Chemical defenses of crucifers: elicitation and metabolism of phytoalexins and indole-3-acetonitrile in brown mustard and turnip

The metabolism of the cruciferous phytoalexins brassinin and cyclobrassinin, and the related compounds indole-3-carboxaldehyde, glucobrassicin, and indole-3-acetaldoxime was investigated in various plant tissues of Brassica juncea and B. rapa. Metabolic studies with brassinin showed that stems of B. juncea metabolized radiolabeled brassinin to indole-3-acetic acid, via indole-3-carboxaldehyde, a detoxification pathway similar to that followed by the "blackleg" fungus (Phoma lingam/Leptosphaeria maculans). In addition, it was established that tetradeuterated brassinin was incorporated into the phytoalexin brassilexin in B. juncea and B. rapa. On the other hand, the tetradeuterated indole glucosinolate glucobrassicin was not incorporated into brassinin, although the chemical structures of brassinins and indole glucosinolates suggest an interconnected biogenesis. Importantly, tetradeuterated indole-3-acetaldoxime was an efficient precursor of phytoalexins brassinin, brassilexin, and spirobrassinin. Elicitation experiments in tissues of Brassica juncea and B. rapa showed that indole-3-acetonitrile was an inducible metabolite produced in leaves and stems of B. juncea but not in B. rapa. Indole-3-acetonitrile displayed antifungal activity similar to that of brassilexin, was metabolized by the blackleg fungus at slower rates than brassinin, cyclobrassinin, or brassilexin, and appeared to be involved in defense responses of B. juncea.     

Phytoalexins and polar metabolites from the oilseeds canola and rapeseed: differential metabolic responses to the biotroph Albugo candida and to abiotic stress

The metabolites produced in leaves of the oilseeds canola and rapeseed (Brassica rapa L.) inoculated with either different races of the biotroph Albugo candida or sprayed with CuCl(2) were determined. This investigation established consistent phytoalexin (spirobrassinin, cyclobrassinin, and rutalexin) and phytoanticipin (indolyl-3-acetonitrile, arvelexin, caulilexin C, and 4-methoxyglucobrassicin) production in canola and rapeseed in response to both biotic and abiotic elicitation. In addition, a wide number of polar metabolites were isolated from infected leaves, including six new phenylpropanoids and two new flavonoids. The extractable chemical components of zoosporangia of A. candida and the anti-oomycete activity of phytoalexins were determined as well. Overall, the results suggest that during the initial stage of the interaction, leaves of B. rapa have a similar response to virulent and avirulent races of A. candida, with respect to the accumulation of chemical defenses. After this stage, despite the higher phytoalexin concentration, the "compatible" races could overcome the plant defense system for further infection, but growth of the "incompatible" races was inhibited. Since results of bioassays showed that cyclobrassinin and brassilexin were more inhibitory to A. candida than rutalexin, the apparent redirection of the phytoalexin pathway towards rutalexin, avoiding cyclobrassinin and brassilexin accumulation might be caused by the pathogen. Alternatively, A. candida might be able to detoxify both cyclobrassinin and brassilexin, similar to necrotrophic plant pathogens. Overall, the correlation between phytoalexin production in infected or stressed leaves and the outcome of the plant-pathogen interaction suggested that A. candida was able to elude the plant defense mechanisms by, for example, redirecting the phytoalexin biosynthetic pathway.     

Glucosinolate profiling of Brassica rapa cultivars after infection by Leptosphaeria maculans and Fusarium oxysporum

The glucosinolate contents of two different cultivars of Brassica rapa (Herfstraap and Oleifera) infected with Leptosphaeria maculans and Fusarium oxysporum were determined. Infection triggered the accumulation of aliphatic glucosinolates (gluconapin, progoitrin, glucobrassicanapin and gluconapoleiferin) and indole glucosinolate (4-hydroxy-glucobrassicin) in Herfstraap and of two indole glucosinolates (glucobrassicin and 4-hydroxy-glucobrassicin) in Oleifera. While total and aliphatic glucosinolates decreased significantly in Oleifera, a large increase was observed in Herfstraap after fungal infection. The indole glucosinolate glucobrassicin accumulated in Oleifera at a higher rate than Herfstraap especially after infection with F. oxysporum. Apparently the interaction between fungus and B. rapa is cultivar and fungal species specific.     

Differential abundance of simple repetitive sequences in species ofBrassica and relatedBrassicaceae

SixBrassica species, known as the triangle of U, and four species from related genera were characterized by DNA fingerprinting with simple repetitive oligonucleotide probes. Our results show that CT-, TCC-, and GTG-repeat motifs are equally abundant in the genomes of the sixBrassica species. In contrast, GATA-, GGAT-, and GACA-multimers are unevenly distributed among different species. As judged from the number and strength of hybridization signals, the highest copy number of all three motifs occurs inBrassica nigra, while the lowest is observed inB. oleracea. The abundance of GATA-and GACA-repeats varies in a coordinate way. The amphidiploid genomes ofB. juncea, B. carinata, andB. napus each harbour intermediate amounts of (GATA)₄ and (GACA)₄-detected repeats as compared to their diploid progenitors, thus supporting the concept of the U triangle. GATA-, GACA-, and GGAT-repeats were also abundant inEruca sativa andSinapis arvensis, but not inRaphanus sativus andSinapis alba. These results support the idea thatBrassica nigra is more closely related toSinapis arvensis than to otherBrassica species such asB. rapa andB. oleracea.     

Distribution of similar self-incompatibility (<Emphasis Type="Italic">S</Emphasis>) haplotypes in different genera,<Emphasis Type="Italic"> Raphanus</Emphasis> and<Emphasis Type="Italic"> Brassica</Emphasis>

The nucleotide sequences of ten SP11 and nine SRK alleles in Raphanus sativus were determined, and deduced amino acid sequences were compared with those of Brassica SP11 and SRK. The amino acid sequence identity of class-I SP11s in R. sativus was about 30% on average, the highest being 52.2%, while that of the S domain of class-I SRK was 77.0% on average and ranged from 70.8% to 83.9%. These values were comparable to those of SP11 and SRK in Brassica oleracea and B. rapa. SP11 of R. sativus S-21 was found to be highly similar to SP11 of B. rapa S-9 (89.5% amino acid identity), and SRK of R. sativus S-21 was similar to SRK of B. rapa S-9 (91.0%). SP11 and SRK of R. sativus S-19 were also similar to SP11 and SRK of B. oleracea S-20, respectively. These similarities of both SP11 and SRK alleles between R. sativus and Brassica suggest that these S haplotype pairs originated from the same ancestral S haplotypes.     

Retention of phytoalexin regulation in legume callus cultures

Legume callus cultures were examined to assess whether regulation of phytoalexin biosynthetic pathways is retained in cultured tissues. Callus tissue cultures ofCanavalia ensiformis (jackbean),Medicago sativa (alfalfa), and nine species ofTrifolium (clover) were established (six clover species for the first time) and maintained on modified Gamborg's B5 medium. Phytoalexins educed in cultures incubated for 48 h with an abiotic elicitor (3.15 mM HgCl₂) were detected by their antifungal activity and were purified by column chromatography and high-performance liquid chromatography. Following crystallization, phytoalexins were identified by ultraviolet and proton nuclear magnetic resonance spectroscopy. None of the treated cultures yielded the same complement of phytoalexins reported for fungal-inoculated leaves of the corresponding plants. Callus from all species exceptT. pratense yielded medicarpin, the only phytoalexin reported in treated leaves of all the corresponding plants. A second phytoalexin, maackiain, was found in treatedT. pratense andT. medium calli; maackiain has been reported in fungal-inoculated leaves of those plant species as well asT. hybridum. The phytoalexins sativan and vestitol were not found in treated callus tissues even though they were reported to be present in fungal-inoculated leaves of the same species. These results suggest that (a) the pathway for medicarpin biosynthesis is of central importance for this group of legumes, (b) some phytoalexin anabolic pathways contain metabolic blocks in cells of cultured tissue, and (c) the mechanism for regulating phytoalexin accumulation in tissues is not lost in culture. Contribution no 8113 of the US Regional Pasture Research Laboratory, USDA-ARS, University Park, PA, USA     

Brassica carinata resynthesized by protoplast fusion

Brassica carinata (bbcc) was resynthesized by protoplast fusion betweenB. nigra (bb) andB. oleracea (cc). In two fusion experiments 64 hybrid plants were obtained and identified to be true hybrids by isoenzyme analysis, nuclear DNA content, chromosome number, and intermediate morphology. Of these plants 56% were normal amphidiploids with 2n=34 chromosomes and a DNA content equivalent to that of naturalB. carinata. The remaining plants were polyploid, morphologically abnormal, and infertile. The majority of the hybrids contained both chloroplasts and mitochondria fromB. nigra, but some plants combined chloroplast and mitochondria from the different progenitors. Hybrids with a DNA content equivalent to that ofB. carinata had a wide range of male fertility (4–98%), but consistently low female fertility. Only a few selfed seed were produced, but these germinated and grew into vigorous plants.Salaries and research support provided by State and Federal funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University. Journal Article No. 296-92     

RFLP mapping of resistance to the blackleg disease [causal agent,Leptosphaeria maculans (Desm.) Ces. et de Not.] in canola (Brassica napus L.)

We report the tagging of genes involved in blackleg resistance, present in the French cultivar Crésor of B. napus, with RFLP markers. A total of 218 cDNA probes were tested on the parental cultivars Crésor (resistant) and Westar (susceptible), and 141 polymorphic markers were used in a segregating population composed of 98 doubled-haploid lines (DH). A genetic map from this cross was constructed with 175 RFLP markers and allowed us to scan for specific chromosomal associations between response to blackleg infection and RFLP markers. Canola residues infested with virulent strains of Leptosphaeria maculans were used as inoculum and a suspension of pycnidiospores from cultures of L. maculans, including the highly virulent isolate Leroy, was sprayed to increase disease pressure. QTL mapping suggested that a single chromosomal region was responsible for resistance in each of the four environments tested. This QTL accounted for a high proportion of the variation of blackleg reaction in each of the assays. A second QTL, responsible for a small proportion of the variation of blackleg reaction, was present in one of four year-site assays. A Mendelian approach, using blackleg disease ratings for classifying DH lines as resistant or susceptible, also allowed us to map resistance in the region of the highly significant LOD scores observed in each environment by interval mapping. Results strongly support the presence of a single major gene, named LmFr ₁ controlling adult plant resistance to blackleg in spring oil-seed rape cultivar Crésor. Several RFLP markers were found associated with LmFr ₁.     

Concerning the identity ofBranchinecta santacrucensis César, 1987

The type series of the speciesBrachinecta santacrucensis César 1987, is compared with specimens ofBranchinecta granulosa Daday 1902 collected near Facundo, Chubut Province, Argentina. The great resemblance between them suggest that both sets of material belong to the same species and therefore,B. santacrucensis andB. granulosa are synonyms.     

Tenualexin,Other Phytoalexins and Indole Glucosinolates from Wild Cruciferous Species

In general, the chemodiversity of phytoalexins, elicited metabolites involved in plant defense mechanisms against microbial pathogens, correlates with the biodiversity of their sources. In this work, the phytoalexins produced by four wild cruciferous species (Brassica tournefortii, Crambe abyssinica (crambe), Diplotaxis tenuifolia (sand rocket), and Diplotaxis tenuisiliqua (wall rocket)) were identified and quantified by HPLC with photodioarray and electrospray mass detectors. In addition, the production of indole glucosinolates, biosynthetic precursors of cruciferous phytoalexins, was evaluated. Tenualexin, (=2‐(1,4‐dimethoxy‐1H‐indol‐3‐yl)acetonitrile), the first cruciferous phytoalexin containing two MeO substituents in the indole ring, was isolated from D. tenuisiliqua, synthesized, and evaluated for antifungal activity. The phytoalexins cyclobrassinin and spirobrassinin were detected in B. tournefortii and C. abyssinica, whereas rutalexin and 4‐methoxybrassinin were only found in B. tournefortii. D. tenuifolia, and D. tenuisiliqua produced 2‐(1H‐indol‐3‐yl)acetonitriles as phytoalexins. Because tenualexin appears to be one of the broad‐range antifungals occurring in crucifers, it is suggested that D. tenuisiliqua may have disease resistance traits important to be incorporated in commercial breeding programs.     

Intersubgenomic heterosis in seed yield potential observed in a new type of Brassica napus introgressed with partial Brassica rapa genome

This paper reports the observation on the intersubgenomic heterosis for seed yield among hybrids between natural Brassica napus (AⁿAⁿCⁿCⁿ) and a new type of B. napus with introgressions of genomic components of Brassica rapa (A^rA^r). This B. napus was selected from the progeny of B. napus × B. rapa and (B. napus × B. rapa) × B. rapa based on extensive phenotypic and cytological observation. Among the 129 studied partial intersubgenomic hybrids, which were obtained by randomly crossing 13 lines of the new type of B. napus in F₃ or BC₁F₃ to 27 cultivars of B. napus from different regions as tester lines, about 90% of combinations exceeded the yield of their respective tester lines, whereas about 75% and 25% of combinations surpassed two elite Chinese cultivars, respectively. This strong heterosis was further confirmed by reevaluating 2 out of the 129 combinations in a successive year and by surveying hybrids between 20 lines of the new type of B. napus in BC₁F₅ and its parental B. napus in two locations. Some DNA segments from B. rapa were identified with significant effects on seed yield and yield components of the new type of B. napus in BC₁F₅ and intersubgenomic hybrids in positive or negative direction. It seems that the genomic components introgressed from B. rapa contributed to improvement of seed yield of rapeseed.     

Toxicity and accumulation of thallium in bacteria and yeast

Thallium sulphate inhibited microbial growth, withBacillus megaterium KM, more sensitive to the metal thanSaccharomyces cerevisiae andEscherichia coli. Inhibition ofB. megaterium KM andS. cerevisiae, but not ofE. coli, was alleviated by increasing the potassium concentration of the medium; inhibition of respiration ofS. cerevisiae, but not ofE. coli, was similarly alleviated. Thallium was rapidly bound, presumably to cell surfaces, byS. cerevisiae andE. coli, and was progressively accumulated by energy-dependent transport systems (probably concerned primarily with potassium uptake) with both organisms. Thallium uptake kinetics suggested more than one transport system operated in yeast, possibly reflecting a multiplicity of potassium transport systems. ApparentK _m andK _i values for competitive inhibition of thallium uptake by potassium indicatedS. cerevisiae to have a higher affinity for thallium uptake than for potassium, whileE. coli had a transport system with a higher affinity for potassium than for thallium. The likely systems for thallium transport are discussed. A mutant ofE. coli with tenfold decreased sensitivity to thallium was isolated and apparently effected surface binding of thallium in amounts equivalent to the wild type organism, but showed no subsequent uptake and accumulation of the metal from buffer, even though it was able to accumulate potassium to normal intracellular concentrations during growth. Abbreviations: Metal are referred to by their recognised atomic symbols (e.g. TI = Thallium; K = potassium; Co = cobalt)     

Plant growth stage effects on the feeding and growth responses of the bertha armyworm,Mamestra configurata to canola and mustard foliage

Mamestra configurata (Walker) (Lep., Noctuidae) larvae were fed excisedBrassica juncea (commercial brown mustard) orB. rapa cv. Tobin (Canola) foliage of three plant growth stages-rosette (stage 2), stem elongation (stage 3) and flowering (stage 4). Relative consumption rates (RCRi) were not significantly different between the plant species. Within theB. juncea treatments, there were no significant growth stage differences in RCRi. However, withinB. rapa, RCRi increased with advancing plant growth stage. Larvae fedB. juncea foliage had significantly reduced relative growth rates (RGRi) compared to larvae fedB. rapa foliage. Within theB. juncea treatments, RGRi decreased with advancing plant growth stage. There were no significant growth stage differences in RGRi in theB. rapa treatments. RGRi was inversely proportional to the levels of isothiocyanate-releasing glucosinolates in theB. juncea treatments. RCRi was inversely proportional to the levels of indolyl glucosinolates in theB. rapa treatments. Levels of total phenols and catechols inB. juncea did not show any trend which could be related to growth stage effects in the insect nutritional indices. InB. rapa, levels of phenols and catechols in stage 3 and 4 foliage were lower than that of stage 2 foliage. Analyses of total nitrogen in field-grown plants showed reductions in percent nitrogen from rosette to flowering stage foliage. The response ofM. configurata to different growth stages of its host plants are discussed in relation to changing levels of allelochemicals and nitrogen.     

Selective antimicrobial action of chitosan against spoilage yeasts in mixed culture fermentations

The effect of chitosan on Saccharomyces cerevisiae (the yeast that carries out alcohol fermentation), Brettanomyces bruxellensis and Brettanomyces intermedius (contaminants of alcohol fermentations), was investigated. The effect of chitosan was tested on each yeast, as well as on mixed cultivations of S. cerevisiae + B. bruxellensis and S. cerevisiae + B. intermedius. Chitosan enhanced the lag period of both strains of Brettanomyces (80 h for B. bruxellensis and 170 h for B. intermedius with 6 and 2 g/l chitosan, respectively). The growth rate of S. cerevisiae was inversely proportional to the chitosan concentration; the former was 50% when 6 g/l polysaccharide was used. Moreover, in mixed cultivations of S. cerevisiae and Brettanomyces strains, it was found that both B. bruxellensis and B. intermedius failed to grow while growth of S. cerevisiae was not affected (using 3 and 6 g/l chitosan, respectively). An interesting collateral result was that the presence of chitosan accelerated the consumption of glucose in the mixed cultivations (60 h instead of 120 h).     

Map-based cloning and characterization of a gene controlling hairiness and seed coat color traits in <Emphasis Type="Italic">Brassica rapa</Emphasis>

A glabrous, yellow-seeded doubled haploid (DH) line and a hairy, black-seeded DH line in Chinese cabbage (B. rapa) were used as parents to develop a DH line population that segregated for both hairiness and seed coat color traits. The data showed that both traits completely co-segregated each other, suggesting that one Mendelian locus controlled both hairiness and seed coat color in this population. A fine genetic map was constructed and a SNP marker that was located inside a Brassica ortholog of TRANSPARENT TESTA GLABRA 1 (TTG1) in Arabidopsis showed complete linkage to both the hairiness and seed coat color gene, suggesting that the Brassica TTG1 ortholog shared the same gene function as its Arabidopsis counterpart. Further sequence analysis of the alleles from hairless, yellow-seeded and hairy, black-seeded DH lines in B. rapa showed that a 94-base deletion was found in the hairless, yellow-seeded DH lines. A nonfunctional truncated protein in the hairless, yellow-seeded DH lines in B. rapa was suggested by the coding sequence of the TTG1 ortholog. Both of the TTG1 homologs from the black and yellow seeded B. rapa lines were used to transform an Arabidopsis ttg1 mutant and the results showed that the TTG1 homolog from the black seeded B. rapa recovered the Arabidopsis ttg1 mutant, while the yellow seeded homolog did not, suggesting that the deletion in the Brassica TTG1 homolog had led to the yellow seeded natural mutant. This was the first identified gene in Brassica species that simultaneously controlled both hairiness and seed coat color traits.     

Origin of new <Emphasis Type="Italic">Brassica</Emphasis> types from a single intergeneric hybrid between <Emphasis Type="Italic">B. rapa</Emphasis> and <Emphasis Type="Italic">Orychophragmus violaceus</Emphasis> by rapid chromosome evolution and introgression

Many novel lines were established from an intergeneric mixoploid between Brassica rapa (2n = 20) and Orychophragmus violaceus (2n = 24) through successive selections for fertility and viability. Pedigrees of individual F₂ plants were advanced to the 10th generation by selfing. Their breeding habit was self-compatible and different from the self-incompatibility of their female parent B. rapa, and these lines were reproductively isolated to different degrees from B. rapa and B. napus. The lines with high productivity showed not only a wide spectrum of phenotypes but also obvious variations in fatty acid profiles of seed oil and glucosinolate contents in seed meal. These lines had 2n = 36, 37, 38, 39 and 40, with 2n = 38 being most frequent (64.56%), and no intact O. violaceus chromosomes were detected by genomic in situ hybridization (GISH) analysis. Amplified fragment length polymorphism (AFLP) analyses revealed a high extent of variation in genomic compositions across all the lines. O. violaceus-specific bands, deleted bands in B. rapa and novel bands for two parents were detected in these lines, with novel bands being the most frequent. The morphological and genetic divergence of these novel types derived from a single hybrid is probably due to rapid chromosomal evolution and introgression, and provides new genetic resources for rapeseed breeding.     

Isozyme variation inBulnesia retama,B. schickendantzii andB. foliosa (Zygophyllaceae)

The genetic variation between two allopatric populations ofBulnesia retama and that ofB. schickendantzii andB. foliosa was investigated. The Peruvian population ofB. retama showed low values of P (0.117) and He (0.057) compared to those in the Argentine population with P = 0.59 and He = 0.269;B. schickendantzii showed P = 0.54 andB. foliosa P = 0.17. Genetic identity between the latter was 0.836 and between the allopatric populations ofB. retama was 0.89; the Peruvian population had reduced allelic variation per locus (A = 1.176) in comparison to the Argentine population (A = 1.955). The values of A, P, He andWright's fixation indices suggest that the Peruvian population could have originated from a single or very few migrants from southern latitudes (founder effect).     

Detection,introgression and localization of genes conferring specific resistance to <Emphasis Type="Italic">Leptosphaeria maculans</Emphasis> from <Emphasis Type="Italic">Brassica rapa</Emphasis> into <Emphasis Type="Italic">B. napus</Emphasis>

Blackleg (stem canker) caused by the fungus Leptosphaeria maculans is one of the most damaging diseases of oilseed rape (Brassica napus). Crop relatives represent a valuable source of “new” resistance genes that could be used to diversify cultivar resistance. B. rapa, one of the progenitors of B. napus, is a potential source of new resistance genes. However, most of the accessions are heterozygous so it is impossible to directly detect the plant genes conferring specific resistance due to the complex patterns of avirulence genes in L. maculans isolates. We developed a strategy to simultaneously characterize and introgress resistance genes from B. rapa, by homologous recombination, into B. napus. One B. rapa plant resistant to one L. maculans isolate was used to produce B. rapa backcross progeny and a resynthesized B. napus plant from which a population of doubled haploid lines was derived after crossing with natural B. napus. We then used molecular analyses and resistance tests on these populations to identify and map the resistance genes and to characterize their introgression from B. rapa into B. napus. Three specific genes conferring resistance to L. maculans (Rlm1, Rlm2 and Rlm7) were identified in B. rapa. Comparisons of genetic maps showed that two of these genes were located on the R7 linkage group, in a region homologous to the region on linkage group N7 in B. napus, where these genes have been reported previously. The results of our study offer new perspectives for gene introgression and cloning in Brassicas.     

The phytoalexins brassilexin and camalexin inhibit cyclobrassinin hydrolase,a unique enzyme from the fungal pathogen Alternaria brassicicola

Alternaria brassicicola is a fungal pathogen of many agriculturally important cruciferous crops. Cyclobrassinin hydrolase (CH) is an enzyme produced by A. brassicicola that catalyzes the transformation of the cruciferous phytoalexin cyclobrassinin into S-methyl[(2-sulfanyl-1H-indolyl-3)methyl]carbamothioate. The purification and characterization of CH was performed using a four-step chromatography method. SDS–PAGE and gel exclusion chromatography indicated that CH is a tetrameric protein with molecular mass of 330 kDa. Sequence analysis and chemical modification of CH with selective reagents suggested that the enzyme mediates hydrolysis of cyclobrassinin using a catalytic amino acid triad. Enzyme kinetic studies using cyclobrassinin and 1-methylcyclobrassinin as substrates revealed that CH displayed positive substrate cooperativity. Investigation of the effect of nine phytoalexins and two derivatives on the activity of CH indicated that six compounds displayed inhibitory activity: brassilexin, 1-methylbrassilexin, dioxibrassinin, camalexin, brassicanal A and sinalexin. The enzyme kinetics of CH strongly suggested that brassilexin and 1-methylbrassilexin are noncompetitive inhibitors of CH activity, and that camalexin is a competitive inhibitor while dioxibrassinin inhibits CH through a mixed mechanism. The phytoalexin brassilexin is the most effective inhibitor of CH (K_i = 32 ± 9 μM). These results suggest that crops able to accumulate higher concentration of brassilexin would display higher resistance levels to the fungus.     

Hairpin-RNA mediated silencing of endogenous FAD2 gene combined with heterologous expression of Crambe abyssinica FAE gene causes an increase in the level of erucic acid in transgenic Brassica carinata seeds

The 3′-UTR of the FAD2 gene from Brassica carinata was cloned by PCR and used to prepare an intron-spliced hairpin RNA (ihpRNA) construct. Compared to that of the wild type (WT) background, this construct, when expressed in B. carinata, resulted in a high degree of FAD2 gene silencing accompanied by strong increases of up to 16 and 10% in oleic acid and erucic acid proportions, respectively. The increase in 18:1 was accompanied by a concomitant proportional reduction in 18:2. A second construct containing ihpRNA targeted to the endogenous FAD2 gene in addition to the heterologous Crambe abyssinica FAE gene under the control of seed specific napin promoter, was used to transform B. carinata. This approach resulted in an even greater increase in erucic acid proportions, by up to 16% in T₁ segregating seeds as compared to that of the WT control. To our knowledge, this is currently the highest accumulation of erucic acid achieved in B. carinata seeds using transgenic approaches, making it an increasingly-attractive alternative to high erucic B. napus cultivars as an industrial oil crop. Database: The nucleotide sequence reported in this paper has been submitted to the EMBL/GenBank under accession number DQ250814.