reducing erucic acid was associated with an increase of most measured bioactive components except α-tocopherol and insoluble-bound sinapic acid, as well as all the tested antioxidant capacities.

Monte Carlo simulation based prediction of blended oil composition containing mustard, rapeseed and soybean oil

High erucic acid rapeseed and mustard seed (HEARM) oils are popular cooking oil in various southeast Asian countries (e.g. Nepal, India, Bangladesh) due to the preference of the local consumer for its strong pungency. Low erucic acid rapeseed (LEAR) oil having lower pungency is frequently mixed with the pungent HEARM oil to increase its sensory appeal to the local consumer. Moreover, these oils are also prone to be adulterated with the cheaper oil available in the local market like soybean oil for economical reasons. In order to detect this fraud in a quick and easy manner, a Monte Carlo simulation based approach was developed for the estimation of blend composition using only the fatty acid composition of the sample. The limits of detection (LODs) of soybean oil in HEARM oil and LEAR oil were 14% and 13%, respectively. The LODs of LEAR oil in HEARM oil and soybean oil were 11 and 9%, respectively. Similarly, the LODs of HEARM oil in LEAR oil and soybean oil were 9% and 3%, respectively. The prediction from the developed method was evaluated both in real oil blends (prepared in the laboratory) and in theoretically simulated blends. The method was applied on forty-nine samples (labeled as mustard/rapeseed oil) collected from the Nepalese market. Among them, twenty-seven samples were found to be adulterated with soybean oil. The predicted adulteration was further supported by their δ-tocopherol content and trans fatty acid content, as an indicator for the adulteration with refined oil. The developed Monte Carlo simulation method is based on a single analytical run of determining the fatty acid composition of the suspected oil blend and thus useful for a quick segregation of samples in routine analysis.

Bioaugmentation with Petroleum-Degrading Consortia Has a Selective Growth-Promoting Impact on Crop Plants Germinated in Diesel Oil-Contaminated Soil

Rhizoremediation is a complex type of green clean-up technology that involves both plants and the rhizosphere-associated microorganisms to decompose hazardous compounds. The success of the strategy strongly depends on plant tolerance towards the pollutant, as well as plant's interactions with the rhizospheric microbes. The microorganisms may be stimulated by the secreted root exudates, which results in an increased breakdown of contaminants in the rhizosphere. The main goal of this study was to establish a potential rhizoremediation combination for a diesel-polluted site. Inoculation of plant roots or seeds with indigenous rhizospheric populations is a common approach in the rhizoremediation. However, we introduced hydrocarbon-degrading consortia (M10, R3, and K52) that were previously isolated from crude oil-contaminated soil instead of indigenous microbes. Bioaugmentation with these petroleum degraders was applied to screen four high biomass crop species (Indian mustard, alfalfa, high erucic acid rapeseed, HEAR, and low erucic acid rapeseed, LEAR) for their tolerance towards diesel oil. At no pollution, a promoting effect of M10 bacteria could be observed on germination and root elongation of all plant species. Moreover, M10 consortiums increased the germination index at 6,000 mg diesel oil per kilogram dry soil in the case of Indian mustard, alfalfa, and HEAR. The latter species was found to increment its dry weight upon bioaugmentation with M10 bacteria and all diesel oil treatments (6,000 and 24,000 mg diesel oil per kilogram dry soil). The initial results indicate HEAR and the M10 bacterial consortium as a promising plant–microbe tandem for a long-term rhizoremediation process.Electronic supplementary materialThe online version of this article (doi:10.1007/s11270-013-1676-0) contains supplementary material, which is available to authorized users.

Assessment of seed quality performance and heterosis for seed quality traits in hybrid high erucic acid rapeseed (HEAR)

Cuthbert, R. D., Crow, G. and McVetty, P. B. E. 2011. Assessment of seed quality performance and heterosis for seed quality traits in hybrid high erucic acid rapeseed (HEAR). Can. J. Plant Sci. 91: 837–846. Hybrids are becoming the dominant cultivar type in Brassica napus L. to utilize the well-documented heterosis for seed yield in canola/rapeseed. While there are numerous reports of heterosis for agronomic traits in B. napus hybrids, there are few reports of heterosis for seed quality traits in this species. The objectives of this study were to determine if high parent or commercial heterosis for any seed quality traits occurs in high erucic acid rapeseed (HEAR) hybrids. Seed quality performance of 45 F1 B. napus HEAR hybrids, derived from crosses of 12 geographically and genetically distinct (based on known pedigrees) HEAR cultivars/lines, was assessed in the current study using seven HEAR cultivars/lines developed by the University of Manitoba (UM) and five proprietary European (EU) HEAR cultivars/lines. Replicated field trials to assess seed oil, protein, sum of oil and protein, glucosinolate, and erucic acid concentrations were conducted over six environments in Manitoba during 2004 and 2005. High parent heterosis and commercial heterosis for seed oil concentration up to 9% and up to 14%, respectively, was observed with hybrids displaying seed oil concentrations as high as 533 g kg−1. Low parent heterosis for protein and glucosinolate concentrations was also observed. Erucic acid concentration displayed commercial heterosis. Many hybrids displayed near zero percent heterosis for any seed quality traits; however, overall seed quality was maintained or increased even in these hybrids. The results of this study indicate that development of HEAR hybrids can be an effective way to improve seed quality components, especially oil concentration, in this crop.

Effect of replacing solvent-extracted canola meal with high-oil traditional canola, high-oleic acid canola, or high-erucic acid rapeseed meals on rumen fermentation, digestibility, milk production, and milk fatty acid composition in lactating dairy cows

The objective of this experiment was to investigate the effects of replacing conventional, solvent-extracted canola meal (control; CTRL) with high oil content; conventional, mechanically extracted canola meal (CMEC); high-oleic, low polyunsaturated fatty acid (FA) canola meal (HOLL); and high-erucic acid, low-glucosinolate rapeseed meal (RPS) on rumen function, digestibility, milk production, and milk FA composition in lactating dairy cows. The experimental design was a replicated 4×4 Latin square with 8 lactating dairy cows. Four of the cows were ruminally cannulated. All oilseed meals were included at approximately 12 to 13% of dietary dry matter (DM). Crude protein and fat concentrations (% of DM) of the meals were 43 and 3.1%, 32.8 and 16.1%, 45.2 and 13.7%, and 34.3 and 17.9% for CTRL, CMEC, HOLL, and RPS, respectively. All diets were formulated to supply net energy of lactation in excess of requirements. The CMEC and RPS diets were predicted to be about 1% deficient in metabolizable protein. Relative to the CTRL, inclusion of high-oil seed meals in the diet lowered ruminal acetate concentration and the molar acetate:propionate ratio and decreased DM intake. Milk yield generally followed DM intake and was lower for CMEC and RPS than the CTRL. Treatments had no effect on milk composition, other than an increase in milk urea nitrogen concentration for HOLL. Fat-corrected milk (3.5%) feed efficiency was increased by HOLL and RPS compared with CTRL. Urinary urea nitrogen losses were increased by HOLL, which, as a consequence, increased the ammonia-emitting potential of manure. The ratio of milk N-to-N intake was greater for CMEC and RPS. Replacing solvent-extracted canola meal with the high-oil meal decreased milk fat 12:0, 14:0, 16:0, and total saturated FA content and enhanced cis-9 18:1 and total monounsaturated FA concentrations. Relative to the CTRL, canola increased total trans FA in milk, whereas inclusion of HOLL in the diet increased trans-11 18:1 and cis-9, trans-11 CLA content. The RPS increased milk fat cis-13 22:1 content from 0.07 to 2.33g/100g of FA. In conclusion, HOLL or RPS, which are likely to come from small-scale biodiesel plants where oil is cold pressed without hexane extraction, fed at levels at or above 12 to 13% of dietary DM may decrease feed intake and milk production, but can be used to alter milk FA composition in lactating dairy cows.

Cloning and Functional Analysis of Enoyl-CoA Reductase Gene BnECR from Oilseed Rape ( Brassica napus L.)

Very-long-chain fatty acids (VLCFAs) are critical components in cuticular waxes, sphingolipids, and triacylglycerols in higher plants. Biosynthesis of VLCFAs is catalyzed by the fatty acyl-CoA elongase, a membrane-bound enzymatic complex containing 3-ketoacyl-CoA synthase (KCS), 3-ketoacyl-CoA reductase (KCR), 3-hydroxacyl-CoA dehydratase (HCD), and trans-2,3-enoyl-CoA reductase (ECR). In this study, the primers were designed based on multiple alignments of trans-2,3-enoyl-CoA reductase (ECR) gene sequences from Arabidopsis thaliana and other plants, and the full-length cDNA, here designated BnECR, and the corresponding genomic sequences were isolated from Brassica napus using rapid amplification of cDNA ends method (GenBank accession numbers FJ899705 and FJ899705). The sequence of BnECR cDNA was 1328 bp in length excluding the poly dA tail, and the corresponding genomic sequence was 2093 bp. BnECR was composed of 4 exons and contained a 163 bp 5′ untranslated region (5′ UTR) and a 233 bp 3′ UTR. The deduced BnECR protein was 310 amino acid in length with a molecular weight of 735.78 kD and an isoelectric point of 9.52. The critical functional sites K 144 and R 145 in AtECR were unchanged in BnECR. The G 225SGGYQIPR/HG 234, which presented a non-classical NADPH-binding motif, was found in the C-terminal of BnECR. NCBI Blastn, multiple alignments and conserved domain search showed that BnECR had the highest homology to A. thaliana AtECR. RT-PCR analysis showed that BnECR was ubiquitously expressed in B. napus and preferentially expressed in the stem. The transcript level of BnECR at mid and late stages of seed development in low erucic acid rapeseed cultivar was obviously lower than that in high erucic acid rapeseed cultivar, suggesting that BnECR was involved in biosynthesis of erucic acid. The 933 bp open reading frame of BnECR was subcloned into the yeast- Escherichia coli shuttle vector pYES2.0. The recombinant plasmid was transformed into Saccharomyces cerevisiae wild type strain By4743 and the mutant strain YDL015c. With galactose as inducer, the transformant was cultured to induce the expression of BnECR. The gas chromatographic result indicated that BnECR was overexpressed effectively in S. cerevisiae, and the content of erucic acid reached 1.34% of the total fatty acid in the recombinant strain, an increase of 52% over the control. Functional complementation of BnECR in an ECR-deficient mutant yeast demonstrated that BnECR mediated the biosynthesis of VLCFAs. The results suggest that BnECR should be functional orthologue of AtECR.

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 (Glycinemax), followed by oilseed rape/canola (Brassicanapus). In Europe the major oilseed crop is oilseed rape (B. napus), followed some way behind by sunflower (Helianthusannuus) and other minor crops like linseed (Linumusitatissimum) or camelina (Camelinasativa). 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.

Assessment of agronomic performance and heterosis for agronomic traits in hybrid high erucic acid rapeseed (HEAR)

Twelve genetically diverse and geographically distinct high erucic acid rapeseed (HEAR) Brassica napus L. cultivars/lines were selected and crossed by hand emasculation in a topcross design to produce 45 F1 hybrid combinations. Seven of the twelve parents were HEAR cultivars/lines developed by the University of Manitoba (UM) and the remaining five parents were proprietary European (EU) HEAR cultivars/lines. Adequate seed was produced to evaluate the hybrids and parents in replicated yield trials in six environments in Manitoba during 2004 and 2005. Vigor, days to first flower, days to maturity, lodging, plant height, and seed yield were assessed. The hybrids were generally more vigorous, taller and more lodging resistant than their parents with similar days to first flower and days to maturity as their earlier parents. The best hybrids in this study displayed high parent heterosis for seed yield of up to 143% and commercial heterosis for seed yield of up to 99%. These hybrids were from the most genetically diverse UM × EU crosses. HEAR hybrids appear very promising approach to increase seed yields in this crop. Key words: Brassica napus L., HEAR, heterosis, hybrids, genetic diversity

Increasing erucic acid content through combination of endogenous low polyunsaturated fatty acids alleles with Ld-LPAAT + Bn-fae1 transgenes in rapeseed (Brassica napus L.)

High erucic acid rapeseed (HEAR) oil is of interest for industrial purposes because erucic acid (22:1) and its derivatives are important renewable raw materials for the oleochemical industry. Currently available cultivars contain only about 50% erucic acid in the seed oil. A substantial increase in erucic acid content would significantly reduce processing costs and could increase market prospects of HEAR oil. It has been proposed that erucic acid content in rapeseed is limited because of insufficient fatty acid elongation, lack of insertion of erucic acid into the central sn-2 position of the triaclyglycerol backbone and due to competitive desaturation of the precursor oleic acid (18:1) to linoleic acid (18:2). The objective of the present study was to increase erucic content of HEAR winter rapeseed through over expression of the rapeseed fatty acid elongase gene (fae1) in combination with expression of the lysophosphatidic acid acyltransferase gene from Limnanthes douglasii (Ld-LPAAT), which enables insertion of erucic acid into the sn-2 glycerol position. Furthermore, mutant alleles for low contents of polyunsaturated fatty acids (18:2 + 18:3) were combined with the transgenic material. Selected transgenic lines showed up to 63% erucic acid in the seed oil in comparison to a mean of 54% erucic acid of segregating non-transgenic HEAR plants. Amongst 220 F(2) plants derived from the cross between a transgenic HEAR line and a non-transgenic HEAR line with a low content of polyunsaturated fatty acids, recombinant F(2) plants were identified with an erucic acid content of up to 72% and a polyunsaturated fatty acid content as low as 6%. Regression analysis revealed that a reduction of 10% in polyunsaturated fatty acids content led to a 6.5% increase in erucic acid content. Results from selected F(2) plants were confirmed in the next generation by analysing F(4) seeds harvested from five F(3) plants per selected F(2) plant. F(3) lines contained up to 72% erucic acid and as little as 4% polyunsaturated fatty acids content in the seed oil. The 72% erucic acid content of rapeseed oil achieved in the present study represents a major breakthrough in breeding high erucic acid rapeseed.

Inheritance and variation of erucic acid content in a transgenic rapeseed (Brassica napus L.) doubled haploid population

Erucic acid (22:1) is a valuable renewable resource for the oleochemical industry. Currently available high erucic acid rapeseed cultivars contain only about 50% erucic acid in the seed oil. A substantial increase of the erucic acid content of the rapeseed oil could increase market prospects. The transgenic line TNKAT, over expressing the rapeseed fatty acid elongase gene (fae1) and expressing the Ld-LPAAT gene from Limnanthes douglasii was crossed with the line 6575-1 HELP (high erucic and low polyunsaturated fatty acid). A from the F1 plants produced population of 90 doubled haploid (DH) lines was tested in a greenhouse with three replicates. Parental lines TNKAT and 6575-1 HELP contained 46 and 50% erucic acid in the seed oil, respectively. In the DH population the erucic acid content ranged between 35 and 59%. The Ld-LPAAT + Bn-fae1.1 transgene showed a 1:1 segregation. The transgenic DH lines contained up to 8% trierucolyglycerol, but surprisingly had a by 2.3% lower erucic acid content compared to the non-transgenic segregants. Results indicated that the ectopically expressed fae1.1 gene may not be functional. The DH population also showed a large quantitative variation for PUFA content ranging from 6 to 28% (TNKAT: 21%, 6575-1 HELP: 8%). Regression analysis showed that in the DH population a 10% reduction in PUFA content led to a 4.2% increase in erucic acid content. Development of locus specific PCR primers for the two resident erucic acid genes fae1.1 (A-genome) and fae1.2 genes (C-genome) of rapeseed allowed sequencing of the respective alleles from TNKAT and 6575-1 HELP. Single nucleotide polymorphisms were only found for the fae1.1 gene. Use of allele specific fae1.1 PCR primers, however, did not reveal a significant effect of the fae1.1 allele from either parent on erucic acid content. The high erucic acid low polyunsaturated fatty acid DH lines and the fae1 locus specific primers developed in the present study should be useful in future studies aimed at increasing erucic acid content in rapeseed.

Cloning and characterization of an acyl‐CoA‐dependent diacylglycerol acyltransferase 1 (DGAT1) gene from Tropaeolum majus, and a study of the functional motifs of the DGAT protein using site‐directed mutagenesis to modify enzyme activity and oil content

A full-length cDNA encoding a putative diacylglycerol acyltransferase 1 (DGAT1, EC 2.3.1.20) was obtained from Tropaeolum majus (garden nasturtium). The 1557-bp open reading frame of this cDNA, designated TmDGAT1, encodes a protein of 518 amino acids showing high homology to other plant DGAT1s. The TmDGAT1 gene was expressed exclusively in developing seeds. Expression of recombinant TmDGAT1 in the yeast H1246MATalpha quadruple mutant (DGA1, LRO1, ARE1, ARE2) restored the capability of the mutant host to produce triacylglycerols (TAGs). The recombinant TmDGAT1 protein was capable of utilizing a range of (14)C-labelled fatty acyl-CoA donors and diacylglycerol acceptors, and could synthesize (14)C-trierucin. Collectively, these findings confirm that the TmDGAT1 gene encodes an acyl-CoA-dependent DGAT1. In plant transformation studies, seed-specific expression of TmDGAT1 was able to complement the low TAG/unusual fatty acid phenotype of the Arabidopsis AS11 (DGAT1) mutant. Over-expression of TmDGAT1 in wild-type Arabidopsis and high-erucic-acid rapeseed (HEAR) and canola Brassica napus resulted in an increase in oil content (3.5%-10% on a dry weight basis, or a net increase of 11%-30%). Site-directed mutagenesis was conducted on six putative functional regions/motifs of the TmDGAT1 enzyme. Mutagenesis of a serine residue in a putative SnRK1 target site resulted in a 38%-80% increase in DGAT1 activity, and over-expression of the mutated TmDGAT1 in Arabidopsis resulted in a 20%-50% increase in oil content on a per seed basis. Thus, alteration of this putative serine/threonine protein kinase site can be exploited to enhance DGAT1 activity, and expression of mutated DGAT1 can be used to enhance oil content.

Relationship of seed quality traits for greenhouse-grown versus field-grown high erucic acid rapesseed: Is seed quality trait selection for greenhouse-grown seed worthwhile?

A study of the relationship of seed quality traits for greenhouse-grown and field-grown seed samples was conducted. Early generation high erucic acid rapeseed (HEAR) cross progeny were grown in the greenhouse, selfed and then the selfed seeds were grown in the field at the University of Manitoba. The oil, protein, erucic acid and glucosinolate concentrations of greenhouse-grown versus field-grown seed samples were compared. There were differences (P ≤ 0.01) between the means of all seed quality traits for greenhouse-grown versus field-grown seed samples. The mean oil, protein and erucic acid concentrations of field-grown seed samples were higher than for greenhouse-grown seed samples. In contrast, the mean glucosinolate concentration of greenhouse-grown seed samples was higher than for field-grown seed samples. Rank correlations between greenhouse-grown seed samples and field-grown seed samples for all seed quality traits were significant but moderate in magnitude. Selection for oil, protein, erucic acid and glucosinolate concentration in greenhouse-grown seed samples dramatically increased the number of high seed quality F4 families in the field and was successful for all seed quality traits. It is concluded that seed quality selection in greenhouse-grown seed samples is worthwhile and that this procedure could lead to greater efficiencies in Brassica plant breeding programs. Key words: Rapeseed (Brassica napus L.), oil, protein, erucic acid, glucosinolates

Breeding high-stearic oilseed rape (Brassica napus) with high- and low-erucic background using optimised promoter-gene constructs

Seed lipids of oilseed rape (Brassica napus) usually contain small proportions (<3%) of stearic acid. The objective of this study was to increase the content of stearic fatty␣acid in rapeseed oil. An antisense down-regulation of the endogenous stearoyl-ACP desaturase (SAD) catalysing the reaction step from stearic to oleic acid in two different genetic backgrounds was studied. The result of down-regulation of the SAD yielded an about 10-fold increase of stearic acid from 3.7% up to 32% in single seeds of transgenic low-erucic acid rapeseed (LEAR), while high-erucic acid rapeseed (HEAR) showed a 4-fold increase of C18:0 from 1% up to 4%. It could be shown in pooled T2 seed material of LEAR rapeseed, that the stearic acid content is highly correlated with the down-regulation of SAD as indicated by the␣stearate desaturation proportion (SDP). The importance of the promoter strength for the alteration of a trait was confirmed in this study as no change in the fatty acid composition of transgenic plants was achieved with gene constructs controlled by the weak FatB4 seed-specific promoter from Cuphea lanceolata.