Redirection of metabolic flux for high levels of omega‐7 monounsaturated fatty acid accumulation in camelina seeds

Seed oils enriched in omega‐7 monounsaturated fatty acids, including palmitoleic acid (16:1?9) and cis‐vaccenic acid (18:1?11), have nutraceutical and industrial value for polyethylene production and biofuels. Existing oilseed crops accumulate only small amounts (<2%) of these novel fatty acids in their seed oils. We demonstrate a strategy for enhanced production of omega‐7 monounsaturated fatty acids in camelina (Camelina sativa) and soybean (Glycine max) that is dependent on redirection of metabolic flux from the typical ?9 desaturation of stearoyl (18:0)‐acyl carrier protein (ACP) to ?9 desaturation of palmitoyl (16:0)‐acyl carrier protein (ACP) and coenzyme A (CoA). This was achieved by seed‐specific co‐expression of a mutant ?9‐acyl‐ACP and an acyl‐CoA desaturase with high specificity for 16:0‐ACP and CoA substrates, respectively. This strategy was most effective in camelina where seed oils with ~17% omega‐7 monounsaturated fatty acids were obtained. Further increases in omega‐7 fatty acid accumulation to 60–65% of the total fatty acids in camelina seeds were achieved by inclusion of seed‐specific suppression of 3‐keto‐acyl‐ACP synthase II and the FatB 16:0‐ACP thioesterase genes to increase substrate pool sizes of 16:0‐ACP for the ?9‐acyl‐ACP desaturase and by blocking C18 fatty acid elongation. Seeds from these lines also had total saturated fatty acids reduced to ~5% of the seed oil versus ~12% in seeds of nontransformed plants. Consistent with accumulation of triacylglycerol species with shorter fatty acid chain lengths and increased monounsaturation, seed oils from engineered lines had marked shifts in thermotropic properties that may be of value for biofuel applications.     

Aspergillus flavus infection and aflatoxin contamination in sorghum seeds and their biological management

In order to establish the current scenario of aflatoxigenic fungal infection and aflatoxin contamination in sorghum seeds across India, 58 seed samples were collected from different agro-climatic regions. Among these, 67.2% samples were infected with Aspergillus spp. and 28% were found contaminated with aflatoxins ranging from 0.0 to 130?μg?kg^?1. Greenhouse studies revealed no correlation between incidence of Aspergillus flavus and aflatoxin content, and its effect on seed quality parameters. Among the 37 A. flavus strains isolated, six were non-aflatoxigenic when analysed through cultural, TLC and ic-ELISA. Seed treatment with biocontrol agents (antagonistic Rhizobacteria and Trichoderma) suppressed the growth of A. flavus under laboratory and significantly enhanced seed quality variables under greenhouse conditions to a various extent. Field trials with selected biocontrol agents showed that talcum powder formulations of Pseudomonas putida Has-1/c, Bacillus spp. 3/a, Trichoderma asperellum M5 and T. asperellum T2 improved seedling emergence, % nutrient accumulation in plants, increased plant biomass and 1000 seed weight. Seeds harvested from treated plants showed significant increase in seed quality variables under laboratory and greenhouse conditions in comparison with control, but there was no significant difference in A. flavus infection and aflatoxin was completely absent in all treatments.     

KAS IV: a 3-ketoacyl-ACP synthase from Cuphea sp . is a medium chain specific condensing enzyme

cDNA clones encoding a novel 3-ketoacyl-ACP synthase (KAS) have been isolated from Cuphea . The amino acid sequence of this enzyme is different from the previously characterized classes of KASs, designated KAS I and III, and similar to those designated as KAS II. To define the acyl chain specificity of this enzyme, we generated transgenic Brassica plants over-expressing the cDNA encoded protein in a seed specific manner. Expression of this enzyme in transgenic Brassica seeds which normally do not produce medium chain fatty acids does not result in any detectable modification of the fatty acid profile. However, co-expression of the Cuphea KAS with medium chain specific thioesterases, capable of production of either 12:0 or 8:0/10:0 fatty acids in seed oil, strongly enhances the levels of these medium chain fatty acids as compared with seed oil of plants expressing the thioesterases alone. By contrast, co-expression of the Cuphea KAS along with an 18:0/18:1-ACP thioesterase does not result in any detectable modification of the fatty acids. These data indicate that the Cuphea KAS reported here has a different acyl-chain specificity to the previously characterized KAS I, II and III. Therefore, we designate this enzyme KAS IV, a medium chain specific condensing enzyme.     

Reduced expression of FatA thioesterases in Arabidopsis affects the oil content and fatty acid composition of the seeds

Acyl–acyl carrier protein (ACP) thioesterases are enzymes that control the termination of intraplastidial fatty acid synthesis by hydrolyzing the acyl–ACP complexes. Among the different thioesterase gene families found in plants, the FatA-type fulfills a fundamental role in the export of the C18 fatty acid moieties that will be used to synthesize most plant glycerolipids. A reverse genomic approach has been used to study the FatA thioesterase in seed oil accumulation by screening different mutant collections of Arabidopsis thaliana for FatA knockouts. Two mutants were identified with T-DNA insertions in the promoter region of each of the two copies of FatA present in the Arabidopsis genome, from which a double FatA Arabidopsis mutant was made. The expression of both forms of FatA thioesterases was reduced in this double mutant (fata1 fata2), as was FatA activity. This decrease did not cause any evident morphological changes in the mutant plants, although the partial reduction of this activity affected the oil content and fatty acid composition of the Arabidopsis seeds. Thus, dry mutant seeds had less triacylglycerol content, while other neutral lipids like diacylglycerols were not affected. Furthermore, the metabolic flow of the different glycerolipid species into seed oil in the developing seeds was reduced at different stages of seed formation in the fata1 fata2 line. This diminished metabolic flow induced increases in the proportion of linolenic and erucic fatty acids in the seed oil, in a similar way as previously reported for the wri1 Arabidopsis mutant that accumulates oil poorly. The similarities between these two mutants and the origin of their phenotype are discussed in function of the results.     

Induction of peroxidase and polyphenol oxidase in Arachis hypogaea in response to treatment with Pseudomonas fluorescens and inoculation with Alternaria alternata

Abstract

The application of talc-formulation through seed, seed treatment plus foliar spray and foliar spray alone significantly reduced the leaf blight incidence both under greenhouse and field conditions. The groundnut plants treated with biocontrol agent and challenge inoculated with Alternaria alternata recorded significantly increased activity of Peroxidase isozyme (PO), Polyphenol oxidase isozymes (PPO) activity. Expression of PO2, PPO1 and PPO2 isoforms were found in all the plants treated with Pf1 while additional PO1, PPO3, PPO4 and PPO5 were observed in Pf1-treated plants followed by challenge inoculation with the pathogen.     

Reducing saturated fatty acids in Arabidopsis seeds by expression of a Caenorhabditis elegans 16:0–specific desaturase

Plant oilseeds are a major source of nutritional oils. Their fatty acid composition, especially the proportion of saturated and unsaturated fatty acids, has important effects on human health. Because intake of saturated fats is correlated with the incidence of cardiovascular disease and diabetes, a goal of metabolic engineering is to develop oils low in saturated fatty acids. Palmitic acid (16:0) is the most abundant saturated fatty acid in the seeds of many oilseed crops and in Arabidopsis thaliana. We expressed FAT–5, a membrane‐bound desaturase cloned from Caenorhabditis elegans, in Arabidopsis using a strong seed‐specific promoter. The FAT‐5 enzyme is highly specific to 16:0 as substrate, converting it to 16:1?9; expression of fat‐5 reduced the 16:0 content of the seed by two‐thirds. Decreased 16:0 and elevated 16:1 levels were evident both in the storage and membrane lipids of seeds. Regiochemical analysis of phosphatidylcholine showed that 16:1 was distributed at both positions on the glycerolipid backbone, unlike 16:0, which is predominately found at the sn‐1 position. Seeds from a plant line homozygous for FAT–5 expression were comparable to wild type with respect to seed set and germination, while oil content and weight were somewhat reduced. These experiments demonstrate that targeted heterologous expression of a desaturase in oilseeds can reduce the level of saturated fatty acids in the oil, significantly improving its nutritional value.     

Comparative evaluation of rumen‐protected fat,coconut oil and various oilseeds supplemented to fattening bulls

The effects of five different dietary fat supplements on fatty acid composition and oxidative stability of subcutaneous and kidney fat were evaluated in 36 Brown Swiss bulls and compared to a low fat diet in a monofactorial design. The following fat supplements were provided as additional fat at 30 g per kg feed dry matter: crystalline rumen‐protected fat, coconut oil, and three types of crushed whole oilseeds (rapeseed, sunflower seed and linseed). Adipose tissues reflected differences (P < 0.05) in dietary fatty acid composition although to a lower extent. Using protected fat, which contained elevated levels of trans fatty acids, and sunflower seed, containing a high proportion of linoleic acid, significantly increased C18:1 trans fatty acid proportion in the adipose tissues. The use of sunflower seed increased conjugated linoleic acid. The oilseeds resulted in lower amounts of C16:0 in favour of C18:0. Except for linseed, all fat supplemented groups improved oxidative stability of adipose tissues as compared with control. This was explained by lower proportions of unsaturated fatty acids in adipose tissue (protected fat), by elevated α‐tocopherol contents (rapeseed, sunflower seed) or by a combination of both (coconut oil). Fat colour remained unaffected by treatments. Compared to other fat supplements oilseeds, especially sunflower seed and rapeseed, can therefore be recommended to be fed to bulls in order to increase the proportions of C18 unsaturated fatty acids in adipose tissues and to maintain or improve oxidative stability.     

High-Oleic Peanut Oils Produced by HpRNA-Mediated Gene Silencing of Oleate Desaturase

The quality of peanut oil largely depends on the quantity of oleic (18:1) and linoleic acids (18:2). These two acids comprise more than 80% of the total fatty acids in peanuts. The oleate desaturase (FAD2) gene is important for maintaining high oleic acid content. A partial conservative sequence of the FAD2 gene from peanut was selected. The sense and antisense 260-bp fragments were amplified and subcloned into pFGC1008 binary expression vectors. A total of 21 transgenic plants were obtained via Agrobacterium-mediated transformation. The resulting down-regulation of the FAD2 gene resulted in a 70% increase in oleic acid content in the seeds of transformed plants compared with a 37.93% increase in untransformed plants. The results demonstrated that the target genes were likely suppressed by hpRNA interference, a pathway capable of achieving phenotypic changes. The silencing of FAD2 enabled the development of peanut oils having novel combinations of oleic acid content that can be used in high-value applications, making this approach a reliable technique for the genetic modification of seed quality and the potential for enhancement of other traits as well.     

Very long chain and hydroxylated fatty acids in offspring of somatic hybrids between Brassica napus and Lesquerella fendleri

 Offspring of somatic hybrids between the zero-erucic acid rapeseed cv Hanna and Lesquerella fendleri were analysed regarding their fatty acid profiles. In the first back-cross generation one plant was found that produced a seed containing up to 16.5% erucic acid and 15% eicosaenoic acid (Line 1), as well as a seed having 4.3% ricinoleic acid (Line 2). This was interpreted as due to a contribution of elongase and hydroxylase genes from the L. fendleri genome since these two fatty acids are not produced in the recipient rapeseed cultivar Hanna. Crosses between Line 1 and cv Hanna resulted in the production of seeds with 35% erucic acid (F₂). Furthermore, crosses between the F₂ plants and the rapeseed cultivar Gulle, producing 35% erucic acid in the seeds, resulted in F₃ seeds with 48% erucic acid. The highest amount of erucic acid, 61.5%, was found in the F₆ generation after crossing Line 1 with a high erucic acid rapeseed line, HEAR, followed by self-fertilisation for two generations. When performing Southern-blot analysis on the F₆ plants, seven of the nine analysed plants hybridised with the L. fendleri species-specific repetitive probe. The presence of the hydroxylase gene was also observed in the F₆ generation of Line 1 according to Southern-blot analysis. Hybridisation with a hydroxylase probe was seen although no hydroxy fatty acids could be detected in any of the F₆ plants. In parallel, Line 2 was crossed with HEAR cv Gulle and self fertilised. No hydroxy fatty acids were detected in the F₂ generation of Line 2 and no specific hybridisation patterns could be found in the Southern-blot analysis. Received: 12 December 1998 / Accepted: 4 January 1999     

Antisense expression of 3-oxoacyl-ACP reductase affects whole plant productivity and causes collateral changes in activity of fatty acid synthase components

Brassica napus cv Westar plants were transformed with 3-oxoacyl-ACP reductase (KR) in antisense orientation, driven by either the cauliflower mosaic virus 35S promoter or a seed-specific acyl carrier protein promoter to determine the effects on plant productivity and on the activity of other fatty acid synthase (FAS) components. In plants with altered KR activity, total seed yield was reduced in all cases. In less severely affected plant lines, seeds had a normal appearance and composition but the yield of seeds was reduced by approximately 50%. In more severely affected lines, reductions in both seed fatty acid content and the number of seeds produced per plant were evident, resulting in a 90% reduction in fatty acid synthesized per plant. These phenotypes were independent of the promoter used. In severely affected lines, a large proportion of seeds showed precocious germination, and these had a reduced oleate content and increased levels of polyunsaturated 18-carbon fatty acids, compared with normal seeds of the same line. This reduction in 18:1 fatty acids was mimicked on imbibition of seeds with a normal appearance, indicating a preferential use of oleate moieties in precocious germination events. The reduction in activity of KR was mirrored for a second fatty acid synthase component, enoyl-ACP reductase, indicating a mechanism to maintain the ratio of fatty acid synthase components throughout embryogenesis.     

Transgenic expression of a Δ12-epoxygenase gene in Arabidopsis seeds inhibits accumulation of linoleic acid

The Crepis palaestina cDNA Cpal2 encodes a Δ12-epoxygenase that can catalyse the synthesis of 12,13-epoxy-cis-9-octadecenoic acid (18:1E) from linoleic acid (18:2). When the Cpal2 gene was expressed under the control of the napin seed-specific promoter in Arabidopsis thaliana (L.) Heynh., the seed lipids accumulated only low levels of 18:1E and also 12,13-epoxy-cis-9,15-octadec-2-enoic acid (18:2E). Despite the fact that the levels of these epoxy fatty acids comprised only up to 6.2% of the total fatty acids, there was a very marked increase in oleic acid (18:1) and decrease in linoleic (18:2) and α-linolenic (18:3) acids in these plants, indicating that endogenous Δ12-desaturation was greatly reduced in these plants. Significant between-line differences in the levels of Cpal2 mRNA were observed during seed development, but were not associated with any major variation in mRNA levels for the endogenous ArabidopsisΔ12-desaturase (Fad2). This suggests that if an unfavourable interaction occurs between the transgenic Δ12-epoxygenase and the endogenous Δ12-desaturase, which decreases the level of desaturation, it occurs at either the translational or post-translational level. We further show that the co-expression of a Δ12-desaturase gene from C. palaestina in Cpal2 transgenic Arabidopsis returns the relative proportions of the C18 seed fatty acids to normal levels and results in an almost twofold increase in total epoxy fatty acids. Received: 11 August 2000 / Accepted: 7 September 2000     

Traits of fatty acid accumulation in dimorphic seeds of the euhalophyte Suaeda salsa in saline conditions

The mechanism on of how salinity affects seed fatty acids accumulation remains unclear in halophytes. The present results revealed that the content of total unsaturated fatty acids in black seeds was higher than in brown seeds in the euhalophyte Suaeda salsa under controlled saline conditions. Salinity (200?mM NaCl) significantly increased the total oil content, unsaturated acid/saturated acid ratio, and content of α-linolenic acid (C18:3) (ALA), especially in brown seeds. The most abundant fatty acid in dimorphic seeds is linoleic acid (C18:2) (>70%). It appears that more ALA accumulated in brown seeds compared to black seeds. The enzyme activity of omega-3 fatty acid desaturase (ω-3 FAD) in brown seeds was much higher than that in black seeds, but salinity had no significant effect on the activity of ω-3 FAD in both brown and black seeds. The relative expression of SsFAD7 was increased by salinity, and the value in brown seeds was much higher than that in black seeds. This means salinity can, salinity can improve the quantity of fatty acids in dimorphic seeds of S. salsa, and the enzyme of ω-3 FAD and SsFAD7 may involve in the accumulation of ALA in dimorphic seeds under salinity.     

A mutation in Arabidopsis cytochrome b5 reductase identified by high-throughput screening differentially affects hydroxylation and desaturation

As a model for analyzing the production of novel fatty acids in oilseeds, we used the genetic and molecular techniques available for Arabidopsis to characterize modifying mutations affecting the accumulation of hydroxy fatty acids in the seeds of Arabidopsis plants that express a transgene for the castor bean fatty acid hydroxylase, FAH12. We developed a high-throughput analytical system and used it to identify three complementation classes of mutations with reduced hydroxy fatty acid accumulation from among Arabidopsis M3 seed samples derived from chemical mutagenesis. We identified one of the mutations by positional cloning as a single base pair change in a gene encoding NADH:cytochrome b5 reductase (CBR1, At5g17770). When expressed in yeast, the mutant form of the enzyme was less active and less stable than the wild-type enzyme. Characterization of homozygous mutant lines with and without the FAH12 transgene (FAH12 cbr1-1 and cbr1-1, respectively) indicated that the only detectable consequence of the cbr1-1 mutation was on desaturase and hydroxylase reactions in the developing seed. The leaf and root fatty compositions, as well as the growth, development and seed production of mutant plants were indistinguishable from wild type. Interestingly, while the cbr1-1 mutation reduced the accumulation of hydroxy fatty acids in seeds by 85%, the effects on 18:1 and 18:2 desaturation reactions were much less (<25% and <60%, respectively). These results suggest that there is competition in developing seeds among the several reactions that utilize reduced cytochrome b5.     

The distribution of caprylate, caprate and laurate in lipids from developing and mature seeds of transgenic Brassica napus L.

The composition and positional distribution of lipids in developing and mature transgenic Brassica napus seeds accumulating up to 7 mol% of caprylate (8:0), 29 mol% caprate (10:0) or 63 mol% of laurate (12:0) were examined. The accumulation of 8:0 and 10:0 resulted from over-expression of the medium-chain-specific thioesterase (Ch FatB2) alone or together with the respective chain-length-specific condensing enzyme (Ch KASIV). Seeds containing high levels of 12:0 were obtained from plants expressing bay thioesterase (BTE) alone or crossed with a line over-expressing the coconut lysophosphatidic acid acyltransferase (LPAAT), an enzyme responsible for the increase in acylation of 12:0 at the sn-2 position. In all instances, 10:0 and 12:0 fatty acids were present in substantial amounts in phosphatidylcholine during seed development with a drastic decrease of 80–90% in mature seeds. At all stages of seed development however, 8:0 was barely detectable in this membrane lipid. Altogether, these results indicate that these transgenic seeds exclude and/or remove the medium-chain fatty acids from their membrane and that this mechanism(s) is more effective with the shorter-chain fatty acids. Furthermore, seeds of 8:0- and 10:0-producing lines had only negligible levels of these fatty acids present in the sn-2 position of the triacylglycerols. In contrast, all 12:0-producing seeds had a substantial amount of this fatty acid in the sn-2 position of the triacylglycerols, suggesting that the endogenous LPAAT is able to acylate 12:0 if no other acyl-CoA species are available. Received: 11 February 2000 / Accepted: 2 May 2000     

Biocontrol of Rhizoctonia Damping-off of Tomato by Bacillus subtilis Combined with Burkholderia cepacia

Bacillus subtilis strain RB14‐C and Burkholderia cepacia strain BY were used in combination to control damping‐off of tomato plants caused by Rhizoctonia solani. Microcosm tests showed complete inhibition of R. solani growth on filter disks buried in soil added with the mixture of both bacteria. Single BY inhibited the fungus, but not completely, and RB14‐C had only slight inhibitory effect on pathogen growth. The efficacy of this combining treatment was checked in pot experiments, where bacteria were applied to the soil in several combinations: RB14‐C and BY together 4 days before seed planting, RB14‐C 4 days and BY 2 days before seed planting, RB14‐C 4 days and BY immediately before seeds. The effect of these treatments on population of R. solani in soil and infection of plants was compared with the activity of single application of each agent. All bacterial treatments significantly decreased damping‐off of tomato plants. The best control was obtained when BY was added 2 days after RB14‐C. In this treatment plant protection was significantly higher than that obtained in other combined applications and obtained by single strains, except BY added to the soil 4 days before seed planting. The lowest suppression indicated BY introduced to the soil before seed planting. RB14‐C only slightly decreased number of R. solani in the soil. In contrast, BY drastically reduced population of the pathogen. However, there was not a clear relation between decrease of pathogen density in soil and the rate of plant infection. The results show that combination of B. subtilis RB14‐C with B. cepacia BY can lead to greater damping‐off suppression than biocontrol exhibited by these strains used separately, but the effect of combining bacterial agents was clearly related to the order in which both agents were introduced.     

The Effect of Mode of Application of Bacillus subtilis RB14-C on its Efficacy as a Biocontrol Agent Against Rhizoctonia solani

Bacillus subtilis RB14‐C, which produces the antibiotic iturin A, was investigated for its effectiveness as a biocontrol agent against Rhizoctonia solani infecting tomato using seed coating and/or direct introduction of the bacteria to the soil. The ability of RB14‐C to colonize plant roots and produce iturin A in soil, depending on the method of bacterial application, was also determined. Seed coating and the combined treatment (soil and seed bacterization) did not protect seedlings against damping‐off caused by R. solani. By contrast, RB14 introduced only to the soil controlled the disease. The total number of RB14‐C bacteria on the roots of plants grown from coated seeds was significantly lower than on the roots of plants grown in soil mixed with the bacteria. In the combined treatment, application of B. subtilis with seeds to soil preinoculated with this bacterium, at first suppressed the population of RB14‐C in the soil. Then the colonization was generally uniform. The concentration of iturin A in non‐planted soil was highest at the beginning of the experiment (i.e. after application of the bacterial suspension) but then decreased, and was undetectable 3 days after incubation. However, after seed planting the antibiotic was produced again around young roots. Bacteria introduced to the soil as a seed coating also released the antibiotic around the seeds.     

Natural variation of GmFATA1B regulates seed oil content and composition in soybean

Soybean (Glycine max) produces seeds that are rich in unsaturated fatty acids and is an important oilseed crop worldwide. Seed oil content and composition largely determine the economic value of soybean. Due to natural genetic variation, seed oil content varies substantially across soybean cultivars. Although much progress has been made in elucidating the genetic trajectory underlying fatty acid metabolism and oil biosynthesis in plants, the causal genes for many quantitative trait loci (QTLs) regulating seed oil content in soybean remain to be revealed. In this study, we identified GmFATA1B as the gene underlying a QTL that regulates seed oil content and composition, as well as seed size in soybean. Nine extra amino acids in the conserved region of GmFATA1B impair its function as a fatty acyl–acyl carrier protein thioesterase, thereby affecting seed oil content and composition. Heterogeneously overexpressing the functional GmFATA1B allele in Arabidopsis thaliana increased both the total oil content and the oleic acid and linoleic acid contents of seeds. Our findings uncover a previously unknown locus underlying variation in seed oil content in soybean and lay the foundation for improving seed oil content and composition in soybean.     

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 F₂ 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 F₂ 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 F₂ plants were confirmed in the next generation by analysing F₄ seeds harvested from five F₃ plants per selected F₂ plant. F₃ 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.