Development of Industrial Oil Crop Crambe abyssinica for Wax Ester Production through Metabolic Engineering and Cross Breeding.

As an important industrial feedstock, wax esters (WEs) have been used as lubricants in a number of technical processes. There is however currently no large-scale biological source for WE production and alteration in metabolic pathways of plant oils for producing WEs could be attractive to the commercial markets. Here, we present the breeding results of long-term studies on successful development of new crambe lines producing WEs through genetic engineering and cross breeding. The transgenic crambe lines producing WEs at over 25% of the total seed oil were first generated by introduction of the jojoba WE biosynthetic genes ScFAR and ScWS. Further improvement of the lines aiming at improving oxidative stability of WEs was achieved through introducing the CaFAD2-RNAi gene into these lines by crossing. The hybrid lines possessed similar agronomic traits to the wild type and a stable level of WEs over several generations, suggesting a high potential of crambe as an industrial crop for WE production.

The Crambe abyssinica plastome: Brassicaceae phylogenomic analysis, evolution of RNA editing sites, hotspot and microsatellite characterization of the tribe Brassiceae.

Crambe abyssinica is an important oilseed crop that accumulates high levels of erucic acid, which is being recognized as a potential oil platform for several industrial purposes. It belongs to the family Brassicaceae, assigned within the tribe Brassiceae. Both family and tribe have been the subject of several phylogenetic studies, but the relationship between some lineages and genera remains unclear. Here, we report the complete sequencing and characterization of the C. abyssinica plastome. Plastome structure, gene order, and gene content of C. abyssinica are similar to other species of the family Brassicaceae. The only exception is the rps16 gene, which is absent in many genera within the family Brassicaceae, but seems to be functional in the tribe Brassiceae, including C. abyssinica. However, the analysis of gene divergence shows that the rps16 is the most divergent gene in C. abyssinica and within the tribe Brassiceae. In addition, species of the tribe Brassiceae also show similar SSR loci distribution, with some regions containing a high number of SSRs, which are located mainly at the single copy regions. Six hotspots of nucleotide divergence among Brassiceae species were located in the single copy regions by sliding window analysis. Brassicaceae phylogenomic analysis, based on the complete plastomes of 72 taxa, resulted in a well-supported and well-resolved tree. The genus Crambe is positioned within the Brassiceae clade together with the genera Brassica, Raphanus, Sinapis, Cakile, Orychophragmus and Sinalliaria. Moreover, we report several losses and gains of RNA editing sites that occurred in plastomes of Brassiceae species during evolution.

High-throughput development of simple sequence repeat markers for genetic diversity research in Crambe abyssinica.

BACKGROUND: The allohexaploid Crambe abyssinica (crambe) is an oilseed crop that has been recognized for its potential value in the chemical industry, particularly in terms of producing high-erucic acid content vegetable oil. However, as an understudied crop, improvement of crambe has been hampered by the lack of genetic and genomic information to enhance its yield, oil quality and resistance against biotic and abiotic stress. Development of molecular markers is therefore of great significance to facilitate genetic improvement of crambe. RESULTS: In this study, high-throughput sequencing was performed to generate sequences for the transcriptome and genome of a widely planted crambe cultivar, Galactica. A total of 186,778 expressed sequence tag (EST) contigs as 8,130,350 genomic contigs were assembled as well. Altogether, 82,523 pairs of primers were designed in the flanking sequences of the simple sequence repeat (SSR) within these contigs. Virtual PCR analysis showed that a fraction of these primers could be mapped onto the genomes of related species of Brassica, including Brassica rapa, B. oleraceae and B. napus. Genetic diversity analysis using a subset of 166 markers on 30 independent C. abyssinica accessions exhibited that 1) 95 % of the designed SSRs were polymorphic among these accessions; 2) the polymorphism information content (PIC) value of the markers ranged from 0.13 to 0.89; 3) the genetic distances (coefficient NEI72) between accessions varied from 0.06 to 0.36. Cluster analysis subsequent on the accessions demonstrated consistency with crambe breeding history. F-statistics analysis revealed a moderate level of genetic differentiation in C. abyssinica (Gst = 0.3934) and a accordingly low estimated gene flow (Nm = 0.7709). CONCLUSION: Application of high-throughput sequencing technology has facilitated SSR marker development, which was successfully employed in evaluating genetic diversity of C. abyssinica as demonstrated in our study. Results showed these molecular markers were robust and provided powerful tools for assessing genetic diversity and estimating crambe breeding history. Moreover, the SSR primers and sequence information developed in the study are freely available to the research community.

Genetic stability of micropropagated plants of Crambe abyssinica Hochst using ISSR markers.

Crambe (Crambe abyssinica) is a non-edible annual herb, which was first cultivated to extract oil for industry, and now has great potential for biodiesel production. The objective of this investigation was to evaluate the genetic stability of micropropagated plants of the C. abyssinica Hochst cultivar 'FMS brilhante' using polymerase chain reaction techniques based on inter-simple sequence repeat (ISSR) molecular markers. The aim was to develop a protocol for the in vitro regeneration of these plants with low genetic variation as compared to the donor plant. For micropropagation, shoot tips from in vitro germinated seedlings were used as explants and were initially cultivated for 90 days on MS medium with 5.0 µM 6-benzylaminopurine (BAP), which at 90 days, led to the highest number of shoots per explant (NSE) (12.20 shoots) being detected. After 120 days, the interaction between BAP concentration and naphthalene acetic acid (NAA) was tested, and the highest NSE was observed following exposure to 0.0/0.5 µM BAP/NAA (11.40 shoots) and 1.0/0.0 µM BAP/NAA (11.00 shoots). The highest proportion of rooting phase were observed following exposure to 0.5 µM NAA (30%). The 13 ISSR primers used to analyze genetic stability produced 91 amplification products, of which only eight bands were polymorphic and 83 were monomorphic for all 10 regenerated crambe plants, compared to the donor plant explant. These results indicate that crambe shoot tips are a highly reliable explant that can be used to micropropagate genetically true-to-type plants or to maintain genetic stability, as verified using ISSR markers.

RNAi targeting putative genes in phosphatidylcholine turnover results in significant change in fatty acid composition in Crambe abyssinica seed oil.

The aim of this study was to evaluate the importance of three enzymes, LPCAT, PDCT and PDAT, involved in acyl turnover in phosphatidylcholine in order to explore the possibility of further increasing erucic acid (22:1) content in Crambe seed oil. The complete coding sequences of LPCAT1-1 and LPCAT1-2 encoding lysophosphatidylcholine acyltransferase (LPCAT), PDCT1 and PDCT2 encoding phosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT), and PDAT encoding phospholipid:diacylglycerol acyltransferase (PDAT) were cloned from developing Crambe seeds. The alignment of deduced amino acid sequences displayed a high similarity to the Arabidopsis homologs. Transgenic lines expressing RNA interference (RNAi) targeting either single or double genes showed significant changes in the fatty acid composition of seed oil. An increase in oleic acid (18:1) was observed, to varying degrees, in all of the transgenic lines, and a cumulative effect of increased 18:1 was shown in the LPCAT-PDCT double-gene RNAi. However, LPCAT single-gene RNAi led to a decrease in 22:1 accumulation, while PDCT or PDAT single-gene RNAi had no obvious effect on the level of 22:1. In agreement with the abovementioned oil phenotypes, the transcript levels of the target genes in these transgenic lines were generally reduced compared to wild-type levels. In this paper, we discuss the potential to further increase the 22:1 content in Crambe seed oil through downregulation of these genes in combination with fatty acid elongase and desaturases.

Detection of induced mutations in CaFAD2 genes by next-generation sequencing leading to the production of improved oil composition in Crambe abyssinica.

Crambe abyssinica is a hexaploid oil crop for industrial applications. An increase of erucic acid (C22:1) and reduction of polyunsaturated fatty acid (PUFA) contents in crambe oil is a valuable improvement. An increase in oleic acid (C18:1), a reduction in PUFA and possibly an increase in C22:1 can be obtained by down-regulating the expression of fatty acid desaturase2 genes (CaFAD2), which code for the enzyme that converts C18:1 into C18:2. We conducted EMS-mutagenesis in crambe, followed by Illumina sequencing, to screen mutations in three expressed CaFAD2 genes. Two novel analysis strategies were used to detect mutation sites. In the first strategy, mutation detection targeted specific sequence motifs. In the second strategy, every nucleotide position in a CaFAD2 fragment was tested for the presence of mutations. Seventeen novel mutations were detected in 1100 one-dimensional pools (11 000 individuals) in three expressed CaFAD2 genes, including non-sense mutations and mis-sense mutations in CaFAD2-C1, -C2 and -C3. The homozygous non-sense mutants for CaFAD2-C3 resulted in a 25% higher content of C18:1 and 25% lower content of PUFA compared to the wild type. The mis-sense mutations only led to small changes in oil composition. Concluding, targeted mutation detection using NGS in a polyploid was successfully applied and it was found that a non-sense mutation in even a single CaFAD2 gene can lead to changes in crambe oil composition. Stacking the mutations in different CaFAD2 may gain additional changes in C18:1 and PUFA contents.

Regeneration and transformation of Crambe abyssinica.

BACKGROUND: Crambe abyssinica (crambe) is a non-food oil seed crop. Its seed oil is widely used in the chemical industry because of the high erucic acid content. Furthermore, it is a potential platform for various feedstock oils for industrial uses based on genetic modification. Here, we describe the development of a series of protocols for all steps required in the process of generating genetically modified crambe. RESULTS: Different explant types from crambe seedlings were tested for shoot regeneration using different hormone-combinations. Cotyledonary nodes on basic medium with 0.5 µM NAA and 2.2 µM BAP gave the highest regeneration percentages. For propagation by tissue culture, explants of stems, petioles, leaves and axillary buds of in vitro plantlets were tested using the optimized medium. Axillary buds showed the highest shoot proliferation efficiency. Cotyledonary nodes were used to test the proper concentration of kanamycin for selection of transformation events, and 10 to 25 mg · L(-1) were identified as effective. The cotyledonary nodes and cotyledons from 7-day-old seedlings were used in Agrobacterium-mediated transformations with two kinds of selection strategies, shifting or consistent. Using the shifting selection method (10 mg · L(-1) kanamycin, 25 mg · L(-1), then back to 10 mg · L(-1)) cotyledonary nodes gave 10% transformation frequency, and cotyledons 4%, while with the consistent method (25 mg · L(-1)) lower frequencies were found, 1% for cotyledonary nodes and 0% for cotyledons). Later, in vitro plant axillary buds were tried as explants for transformation, however, transformation frequency was low ranging from 0.5 to 2%. Overall, testing six different vectors and two kinds of Agrobacterium strains, the average transformation frequency using the shifting method was 4.4%. Determining T-DNA insertion numbers by Southern blotting showed that approximately 50% of the transgenic lines had a single-copy insertion. CONCLUSIONS: Present research revealed the potential of using crambe meristematic tissue for genetic transformation and in vitro propagation. The most efficient method of transformation used cotyledonary node explants from 7-days-old seedlings with a shifting kanamycin selection. Meristematic tissues (cotyledonary node or axillary bud) had the highest ability for shoot proliferation. Single-copy T-DNA insert lines could be efficiently and reproducibly generated.

Production and genetic characterization of interspecific hybrids among Crambe abyssinica, Crambe hispanica and Crambe kralikii.

In this paper, interspecific crosses among Crambe abyssinica, Crambe hispanica, and Crambe kralikii were reported. In the C. hispanica x C. abyssinica (H x A) cross, 118 F1 hybrids were produced without embryo rescue, while 5 F1 hybrids were obtained with embryo rescue, when C. hispanica was used as the female parent. In the reciprocal cross (A x H), 232 hybrids were obtained without embryo rescue. From more than 1000 C. kralikii flowers pollinated with pollen grains of C. abyssinica (K x A), only 2 F1 hybrids were obtained with embryo rescue, whereas the reciprocal cross produced no hybrids, even with embryo rescue. The hybrids were confirmed at the morphological, cytological, and molecular levels. In the combinations of A x H and H x A, many BC1 hybrids were obtained without embryo rescue. In contrast, in the K x A cross, only 7 BC1 plants were obtained with embryo rescue, while no seed set was achieved under self-pollination or in backcrosses without embryo rescue. In the H x A F1 hybrids, the pollen stainability was 65.4-86.0%, with an average of 76.9%. In comparison, the pollen viability of hybrids in the reciprocal cross (A x H) ranged from 66.2 to 81.1%, with an average of 75.4%. Fertile pollen grains were not found in the K x A F1 hybrids. All F1 hybrids of the 3 crosses (H x A, A x H, and K x A) had the expected 2n = 75 chromosomes. AFLP analyses indicated that all F1 hybrids and their progenies had typical bands of the parents. These hybrids and progenies are anticipated to be valuable for future C. abyssinica improvement in breeding programs.

Bottlenecks in erucic acid accumulation in genetically engineered ultrahigh erucic acid Crambe abyssinica.

Erucic acid is a valuable industrial fatty acid with many applications. The main producers of this acid are today high erucic rapeseed (Brassica napus) and mustard (Brassica juncea), which have 45%-50% of erucic acid in their seed oils. Crambe abyssinica is an alternative promising producer of this acid as it has 55%-60% of erucic acid in its oil. Through genetic modification (GM) of three genes, we have previously increased the level of erucic acid to 71% (68 mol%) in Crambe seed oil. In this study, we further investigated different aspects of oil biosynthesis in the developing GM Crambe seeds in comparison with wild-type (Wt) Crambe, rapeseed and safflower (Carthamus tinctorius). We show that Crambe seeds have very low phosphatidylcholine-diacylglycerol interconversion, suggesting it to be the main reason why erucic acid is limited in the membrane lipids during oil biosynthesis. We further show that GM Crambe seeds have slower seed development than Wt, accompanied by slower oil accumulation during the first 20 days after flowering (DAF). Despite low accumulation of erucic acid during early stages of GM seed development, nearly 86 mol% of all fatty acids accumulated between 27 and 50 DAF was erucic acid, when 40% of the total oil is laid down. Likely bottlenecks in the accumulation of erucic acid during early stages of GM Crambe seed development are discussed.

Functional analysis of the omega-6 fatty acid desaturase (CaFAD2) gene family of the oil seed crop Crambe abyssinica.

BACKGROUND: Crambe abyssinica produces high erucic acid (C22:1, 55-60%) in the seed oil, which can be further increased by reduction of polyunsaturated fatty acid (PUFA) levels. The omega-6 fatty acid desaturase enzyme (FAD2) is known to be involved in PUFA biosynthesis. In crambe, three CaFAD2 genes, CaFAD2-C1, CaFAD2-C2 and CaFAD2-C3 are expressed. RESULTS: The individual effect of each CaFAD2 gene on oil composition was investigated through studying transgenic lines (CaFAD2-RNAi) for differential expression levels in relation to the composition of seed-oil. Six first generation transgenic plants (T1) showed C18:1 increase (by 6% to 10.5%) and PUFA reduction (by 8.6% to 10.2%). The silencing effect in these T1-plants ranged from the moderate silencing (40% to 50% reduction) of all three CaFAD2 genes to strong silencing (95% reduction) of CaFAD2-C3 alone. The progeny of two T1-plants (WG4-4 and WG19-6) was further analysed. Four or five transgene insertions are characterized in the progeny (T2) of WG19-6 in contrast to a single insertion in the T2 progeny of WG4-4. For the individual T2-plants of both families (WG19-6 and WG4-4), seed-specific silencing of CaFAD2-C1 and CaFAD2-C2 was observed in several individual T2-plants but, on average in both families, the level of silencing of these genes was not significant. A significant reduction in expression level (P < 0.01) in both families was only observed for CaFAD2-C3 together with significantly different C18:1 and PUFA levels in oil. CONCLUSIONS: CaFAD2-C3 expression is highly correlated to levels of C18:1 (r = -0.78) and PUFA (r = 0.75), which suggests that CaFAD2-C3 is the most important one for changing the oil composition of crambe.

Development of ultra-high erucic acid oil in the industrial oil crop Crambe abyssinica.

Erucic acid (22 : 1) is a major feedstock for the oleochemical industry. In this study, a gene stacking strategy was employed to develop transgenic Crambe abyssinica lines with increased 22 : 1 levels. Through integration of the LdLPAAT, BnFAE1 and CaFAD2-RNAi genes into the crambe genome, confirmed by Southern blot and qRT-PCR, the average levels of 18 : 1, 18 : 2 and 18 : 3 were markedly decreased and that of 22 : 1 was increased from 60% in the wild type to 73% in the best transgenic line of T4 generation. In single seeds of the same line, the 22 : 1 level could reach 76.9%, an increase of 28.0% over the wild type. The trierucin amount was positively correlated to 22 : 1 in the transgenic lines. Unlike high erucic rapeseed, the wild-type crambe contains 22 : 1 in the seed phosphatidylcholine and in the sn-2 position of triacylglycerols (5% and 8%, respectively). The transgenic line with high 22 : 1 had decreased 22 : 1 level in phosphatidylcholine, and this was negatively correlated with the 22 : 1 level at the sn-2 position of TAG. The significances of this study include (i) achieving an unprecedented level of 22 : 1 in an oil crop; (ii) disclosing mechanisms in the channelling of a triacylglycerol-specific unusual fatty acid in oil seeds; (iii) indicating potential limiting factors involved in the erucic acid biosynthesis and paving the way for further increase of this acid and (iv) development of an added value genetically modified oil crop having no risk of gene flow into feed and food crops.

Identifying genes and gene networks involved in chromium metabolism and detoxification in Crambe abyssinica.

Chromium pollution is a serious environmental problem with few cost-effective remediation strategies available. Crambe abyssinica (a member of Brassicaseae), a non-food, fast growing high biomass crop, is an ideal candidate for phytoremediation of heavy metals contaminated soils. The present study used a PCR-Select Suppression Subtraction Hybridization approach in C. abyssinica to isolate differentially expressed genes in response to Cr exposure. A total of 72 differentially expressed subtracted cDNAs were sequenced and found to represent 43 genes. The subtracted cDNAs suggest that Cr stress significantly affects pathways related to stress/defense, ion transporters, sulfur assimilation, cell signaling, protein degradation, photosynthesis and cell metabolism. The regulation of these genes in response to Cr exposure was further confirmed by semi-quantitative RT-PCR. Characterization of these differentially expressed genes may enable the engineering of non-food, high-biomass plants, including C. abyssinica, for phytoremediation of Cr-contaminated soils and sediments.

Expression profiling of Crambe abyssinica under arsenate stress identifies genes and gene networks involved in arsenic metabolism and detoxification.

BACKGROUND: Arsenic contamination is widespread throughout the world and this toxic metalloid is known to cause cancers of organs such as liver, kidney, skin, and lung in human. In spite of a recent surge in arsenic related studies, we are still far from a comprehensive understanding of arsenic uptake, detoxification, and sequestration in plants. Crambe abyssinica, commonly known as 'abyssinian mustard', is a non-food, high biomass oil seed crop that is naturally tolerant to heavy metals. Moreover, it accumulates significantly higher levels of arsenic as compared to other species of the Brassicaceae family. Thus, C. abyssinica has great potential to be utilized as an ideal inedible crop for phytoremediation of heavy metals and metalloids. However, the mechanism of arsenic metabolism in higher plants, including C. abyssinica, remains elusive. RESULTS: To identify the differentially expressed transcripts and the pathways involved in arsenic metabolism and detoxification, C. abyssinica plants were subjected to arsenate stress and a PCR-Select Suppression Subtraction Hybridization (SSH) approach was employed. A total of 105 differentially expressed subtracted cDNAs were sequenced which were found to represent 38 genes. Those genes encode proteins functioning as antioxidants, metal transporters, reductases, enzymes involved in the protein degradation pathway, and several novel uncharacterized proteins. The transcripts corresponding to the subtracted cDNAs showed strong upregulation by arsenate stress as confirmed by the semi-quantitative RT-PCR. CONCLUSIONS: Our study revealed novel insights into the plant defense mechanisms and the regulation of genes and gene networks in response to arsenate toxicity. The differential expression of transcripts encoding glutathione-S-transferases, antioxidants, sulfur metabolism, heat-shock proteins, metal transporters, and enzymes in the ubiquitination pathway of protein degradation as well as several unknown novel proteins serve as molecular evidence for the physiological responses to arsenate stress in plants. Additionally, many of these cDNA clones showing strong upregulation due to arsenate stress could be used as valuable markers. Further characterization of these differentially expressed genes would be useful to develop novel strategies for efficient phytoremediation as well as for engineering arsenic tolerant crops with reduced arsenic translocation to the edible parts of plants.

Cloning and functional characterization of the fatty acid elongase 1 (FAE1) gene from high erucic Crambe abyssinica cv. Prophet.

A genomic fatty acid elongation 1 (FAE1) clone was isolated from Crambe abyssinica. The genomic clone corresponds to a 1521-bp open reading frame, which encodes a protein of 507 amino acids. In yeast cells expression of CrFAE led to production of new very long chain monounsaturated fatty acids such as eicosenoic (20:1(delta11)) and erucic (22:1(delta13)) acids. Seed-specific expression in Arabidopsis thaliana resulted in up to a 12-fold increase in the proportion of erucic acid. On the other hand, in transgenic high-erucic Brassica carinata plants, the proportion of erucic acid was as high as 51.9% in the best transgenic line, a net increase of 40% compared to wild type. These results indicate that the CrFAE gene encodes a condensing enzyme involved in the biosynthesis of very long-chain fatty acids utilizing monounsaturated and saturated acyl substrates, with a strong capability for improving the erucic acid content.

[Cytological observation on intergeneric hybrid between Brassica. Chinensis and Crambe abyssinica].

The intergeneric hybrid from a cross between Brassica. Chinensis and Crambe abyssinica was observed with 2n=55 chromosomes in the original progenies. After several generations of in-vitro propagation by tissue culture, the chromosomes of the intergeneric hybrid were remarkably reduced, varying from 25 to 28, averaged at 26.In meiosis of the PMC of the hybrid, the average configuration of chromosome pairing was 0.06 III + 11.26 II + 3.80 I. The number of bivalents varied from 8 to 13. The majority of PMC cells showed 10 II, 11 II and 12 II bivalents with frequencies of 24.58%, 23.91% and 30.98% respectively. The number of univalents varied from 0 to 8. The reduction of chromosomes in the hybrid and the high numbers of bivalents were possibly due to the chromosome of Crambe abyssinica eliminating and the genome of Brassica. Chinensis doubling in the hybrid cells. Triade cells, chromosome lagging, and chromosome bridges were observed in anaphase II.

GISH analysis of disomic Brassica napus-Crambe abyssinica chromosome addition lines produced by microspore culture from monosomic addition lines.

Two Brassica napus-Crambe abyssinica monosomic addition lines (2n=39, AACC plus a single chromosome from C. abyssinca) were obtained from the F(2) progeny of the asymmetric somatic hybrid. The alien chromosome from C. abyssinca in the addition line was clearly distinguished by genomic in situ hybridization (GISH). Twenty-seven microspore-derived plants from the addition lines were obtained. Fourteen seedlings were determined to be diploid plants (2n=38) arising from spontaneous chromosome doubling, while 13 seedlings were confirmed as haploid plants. Doubled haploid plants produced after treatment with colchicine and two disomic chromosome addition lines (2n=40, AACC plus a single pair of homologous chromosomes from C. abyssinca) could again be identified by GISH analysis. The lines are potentially useful for molecular genetic analysis of novel C. abyssinica genes or alleles contributing to traits relevant for oilseed rape (B. napus) breeding.

Cytogenetic characterization and fae1 gene variation in progenies from asymmetric somatic hybrids between Brassica napus and Crambe abyssinica.

Sexual progenies of asymmetric somatic hybrids between Brassica napus and Crambe abyssinica were analyzed with respect to chromosomal behavior, fae1 gene introgression, fertility, and fatty-acid composition of the seed. Among 24 progeny plants investigated, 11 plants had 38 chromosomes and were characterized by the occurrence of normal meiosis with 19 bivalents. The other 13 plants had more than 38 chromosomes, constituting a complete chromosomal set from B. napus plus different numbers of additional chromosomes from C. abyssinica. The chromosomes of B. napus and C. abyssinica origin could be clearly discriminated by genomic in situ hybridization (GISH) in mitotic and meiotic cells. Furthermore, meiotic GISH enabled identification of intergenomic chromatin bridges and of asynchrony between the B. napus and C. abyssinca meiotic cycles. Lagging, bridging and late disjunction of univalents derived from C. abyssinica were observed. Analysis of cleaved amplified polymorphic sequence (CAPS) markers derived from the fae1 gene showed novel patterns different from the B. napus recipient in some hybrid offspring. Most of the progeny plants had a high pollen fertility and seed set, and some contained significantly greater amounts of seed erucic acid than the B. napus parent. This study demonstrates that a part of the C. abyssinica genome can be transferred into B. napus via asymmetric hybridization and maintained in sexual progenies of the hybrids. Furthermore, it confirms that UV irradiation improves the fertility of the hybrid and of its sexual progeny via chromosomal elimination and facilitates the introgression of exotic genetic material into crop species.

Development of rapeseed with high erucic acid content by asymmetric somatic hybridization between Brassica napus and Crambe abyssinica.

PEG-induced asymmetric somatic hybridization between Brassica napus and Crambe abyssinica was carried out. C. abyssinica is an annual cruciferous oil crop with a high content of erucic acid in the seed oil valuable for technical purposes. UV-irradiated mesophyll protoplasts of C. abyssinica cv 'Carmen' and cv 'Galactica' were fused with hypocotyl protoplasts of different genotypes of B. napus cv 'Maplus' and breeding line '11502'. Shoot regeneration frequency varied between 6.1% and 20.8% among the different doses of UV-irradiation, ranging from 0.05 J/cm(2) to 0.30 J/cm(2). In total, 124 shoots were regenerated, of which 20 asymmetric somatic hybrids were obtained and verified by nuclear DNA content and AFLP analysis. AFLP data showed that some of the characteristic bands from C. abyssinica were present in the hybrids. Cytological analysis of these hybrids showed that 9 out of 20 asymmetric hybrids had 38 chromosomes, the others contained 40-78 chromosomes, having additional chromosomes between 2 and 40 beyond the 38 expected for B. napus. The investigation into the fertility of asymmetric somatic hybrids indicated that the fertility increased with increasing UV-doses ranging from 0.05 J/cm(2) to 0.15 J/cm(2). All of the hybrids were cultured to full maturity, and could be fertilized and set seeds after self-pollination or backcrosses with B. napus. An analysis of fatty acid composition in the seeds was conducted and found to contain significantly greater amounts of erucic acid than B. napus. This study indicates that UV-irradiation could be used as a tool to produce asymmetric somatic hybrids and to promote the fertility of the hybrids.