Transfer of Brassica tournefartii (TT) genes to allotetraploid oilseed Brassica species (B. juncea AABB, B. napus AACC, B. carinata BBCC): homoeologous pairing is more pronounced in the three-genome hybrids (TACC, TBAA, TCAA, TCBB) as compared to allodiploids (TA, TB, TC)

For the transfer of genes from B. tournefortii (TT) to the allotetraploid oilseed brassicas, B. juncea AABB, B. carinata BBCC and B. napus AACC, B. tournefortii was first crossed with the three basic diploid species, B. campestris (AA), B. nigra (BE) and B. oleracea (CC), to produce the allodiploids TA, TB and TC. These were tetraploidized by colchicine treatment to produce the allotetraploids TTAA, TTBB and TTCC, which were further crossed with B. juncea and B. napus to produce three-genome hybrids with substitution-type genomic configurations: TACC, TBAA and TCAA. These hybrids along with another hybrid TCBB produced earlier, the three allodiploids, their allotetraploids and the four diploid parent species were studied for their male meiotic behaviour. The diploid parent and the allotetraploids (TTAA, TTBB and TTCC) showed regular meiosis although the pollen viability was generally low in the allotetraploids. In the allodiploids (TA, TB and TC) only some end-to-end associations were observed without any clearly discernible chiasmata or exchange points. Chromosomes involved in end-to-end associations were randomly distributed at the metaphase/anaphase-I stages. In contrast, the three-genome hybrids (TACC, TBAA, TCAA and TCBB) showed normal bivalents whose number exceeded the expected bivalent values. Bivalents arising out of homoeologous pairing were indistinguishable from normal pairs by their disjunction pattern but could be distinguished on the basis of the heteromorphy of the homoeologous chromosomes. The three-genome hybrids could be backcrossed to allotetraploid oilseed brassicas as they had some fertility. In contrast, the allodiploids could neither be selfed nor back-crossed. On the basis of their meiotic stability, in terms of more pronounced homoeologous pairing and fertility for backcrossing, the three-genome configurations provide the best possible situation for the introgression of alien genes from the secondary gene pool to the allotetraploid oilseed crops B. juncea, B. napus and B. carinata.     

Resynthesis of Brassica carinata by protoplast fusion and recovery of a novel cytoplasmic hybrid

Brassica carinata (2n=34, BBCC), was synthesized by fusing dark grown etiolated hypocotyl protoplasts of B. nigra (2n=16, BB) with green mesophyll protoplasts of B. oleracea (2n=18,CC) using polyethylene glycol. Heterokaryons could be microscopically distinguished from the parental types by their dark green chloroplasts in the colourless hypocotyl protoplast background. The mean heterokaryotic fusion frequency estimated on the basis of this morphological distinction was about 16%. A total of 626 calli were obtained, of which 92 regenerated shoots after transfer to zeatin (2 mg/l) supplemented MS medium. Of these, 81 calli differentiated into plants morphologically similar to naturally occurring B. carinata and 11 calli yielded plants resembling parental types. Meiosis in seven hybrid plants showed the chromosome number to be 2n=34 the sum of B. nigra and B. oleracea chromosomes. Molecular confirmation of the amphidiploid nature of hybrids was obtained by probing with a B. juncea derived genomic clone. The use of chloroplast and mitochondrial specific gene probes, revealed that one plant was a cytoplasmic hybrid having cp DNA sequences of both B. oleracea and B. nigra and mt DNA sequences of B. nigra.Abbreviations PEG Polyethylene glycol - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA Naphthaleneacetic acid - IBA Indole-3-butyric acid - BAP 6-Benzylaminopurine - MS Murashige and Skoog (1962)     

Synthesis of Brassica carinata from Brassica nigra x Brassica oleracea hybrids obtained by ovary culture

Summary Brassica carinata was synthesized by hybridization between its diploid progenitor species B. nigra and B. oleracea followed by chromosome doubling. Up to 40 times more hybrids were obtained, using ovary culture than with conventional hand pollination. Two hybrids from the cross B. nigra x B. oleracea var alboglabra were raised to maturity. High chromosome pairing was observed in both the hybrids and the amphidiploid. A range of variability for desirable characters is reported in the synthesized amphidiploids.     

Somatic cell hybridization of ’oxy’ CMS Brassica juncea (AABB) with B. oleracea (CC) for correction of chlorosis and transfer of novel organelle combinations to allotetraploid brassicas

Alloplasmic lines of cultivated Brassica species with B. oxyrrhina cytoplasm are male-sterile and suffer from severe chlorosis. We developed male-sterile lines corrected for chlorosis by fusing protoplasts of CMS B. juncea (AABB) with ’oxy’ cytoplasm and normal B. oleracea (CC). A large number of male-sterile AABBCC somatic hybrids with desirable organelle combinations, i.e. chloroplasts of B. oleracea and mitochondria with recombinant genomes, were recovered. While no recombination was observed in the chloroplast genome, the mitochondrial genome showed extensive recombination that resulted in the appearance of totally novel banding patterns in some of the hybrids. Hybrids with a parental-type mitochondrial genome as well as recombinant patterns close to either of the parental types were also obtained. Using AABBCC somatic hybrids as bridging material, we transferred the desirable organelle combinations to B. juncea (AABB), B. napus (AACC), and B. carinata (BBCC). Many of these lines are now at advanced stages of backcrossing and show stable inheritance of the CMS character and do not suffer from chlorosis. Received: 9 August 1999 / Accepted: 14 September 1999     

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     

Synthesis of hexaploid (AABBCC) somatic hybrids: a bridging material for transfer of ‘tour’ cytoplasmic male sterility to different Brassica species

Most of the alloplasmic cytoplasmic male sterility (CMS) systems are known to be associated with a number of floral abnormalities that result from nuclear-cytoplasmic incompatibilities. One such system, tour, which is derived from Brassica tournefortii, induces additional floral abnormalities and causes chlorosis in Brassica spp. While the restorer for this CMS has been reported to be present in B. napus, in B. juncea, where the abnormalities are more pronounced, no restorer has yet been identified. Rectification of these floral abnormalities through mitochondrial recombinations and chloroplast replacement might result in the improvement of this CMS system. As organelle recombinations can possibly be achieved only by somatic cell hybridization, fusion experiments were carried out between hygromycin-resistant B. juncea AABB carrying tour cytoplasm and phosphinotricin-resistant, normal B. oleracea CC to generate AABBCC hexaploid somatic hybrids. The presence of selectable marker genes facilitated the selection of hybrids in large numbers. The resulting hybrids showed wide variation in floral morphology and organelle composition. Regenerants with normal, male-sterile flowers having recombinant tour-or oleracea-type mitochondria and oleracea-type chloroplasts were obtained. Hybrids with male-fertile flowers were also obtained that had recombined tour mitochondria. The AABBCC hexaploid hybrids synthesized in the present study were successfully utilized as a bridging material for transferring variability in the organelle genome simultaneously to all the digenomic Brassica species, and all of these hybrids are now being stabilized through repeated backcrosses to the allopolyploid crop brassicas.     

Construction of Brassica B genome synteny groups based on chromosomes extracted from three different sources by phenotypic, isozyme and molecular markers

The three B genomes of Brassica contained in B. nigra, B. carinata and B. juncea were dissected by addition in B. napus. Using phenotypic, isozyme and molecular markers we characterized 8 alien B-genome chromosomes from B. nigra and B. carinata and 7 from B. juncea by constructing synteney groups. The alien chromosomes of the three different sources showed extensive intragenomic recombinations that were detected by the presence of the same loci in more than one synteny group but flanked by different markers. In addition, intergenomic recombinations were observed. These were evident in euploid AACC plants of the rapeseed phenotype derived from the addition lines carrying a few markers from the B genome due to translocations and recombinations between non-homoeologous chromosomes. The high plasticity of the Brassica genomes may have been an powerful factor in directing their evolution by hybridization and amphiploidy.     

Intergeneric hybrids between Enarthrocarpus lyratus,a wild species,and crop brassicas

Summary Attempts were made to produce intergeneric hybrids between Enarthrocarpus lyratus, a wild species, and several species of crop brassicas: B. campestris, B. nigra, B. oleracea, B. juncea, B. napus and B. Carinata. Hybrids using E. lyratus as female parent were realized by means of embryo rescue in four combinations — E. lyratus x B. campestris, E. lyratus x B. oleracea, E. lyratus x B. napus and E. lyratus x B. carinata. Reciprocal crosses showed strong pre-fertilization barriers and yielded no hybrids except in one combination — B. Juncea x E. Lyratus — in which a single hybrid could be realized. All of the hybrids were multiplied in vitro through the multiplication of axillary shoots. Morphological and cytological studies confirmed hybridity. All hybrids were completely pollen sterile except for E. lyratus x B. carinata, which showed 2% pollen fertility. Attempts to double the chromosome number through the in vitro application of colchicine to axillary meristems of F₁ hybrids were successful in only one hybrid, E. lyratus x B. oleracea. Cytological studies of the hybrids indicated the presence of a partial homology between the genomes of E. lyratus and crop brassicas. Backcross progenies were raised from all of the five F₁ hybrids to develop malesterile alloplasmic lines.     

Production of hybrids, amphiploids and backcross progenies between a cold-tolerant wild species, Erucastrum abyssinicum and crop brassicas

Three intergeneric hybrids were produced between a cold-tolerant wild species, Erucastrum abyssinicum and three cultivated species of Brassica, B. juncea, B. carinata and B. oleracea, through ovary culture. The hybrids were characterized by morphology, cytology and DNA analysis. Amphiploidy was induced in all the F₁ hybrids through colchicine treatment. Stable amphiploids and backcross progenies were obtained from two of the crosses, E. abyssinicum x B. juncea and E. abyssinicum x B. carinata. The amphiploid, E. abyssinicum x B. juncea was successfully used as a bridge species to produce hybrids with B. napus, B. campestris and B. nigra. These hybrids and backcross progenies provide useful genetic variability for the improvement of crop brassicas.     

Production and characterization of interspecific hybrids between Brassica maurorum and crop brassicas

 Seven interspecific hybrids were produced between Brassica maurorum (♀), a wild species resistant to Alternaria blight and white rust, and all the monogenomic (B. campestris, B. nigra and B. oleracea) and digenomic (B. juncea, B. napus and B. oleracea) crop brassicas (♂) through embryo rescue. The hybrids were confirmed by means of morphological and cytological studies. All the hybrids were pollen-sterile. Amphidiploids were induced in three of the hybrids: B. maurorum×B. napus, B. maurorum×B. carinata, B. maurorum×B. nigra. The hybrids were also confirmed through DNA analyses for nuclear and organelle genomes using RAPD and RFLP techniques. Received: 31 July 1998 / Accepted: 14 August 1998     

Incorporation of hygromycin resistance in Brassica nigra and its transfer to B. napus through asymmetric protoplast fusion

Summary With the idea to develop a selection system for asymmetric somatic hybrids between oilseed rape (Brassica napus) and black mustard (B. nigra), the marker gene hygromycin resistance was introduced in this last species by protoplast transformation with the disarmed Agrobacterium tumefaciens strain C58 pGV 3850 HPT. The B. nigra lines used for transformation had been previously selected for resistance to two important rape pathogens (Phoma lingam, Plasmodiophora brassicae). Asymmetric somatic hybrids were obtained through fusion of X-ray irradiated (mitotically inactivated) B. nigra protoplasts from transformed lines as donor with intact protoplasts of B. napus, using the hygromycin resistance as selection marker for fusion products. The somatic hybrids hitherto obtained expressed both hygromycin phosphotransferase and nopaline synthase genes. Previous experience with other plant species had demonstrated that besides the T-DNA, other genes of the donor genome can be co-transferred. In this way, the produced hybrids constitute a valuable material for studying the possibility to transfer agronomically relevant characters — in our case, diseases resistances — through asymmetric protoplast fusion.     

Analysis of chloroplast and mitochondrial segregation in three different combinations of somatic hybrids produced within Brassicaceae

Summary Mitochondrial and chloroplast DNA were characterized in three different combinations of somatic hybrids produced between different species within Brassicaceae. The fusions were made between B. campestris and B. oleracea, B. napus and B. nigra and between B. napus and Eruca sativa. The combinations represent interspecific hybridizations, but the phylogenetic distance between the species used in each instance is different. Whereas the B. campestris (+) B. oleracea and the B. napus (+)B. nigra hybrids are both examples of intrageneric hybrids, B. campestris is more closely related to B. oleracea than B. napus is to B. nigra. The fusion of B. napus and E. sativa represents an intergeneric hybridization. Since hybrids were produced with reproducible and uniform fusion and culture methods, a comparison of chloroplast and mitochondrial segregation and mitochondrial DNA (mt-DNA) rearrangements could be made between the combinations. The segregation of both chloroplasts and mitochondria was biased in the B. napus (+)B. nigra and the B. napus (+)E. sativa combination. The nonrandom segregation of chloroplasts and mitochondria could be due to the different ploidy levels of the fusion partners and/or reflect differences in organelle replication rate. Furthermore, segregation of mitochondria was correlated to the differences in phylogenetic distance between the species used in the fusions. However, mitochondrial segregation, in contrast to chloroplast segregation, could in all combinations also have been affected by the cell type used as protoplast source in the fusions. All different chloroplast types could be established within each combination. Hybrids containing chloroplast from one parent together with mitochondria from the other parent were found in two of the combinations, although the majority of the hybrids had mt-DNA that was altered compared to the parental species. The rearranged mt-DNA found in most hybrids was an effect of the heteroplasmic state following protoplast fusion rather than of the tissue culture methods, since no mt-DNA rearrangements were found in B. napus plants regenerated from protoplast culture. The mtDNA restriction patterns of the hybrids with rearranged mt-DNA indicated that specific regions of the mt-DNA were involved in the rearrangements following protoplast fusion.     

Accessing and exploiting genes of breeding value of distant relatives of crop Brassicas

The Brassicas are an important group of crops in India yielding edible oils and many vegetables. For improving cultivated Brassicas, the wild relatives are of considerable value. The Brassica group of seed oil and vegetables comprises six cultivated species, out of which three are diploids and three are digenomic tetraploids. Brassica juncea is the major seed oil crop in India which can be improved for several traits by incorporating genes from its distant relatives. The early work in India relating to genome manipulation consisted of synthesis of B. juncea by crossing B. campestris with B. nigra, experimental resynthesis of Brassica species and non-homologous pairing and genetic exchange at the interspecific level. The alloploid species B. napus and B. carinata have not been successful in India due to agrometereological limitations. However, synthetic forms of B. napus have been produced which have a desirable maturity period with good yield potential. Also, through non-homologous pairing, pod shatter resistant B. napus has been obtained, B. napus ordinarily suffers from pod shattering. Similarly, synthetic forms of B. carinata have been derived from reciprocal crosses between morphotypes of B. oleracea and B. nigra and also through protoplast fusion of B. nigra with B. oleracea. Molecular analysis has revealed that one of the somatic hybrids had a novel cytoplasmic combination which carried B. nigra mitochondrial and B. oleracea chloroplast genomes. A range of wild and weedy species related to crop Brassicas possess extensive genetic variability. Work for utilizing this variability included hybridization between wild and crop species, analysis of chromosome pairing and induction of alloploidy. Among Brassicas of interest to India, protoplast culture and regeneration has been successful in the case of B. oleracea, B. juncea, B. nigra and B. carinata (cultivated species) and Eruca sativa and Diplotaxis muralis (related wild species). Polyethylene glycol mediated protoplast fusion has been the most commonly used method in India for producing somatic hybrids involving Brassicas. The eight somatic hybrids produced and studied showed that in the majority of cases the fusions led to symmetric hybrids combining the complete genomes of the donor species. For developing suitable male sterile lines, B. juncea, B. campestris and B. napus nuclei have been combined with the cytoplasm of six wild species and stable male steriles have been developed. Protoplast fusion methodology has been used extensively for improving these CMS by manipulating cytoplasmic organelles, including production of new combinations of cp and mt.     

Synthesis of male sterile,triazine-resistant Brassica napus by somatic hybridization between cytoplasmic male sterile B. oleracea and atrazine-resistant B. campestris

Summary Fusion of leaf protoplasts from an inbred line of Brassica oleracea ssp. botrytis (cauliflower, n=9) carrying the Ogura (R1) male sterile cytoplasm with hypocotyl protoplasts of B. campestris ssp. oleifera (cv Candle, n=10) carrying an atrazine-resistant (ATR) cytoplasm resulted in the production of synthetic B. napus (n=19). Thirty-four somatic hybrids were produced; they were characterized for morphology, phosphoglucose isomerase isoenzymes, ribosomal DNA hybridization patterns, chromosome numbers, and organelle composition. All somatic hybrids carried atrazine-resistant chloroplasts derived from B. campestris. The mitochondrial genomes in 19 hybrids were examined by restriction endonuclease and Southern blot analyses. Twelve of the 19 hybrids contained mitochondria showing novel DNA restriction patterns; of these 12 hybrids, 5 were male sterile and 7 were male fertile. The remaining hybrids contained mitochondrial DNA that was identical to that of the ATR parent and all were male fertile.     

Production and cytogenetics of intergeneric hybrids between the three cultivated Brassica diploids and Orychophragmusviolaceus

It has been proposed that both complete and partial separation of the parental genomes during mitosis and meiosis occurs in the intergeneric hybrids between Orychophragmus violaceus (2n=24) and the three cultivated Brassica tetraploids (B. napus, B. carinata and B. juncea). The hypothesis has been that this and the variations in chromosome numbers of these hybrids and their progenies result from the different roles of the A, B and C genomes originating from Brassica. To test this hypothesis, we produced hybrids between O. violaceus and the cultivated Brassica diploids. The hybrids with B. oleracea (2n=18, CC) had an intermediate morphology, but their petals were purple like those of O. violaceus. They were sterile and had the expected chromosome number (2n=21) in their mitotic and meiotic cells. The hybrid with B. campestris (2n=20, AA) was morphologically intermediate, except for its partial fertility and its yellow petals, which were similar to those of B. campestris. It was mixoploid (2n=23–42), and cells with 2n=34 were most frequent. Partial separation of parental genomes during mitosis, leading to the addition of O. violaceus chromosomes to the B. campestris complement, was proposed to explain the findings in the mitotic and meiotic cells of the hybrid and its progeny. In crosses with B. nigra (2n=16, BB), the majority of the F₁ plants were of the maternal type (2n=16), a small fraction had B. nigra morphology but were mixoploids (2n=16–18), predominantly with 2n=16 cells and three plants, each with a specific morphology, were mixoploids consisting of cells with varying ranges of chromosome numbers (2n=17–26, 11–17 and 14–17). The origin of these different types of plants was inferred to be a result of the complete and partial separation of parental genomes and the loss of O. violaceus chromosomes. Our findings in the three crosses suggest that the A genome was more influential than the C genome with respect to complete genome separation during mitosis and meiosis of the hybrids with B. napus. Possible complete and partial genome separation during mitotic divisions of the hybrids with B. carinata was mainly attributed to the role of the B genome. The combined roles of the A and B genomes would thus contribute to the most variable chromosome numbers of mitotic and meiotic cells in the hybrids with B. juncea and their progenies. The possible cytological mechanisms pertaining to these hybrids and the potential of genome separation in the production of Brassica aneuploids and homozygous plants are discussed. Received: 8 February 1998 / Accepted: 12 March 1999     

Brassica naponigra,a somatic hybrid resistant to Phoma lingam

Summary Brassica napus and B. nigra were combined via protoplast fusion into the novel hybrid Brassica naponigra. The heterokaryons were identified by fluorescent markers and selected by flow sorting. Thirty hybrid plants were confirmed by isozyme analysis to contain both B. nigra and B. napus chromosomes; of these, 20 plants had the sum of the parental chromosome numbers. A non-random segregation of the chloroplasts was found in the hybrids. Of 14 hybrid plants investigated, all had the B. napus type of chloroplast. The resistance to Phoma lingam found in the B. nigra cultivar used in the fusion experiments was expressed in 26 of the hybrid plants. The hybrids obtained in this study contain all of the three Brassica genomes (A, B and C) and have thus created unique possibilities for genetic exchanges between the genomes. Since most of the plants were fertile as well as resistant to P. lingam, they have been incorporated into conventional rapeseed breeding programs.     

Transfer of hygromycin resistance into Brassica napus using total DNA of a transgenic B. nigra line

The successful transfer of a marker gene (hpt gene) from Brassica nigra into B. napus via direct gene transfer was demonstrated. Total DNA was isolated from a hygromycin-resistant callus line, which contained three to five copies of the hpt gene. This line had been produced via direct gene transfer with the hygromycin resistance-conferring plasmid pGL2. The treatment of B. napus protoplasts with genomic DNA of B. nigra (Hyg^R) resulted in relative transformation frequencies of 0.1–0.4%. Similar transformation rates were obtained in direct gene transfer experiments using B. napus protoplasts and plasmid pGL2.     

Unique chromosome behavior and genetic control in Brassica×Orychophragmus wide hybrids: a review

Researchers recognized early that chromosome behavior, as other morphological characters, is under genetic control and gave some cytogenetical examples such as the homoeologous chromosome pairing in wheat. In the intergeneric sexual hybrids between cultivated Brassica species and another crucifer Orychophragmus violaceus, the phenomenon of parental genome separation was found under genetic control during mitosis and meiosis. The cytogenetics of these hybrids was species-specific for Brassica parents. The different chromosome behavior of hybrids with three Brassica diploids (B. rapa, B. nigra and B. oleracea) might contribute to the different cytology of hybrids with three tetraploids (B. napus, B. juncea and B. carinata). The finding that genome-specific retention or loss of chromosomes in hybrids of O. violaceus with B. carinata and synthetic Brassica hexaploids (2n=54, AABBCC) is likely related to nucleolar dominance gives new insight into the molecular mechanisms regarding the cytology in these hybrids. It is proposed that the preferential expressions of genes for centromeric proteins from one parent (such as the well presented centromeric histone H3) are related with chromosome stability in wide hybrids and nucleolar dominance is beneficial to the production of centromere-specific proteins of the rRNAs-donor parent and to the stability of its chromosomes.     

Investigations on the artificial synthesis of amphidiploids of Brassica tournefortii Gouan with the other elementary species of Brassica. I. Genomic relationships

With the object of studying the genomic relationships of Brassica tournefortii Gouan with the other elementary species of Brassica viz. B. campestris (2n=20, A genome), B. oleracea (2n=18, C genome) and B. nigra (2n=16, B genome), it has been hybridized with them. The percentage of F₁ hybrids formed, their morphology and meiotic behaviour have been described. Based upon crossability relationships and meiotic pairing in the F₁ hybrids, it is inferred that the D genome of B. tournefortii is more closely related to the A genome than to the B and C genomes. It may have been derived from the A genome which likewise shows a strong genetic isolation from B and C. The species has developed a strong genetic barrier in the course of its evolution and shows little crossability, high hybrid sterility and no gene flow with any of the other elementary species. The fact that it has not formed any natural amphidiploids with the elementary species which otherwise are formed in all combinations, is more evidence that it originated more recently than the A genome. It is presumed that B. tournefortii, being more distantly related to B. nigra than to other elementary species, may form stable artificial aphid resistant amphidiploids with the former.     

Effects of parental ploidy level and genetic divergence on chromosome elimination and chloroplast segregation in somatic hybrids within Brassicaceae

Summary Chromosome and organelle segregation after the somatic hybridization of related species with different degrees of genetic divergence were studied by comparing the interspecific somatic hybrids Brassica oleracea (CC) (+) B. campestris (AA), B. napus (AACC) (+) B. oleracea (CC) B. napus (AACC) (+) B. nigra (BB) and B. napus (AACC) (+) B. juncea (AABB) with the intergeneric somatic hybrids B. napus (AACC) (+) Raphanus sativus (RR) and B. napus (AACC) (+) Eruca sativa (EE). Within each combination, some hybrids were found whose DNA content was equal to the sum of parental chromosomes, others had a relatively higher DNA content and in most of the cases, some had a relatively lower content. However, the frequency distribution in these three classes differed significantly between the combinations. A positive correlation between the frequency of hybrids with eliminated chromosomes and the genetic distance between the species in each combination was found. Furthermore, by combining species with different ploidy levels we found a significantly higher degree of chromosome elimination compared to combinations of species with the same ploidy level. In the B. napus (+) B. Nigra, B. napus (+) R. sativus and B. napus (+) E. sativa combinations chromosomes from the B, R and E genomes appeared to be preferentially sorted out, as indicated by the fact that some of the nuclear markers from these genomes were missing in 7–46% of the plants, whereas no plants were lacking B. napus nuclear markers. Fertile hybrids were found in all but the B. napus (+) R. sativus fusion combination; the latter hybrids were male sterile, but female fertile. Hybrids between the A and C genomes were more fertile than hybrids obtained between the distantly related AC and B, R or E genomes, respectively. Analysis of the chloroplast RFLP pattern revealed that chloroplasts in the B. oleracea (+) B. campestris hybrids segregated randomly. A slightly biased segregation, favouring B. napus chloroplasts, was found in the B. napus (+) B. oleracea combination, whereas B. napus chloroplasts were strongly selected for in the B. napus (+) B. juncea, B. napus (+) B. nigra, B. napus (+) R. sativus and B. napus (+) E. sativa somatic hybrids.