Fatty acid composition of resynthesized Brassica napus and trigenomic Brassica void of genes for erucic acid in their A genomes

The fatty acid composition of seed oil of four interspecific hybrids, resulting from crosses between zero erucic acid Brassica rapa (AA), and high erucic acid Brassica alboglabra/Brassica oleracea (CC) and Brassica carinata (BBCC), void of erucic acid genes in their A‐genomes was examined. The erucic acid content in resynthesized Brassica napus (AACC) lines derived from these crosses was only about half that of the high erucic acid CC genome parents, indicating equal contributions of the two genomes to oil (fatty acid) synthesis and accumulation. The differences in C18 fatty acid synthesis between the parents were also evident in the resulting resynthesized B. napus plants. Hexaploid Brassica plants of the genomic constitution AABBCC, in which the AA genome was incapable of erucic acid synthesis, had lower erucic acid contents than the B. carinata (BBCC) parent. This is plausible considering the fact that the zero erucic acid AA genome contributes to oil synthesis in AABBCC plants, thus reducing erucic acid content.     

Development of yellow-seeded Brassica napus of double low quality

Two yellow‐seeded white‐petalled Brassica napus F₇ inbred lines, developed from interspecific crosses, containing 26–28% emcic acid and more than 40 μmol glucosinolates (GLS)/g seed were crossed with two black/dark brown seeded B. napus varieties of double low quality and 287 doubled haploid (DH) lines were produced. The segregation in the DH lines indicated that three to four gene loci are involved in the determination of seed colour, and yellow seeds are formed when all alleles in all loci are in the homozygous recessive state. A dominant gene governed white petal colour and is linked with an erucic acid allele that, in the homozygous condition, produces 26–28% erucic acid. Four gene loci are involved in the control of total GLS content where low GLS was due to the presence of recessive alleles in the homozygous condition in all loci. From the DH breeding population a yellow‐seeded, yellow‐petalled, zero erucic acid line was obtained. This line was further crossed with conventional B. napus varieties of double low quality and, following pedigree selection, a yellow seeded B. napus of double low quality was obtained. The yellow seeds had higher oil plus protein content and lower fibre content than black seeds. A reduction of the concentration of chromogenic substances was found in the transparent seed coat of the yellow‐seeded B. napus.     

Glucosinolate content in interspecific crosses of Brassica carinata with B. juncea and B. napus

Brassica carinata A. Braun is a highly productive oilseed crop in the Ethiopian highlands, but the seed has a high 2-propenyl glucosinolate content, which is undesirable. The objective of this study was to introgress, through interspecific crosses, genes for low 2-propenyl glucosinolate content from the B genome of B. juncea and C genome of B. napus into the B. carinata B and C genomes and thus develop low glucosinolate B. carinata. The cross [(B. carinata×B. juncea) ×B. carinata] yielded plants that contained only ～ 20 μmoles of 2-propenyl glucosinolate, which was an 85% reduction compared with levels in B. carinata seed. Plants of the [(B. carinata×B. napus) ×B. carinata] cross had normal high concentrations of 2-propenyl glucosinolate. Backcross plants of both interspecific crosses also contained 3-butenyl and 2-hydroxy-3-butenyl glucosinolates. The results of these crosses suggested that genes for glucosinolate synthesis were located on B genome chromosomes of B. carinata because B. napus C genome introgressions did not result in reductions of total glucosinolate contents. The total alkenyl glucosinolate content of one F₃ family of the B. juncea backcross was similar to that of the B. juncea parent. It was concluded that through further selection in this family, B. carinata plants could be identified that would be basically free of 2-propenyl glucosinolate, and have a low total alkenyl glucosinolate content.     

Alloplasmic effects of <Emphasis Type="Italic">Brassica napus</Emphasis> and <Emphasis Type="Italic">B. juncea</Emphasis> on seed characteristics of <Emphasis Type="Italic">B. carinata</Emphasis>

Effects of Brassica napus (N) and B. juncea (J) cytoplasm on seed characteristics of B. carinata (C) were examined. Alloplasmic lines of B. carinata were produced from N × C and J × C hybrids by recurrent backcrossing to the BC₈ generation. Fourteen sets of reciprocal crosses were used. Compared with their euplasmic sibs, alloplasmic B. carinata line seeds with B. napus cytoplasm showed reduced dormancy, higher seed weight, lower germination rate at high temperatures, higher germination rate at low temperatures, and had lower erucic acid and higher linoleic acid contents. Alloplasmic B. carinata line seeds with B. juncea cytoplasm had higher seed weight but lower germination rate than their corresponding euplasmic sibs. These results showed a cytoplasmic effect on seed development, and an influence on seed weight, dormancy, and fatty acid composition. B. carinata was more deleteriously affected by cytoplasm from B. napus than by cytoplasm of B. juncea.     

Doubled haploid lines of Brassica carinata with modified erucic acid content through mutagenesis by EMS treatment of isolated microspores

Brassica carinata is a potential oilseed crop for the Mediterranean area. Chemical mutagenesis has been applied to microspores of B. carinata with the purpose of identifying lines with altered erucic acid content. From a population of nearly 400 doubled haploid plants recovered, nine lines have been identified that exhibit promising useful changes in erucic acid concentration in the seed oil. Three lines showed erucic acid contents below 25%, with a minimum of 17.1%, and in six lines the level of this fatty acid was greater than 52%. Changes in other fatty acids are also described and discussed.     

Inheritance of seed colour in Brassica campestris L. and breeding for yellow-seeded B. napus L.

Summary Seed colour inheritance was studied in five yellow-seeded and one black-seeded B. campestris accessions. Diallel crosses between the yellow-seeded types indicated that the four var. yellow sarson accessions of Indian origin had the same genotype for seed colour but were different from the Swedish yellow-seeded breeding line. Black seed colour was dominant over yellow. The segregation patterns for seed colour in F₂ (Including reciprocals) and BC₁ (backcross of F₁ to the yellow-seeded parent) indicated that the black seed colour was conditioned by a single dominant gene. Seed colour was mainly controlled by the maternal genotype but influenced by the interplay between the maternal and endosperm and/or embryonic genotypes. For developing yellow-seeded B. napus genotypes, resynthesized B. napus lines containing genes for yellow seed (Chen et al., 1988) were crossed with B. napus of yellow/brown seeds, or with yellow-seeded B. carinata. Yellow-seeded F₂ plants were found in the crosses that involved the B. napus breeding line. However, this yellow-seeded character did not breed true up to F₄. Crosses between a yellow-seeded F₃ plant and a monogenomically controlled black-seeded B. napus line of resynthesized origin revealed that the black-seeded trait in the B. alboglabra genome was possibly governed by two independently dominant genes with duplicated effect. Crossability between the resynthesized B. napus lines as female and B. carinata as male was fairly high. The sterility of the F₁ plants prevented further breeding progress for developing yellow-seeded B. napus by this strategy.     

Development of Yellow Seeded Brassica napus Through Interspecific Crosses

Yellow seeded Brassica napus was developed through interspecific crosses with the two mustard species, B. juncea and B. carinata. The objective of these two interspecific crosses was the introgression of genes for yellow seed colour from the A genome of B. juncea and C genome of B. carinata into the A and C genomes of B. napus, respectively. The interspecific F₁ generations were backcrossed to B. napus in an attempt to eliminate B genome chromosomes and to improve fertility. Backcross F₂ plants of the (B. napus×B. juncea) ×B. napus cross were then crossed with backcross F₂ plants of the (B. napus×B. carinata) ×B. napus cross. The objective of this intercrossing was to combine the A and C genome yellow seeded characteristics of the two backcross populations into one genotype. The F₂ generation of the backcross F₂ intercrosses was grown in the field, plants were individually harvested and visually rated for seed colour. Ninety-one yellow seeded plants were identified among the 4858 plants inspected. This result indicated that the interspecific crossing scheme was successful in developing yellow seeded B. napus.     

Genomic in situ hybridization analysis of Brassica napus × Orychophragmus violaceus hybrids and production of B. napus aneuploids

To further utilize the valuable germplasm Orychophragmus violaceus for Brassica genetics and breeding, a B. napus × O. violaceus cross was repeated with embryo rescue. All F₁ plants except one B. napus haploid were mixoploids (2n = 17–39 in ovaries) with 2n = 31, 37, 38 and 39 as the maximal chromosome numbers in individuals, but the higher numbers mostly appeared in pollen mother cells (PMCs) with a preponderance of 2n = 30, 37 and 38. Only one chromosome and one chromosome segment of O. violaceus were detected at a low frequency in some ovary cells and PMCs with 2n = 37, 38 and 39 as determined by genomic in situ hybridization analysis. The fatty acid profiles of seeds from the majority of the F₁ and F₂ plants were similar to those of female B. napus cv. ‘Oro’, but some were obviously different in the percentages of oleic, linoleic and erucic acids, and some F₂ plants (2n = 38) with good seed set had high percentages of oleic (>70.0%) or linoleic (to 38.3%) acids and low erucic acid (<1%). Subsequently, many kinds of B. napus aneuploids (2n = 28, 30, 34, 36, 37, 39 and 42), without O. violaceus chromosomes, were derived from F₂ progeny and microspores of partial F₁ plants. Finally, the cytological mechanisms behind the variations in chromosome numbers were discussed together with the implications of these aneuploids for Brassica genome research and of the plants with altered fatty acid profiles for improving the oil quality of B. napus.     

Independent Inheritance of Erucic Acid Content and Flower Colour in the C-Genome of Brassica napus L.

The synthetic Brassica napus L. line No7076 was obtained from a cross between yellow-flowered and zero-erucic turnip rape (B. campestris) Sv85-38301 and white-flowered and high-erucic (41.4%) B. oleracea ssp. alboglabra No6510. This synthetic B. napus is pale-flowered and has an average erucic acid content of 25.8 %. It was crossed with the yellow-flowered and zero-erucic B. napus line SvS4-2S053 and segregation of the erucic acid content and flower colour was studied in F₁ and F₂ generations. The high erucic acid content was controlled by a single gene in the C-genome and was additively inherited. Strong evidence was obtained in support of independent segregation of the erucic-arid content and the flower colour characters controlled by the C-genome of B. napus.     

Production of yellow-seeded Brassica napus through interspecific crosses

Yellow‐seeded Brassica napus was developed from interspecific crosses between yellow‐seeded Brassica rapa var.‘yellow sarson’ (AA), black‐seeded Brassica alboglabra (CC), yellow‐seeded Brassica carinata (Bbcc) and black‐seeded B. napus (AACC). Three different interspecific crossing approaches were undertaken. Approaches 1 and 2 were designed directly to develop yellow‐seeded B. napus while approach 3 was designed to produce a yellow‐seeded CC genome species. Approaches 1 and 2 differed in the steps taken after trigenomic interspecific hybrids (ABC) were generated from B. carinata×B. rapa crosses. The aim of approach 1 was to transfer the yellow seed colour genes from the A to the C genome as an intermediate step in developing yellow‐seeded B. napus. For this purpose, the ABC hybrids were crossed with black‐seeded B. napus and the three‐way interspecific hybrids were self‐pollinated for a number of generations. The F₇ generation resulted in the yellowish‐brown‐seeded B. napus line, No. 06. Crossing this line with the B. napus line No. 01, resynthesized from a black‐seeded B. alboglabra x B. rapa var.‘yellow sarson’ cross (containing the yellow seed colour genes in its AA genome), yielded yellow‐seeded B. napus. This result indicated that the yellow seed colour genes were transferred from the A to the C genome in the yellowish‐brown seed colour line No. 06. In approach 2, trigenomic diploids (AABBCC) were generated from the above‐mentioned trigenomic haploids (ABC). The seed colour of the trigenomic diploid was brown, in contrast to the yellow seed colour of the parental species. Trigenomic diploids were crossed with the resynthesized B. napus line No. 01 to eliminate the B genome chromosomes, and to develop yellow‐seeded B. napus with the AA genome of ‘yellow sarson’ and the CC genome of B. carinata with yellow seed colour genes. This interspecific cross failed to generate any yellow‐seeded B. napus. Approach 3 was to develop yellow‐seeded CC genome species from B. alboglabra×B. carinata crosses. It was possible to obtain a yellowish‐brown seeded B. alboglabra, but crossing this B. alboglabra with B. rapa var.‘yellow sarson’ failed to produce yellow seed in the resynthesized B. napus. The results of approaches 2 and 3 demonstrated that yellow‐seeded B. napus cannot be developed by combining the yellow seed colour genes of the CC genome of yellow‐seeded B. carinata and the AA genome of ‘yellow sarson’.     

Interspecific hybridization between Abelia × grandiflora ‘Francis Mason’ and A. schumannii via ovule culture

Ethiopian mustard (Brassica carinata Braun) is a potential oil crop for the Mediterranean area. The objective of this study was to develop an efficient system of mutagenesis using ultraviolet (UV) light irradiation of isolated microspores from Brassica carinata. From the survival curve based on embryo yield after irradiation of the microspores with UV light, the LD50 was estimated to be an exposure of 8 min. Total content of glucosinolates and fatty acid composition were analysed in the seeds of the doubled haploid homozygous plants with the purpose of selecting lines with modified glucosinolate and erucic acid contents. Three groups of doubled haploid lines exhibiting low and high glucosinolate contents, and high erucic acid content have been identified from a population of 270 doubled haploid lines. In eight lines, the content of glucosinolates was reduced from an average of 80.6 mol g^-1 seed to 37.5 mol g^-1 seed, whereas in four lines, the content of glucosinolates was increased up to 99.2 mol g^-1 seed. In six additional lines, the content of erucic acid was increased from 42.8% in the nontreated lines to 49.5% of the totalfatty acid composition in some of the mutant lines. All lines showed stablelevels of erucic acid in two generations, the M₂ and M₃.     

Erucic acid heredity in Brassica juncea - some additional information

Genetic studies were undertaken to reassess erucic acid heredity in Brassica juncea. Analysis of segregation in F₂ and BC₁ generations from two zero × high erucic acid crosses indicated that higher erucic acid in B. juncea was controlled by two dominant genes with additive effects, whereas segregation in a cross involving ‘CCWF 16′, a genotype having intermediate erucic acid (25.6%), and a zero erucic acid strain, indicated monogenic dominant control for intermediate erucic acid content. The B. juncea strain ‘CCWF 16’ was developed by hybridizing high‐erucic acid B. juncea cv.‘WF‐1’ with a ‘0’ erucic B. rapa cv.‘Candle’ followed by backcrossing with ‘WF‐1’ and half‐seed selection for low erucic acid in each backcross generation. This strategy resulted in substitution of the high erucic acid allele present in the A genome of B. juncea (AABB) by the zero erucic acid allele associated with ‘A’ genome of ‘Candle’. The intermediate erucic acid content in ‘CCWF 16’ was thus attributed to a gene present in the ‘BB’ genome. Experimental data clearly suggested that the gene (E₂) associated with the A genome had a greater contribution to the total erucic acid content in B. juncea than the gene (E₁) located on the B genome. This provided experimental evidence for a previous suggestion of unequal contributions of two dominant genes (E₁= 12%, E₂= 20%) to high erucic acid content in conventional digenomic Brassica species.     

A comparison of traditional and haploid-derived breeding populations of oilseed rape (Brassica napus L.) for fatty acid composition of the seed oil

Summary Microspore embryogenesis technology allows plant breeders to efficiently generate homozygous micros-pore-derived breeding populations of oilseed rape (Brassica napus L.) without traditional generations of inbreeding. This study was conducted to compare the frequency distribution of microspore-derived population and single seed descent populations with respect to fatty acids of seed oil. Both microspore-derived populations and single seed descent populations were produced from each of three crosses made between selected parents containing contrasting amount of erucic, oleic, linoleic and linolenic acids. The fatty acid content of F₃ plants derived lines (F₅ seed) developed by single seed descent was compared to that of microspore-derived populations. The means, ranges and distribution pattern of seed fatty acid contents were similar in both populations for each fatty acid studied, although a few heterozygous lines were observed in the single seed descent populations. The results indicated that microspore-derived population form random, homozygous F₁ plant derived gametic arrays for all fatty acids evaluated. Selection for altered fatty acid composition in microspore-derived and single seed descent homozygous populations should be equally efficient, in the absence of linkage of traits investigated.     

Interspecific transfer of Brassica juncea-type high blackleg resistance to Brassica napus

Summary Complete resistance to Leptosphaeria maculans, the cause of blackleg of oilseed rape (Brassica napus), was transferred from B. juncea to B. napus through an interspecific cross. B. juncea-type complete resistance (JR) was recognized first in one F₃ progeny (Onap^JR) by the absence of leaf-lesions on seedlings and canker-free adult plants. The commercially important characters of B. napus were retained in advanced lines of Onap^JR, which combined JR with low erucic acid levels (<0.5%), high seed yield and variable maturity dates.JR appeared to be inherited as a major gene or genes. Segregation for resistance and susceptibility contintied to occur during later generations of selection of Onap^JR. JR was readily transferred from Onap^JR to other suitable B. napus cultivars or lines with partial resistance to blackleg and resulted in highly vigorous carly generation selections adapted to cold, wet situations along with complete resistance to blackleg.     

Inheritance of reduced linolenic acid content in the Ethiopian mustard mutant N2-4961

The zero erucic acid Ethiopian mustard lines developed so far are characterized by an exceptionally high linolenic acid content in the seed oil. The mutant line N2‐4961, expressing low linolenic acid content in a high erucic acid background, was developed through chemical mutagenesis. The objective of this research was to study the inheritance of low linolenic acid content in this mutant. Line N2‐4961 was reciprocally crossed with its parent line C‐101 and the linolenic acid content of the reciprocal F₁, F₂ and BC₁ generations was studied. No maternal, cytoplasmic or dominance effects were detected in the analysis of F₁ seeds and F₁ plants from reciprocal crosses. Linolenic acid content segregated in 1: 2: 1 ratios in all the F₂ populations studied, suggesting monogenic inheritance. This was confirmed with the analysis of the reciprocal backcross generation. The simple inheritance of low linolenic acid content in N2‐4961 will facilitate the transference of this trait to zero erucic acid lines of Ethiopian mustard.     

Isolation of induced mutants in Ethiopian mustard (Brassica carinata Braun) with low levels of erucic acid

Ethiopian mustard (Brassica carinata Braun) is a potential oil crop for the rain-fed Mediterranean area. However, its usage is limited by the high erucic and high glucosinolate content of the oil and meal, respectively. In the course of a mutagenesis programme, an agronomically good line of Ethiopian mustard was treated with EMS in order to widen the natural variability of nutritional traits in this species. As a result of this programme several low erucic mutants were isolated; two of these mutants showed erucic acid values in the M4 generation in the range 5–10% of total fatty acids. Near-infrared reflectance spectroscopy (N1RS) was successfully applied as a rapid screening method for erucic acid in this breeding programme.     

Marker-assisted increase of genetic diversity in a double-low seed quality winter oilseed rape genetic background

Generation of novel genetic diversity for maximization of heterosis in hybrid production is a significant goal in winter oilseed rape breeding. Here, we demonstrate that doubled haploid (DH) production using microspore cultivation can simultaneously introgress favourable alleles for double‐low seed quality (low erucic acid and low‐glucosinolate content) into a genetically diverse Brassica napus genetic background. The DH lines were derived from a cross between a double‐low quality winter rapeseed variety and a genetically diverse semisynthetic B. napus line with high erucic acid and high glucosinolates (++ quality). Twenty‐three low‐glucosinolate lines were identified with a genome component of 50–67% derived from the ++ parent. Four of these lines, with a genome component of 50–55% derived from the ++ parent, also contained low erucic acid. Heterosis for seed yield was confirmed in test‐crosses using these genetically diverse lines as pollinator. The results demonstrate the potential of marker‐assisted identification of novel genetic pools for breeding of double‐low quality winter oilseed rape hybrids.     

Broadening the genetic base of <Emphasis Type="Italic">Brassica napus</Emphasis> canola by interspecific crosses with different variants of <Emphasis Type="Italic">B. oleracea</Emphasis>

Broadening the genetic base of the C genome of Brassica napus canola by use of B. oleracea is important. In this study, the prospect of developing B. napus canola lines from B. napus?×?B. oleracea var. alboglabra, botrytis, italica and capitata crosses and the effect of backcrossing the F₁’s to B. napus were investigated. The efficiency of the production of the F₁’s varied depending on the B. oleracea variant used in the cross. Fertility of the F₁ plants was low—produced, on average, about 0.7 F₂ seeds per self-pollination and similar number of BC₁ seeds on backcrossing to B. napus. The F₃ population showed greater fertility than the BC₁F₂; however, this difference diminished with the advancement of generation. The advanced generation populations, whether derived from F₂ or BC₁, showed similar fertility and produced similar size silique with similar number of seeds per silique. Progeny of all F₁’s and BC₁’s stabilized into B. napus, although B. oleracea plant was expected, especially in the progeny of F₁ (ACC) owing to elimination of the A chromosomes during meiosis. Segregation distortion for erucic acid alleles occurred in both F₂ and BC₁ resulting significantly fewer zero-erucic plants than expected; however, plants with?≤?15% erucic acid frequently yielded zero-erucic progeny. No consistent correlation between parent and progeny generation was found for seed glucosinolate content; however, selection for this trait was effective and B. napus canola lines were obtained from all crosses. Silique length showed positive correlation with seed set; the advanced generation populations, whether derived from F₂ or BC₁, were similar for these traits. SSR marker analysis showed that genetically diverse canola lines can be developed by using different variants of B. oleracea in B. napus?×?B. oleracea interspecific crosses.     

Impacts of erucic acid and glucosinolate content on genetic relationships between protein content and fatty acids of rape seed across environments

Impacts of erucic acid content (EAC) and glucosinolate content (GSLC) on the genetic correlations between protein content (PC) and oil content (OC) or PC and fatty acid contents (FAC) in rape seed (Brassica napus L.) was analyzed by using unconditional and conditional methods related to genetic effects from the diploid embryo nuclear genes, cytoplasm genes and diploid maternal plant nuclear genes. A diallel mating design in two environments was conducted by using eight varieties along with their F₁ and F₂. It was found that there were significant relationships between PC and EAC or PC and GSLC of rape seed, and the conditional analysis method could be used to exclude the influences of EAC or GSLC for further revealing the actual genetic relationships between PC and OC or PC and FAC. The results from conditional analysis showed that when PC was conditioned on EAC or GSLC the conditional phenotypic and genotypic relationships between PC|EAC and oleic acid content or PC|GSLC and OC were changed to significantly positive, while those between PC|EAC and eicosenoic acid content or PC|GSLC and linolenic acid content became significantly negative. Thus, the levels of EAC and GSLC of rape seed could affect the correlations between PC and OC or PC and FAC. For the conditional genetic relationship analysis of different genetic systems, visible changes were found for many genetic correlation components from the embryo, cytoplasm and maternal plant between PC and OC or PC and FAC after eliminating the influences of EAC or GSLC, especially for conditional embryo dominance, cytoplasmic, maternal additive main covariances and conditional embryo dominance interaction covariance.     

Evaluation of microspore-derived embryos as models for studying lipid biosynthesis in seed of rapessed (Brassica napus L.)

Summary Microspore culture of rapeseed (Brassica napus L.) has provided a powerful tool not only for breeding but also in developmental studies. In this study, microspore-derived embryos (MDE) of B. napus were evaluated as a model in seed for studying accumulations of triacylglyceride (TAG) fatty acids in both a low and high erucic acid rapeseed line; and accumulations of TAG and free fatty acids (FFA) in a high erucic acid rapessed line. The accumulation patterns confirmed that MDE had a similar TAG fatty acid profile to seed during the embryo development within each genotype. The oil accumulation in MDE after 36 days in culture (DIC) approached levels similar to those in zygotic seed 25 days after flowering (DAF). Significant differences were detected in contents of both total free fatty acids and specific free fatty acids between MDE and seed. During the developmental period, total free fatty acids changed from 16% to 2.1% in MDE, but from 10.5% to 0.1% in seed. MDE had much higher percentage of free linolenic and erucic acids than seed, particularly during the late developmental stages. The current study indicated that MDE can be used as a model to study TAG and TAG fatty acids in seed but caution must be taken to study free fatty acid metabolism.