Transfer of disease resistance within the genus Brassica through asymmetric somatic hybridization

Summary Asymmetric somatic hybrid plants between Brassica napus L. (oilseed rape genome AACC) and a transgenic line of Brassica nigra L. Koch (black mustard genome BB) were tested for their resistance against rapeseed pathogens Phoma lingam (black leg disease) and Plasmodiophora brassicae (club root disease). The transgenic B. nigra line used (hygromycin-resistant, donor) is highly resistant to both fungi, whereas B. napus (recipient) is highly susceptible. The asymmetric somatic hybrids were produced using the donor-recipient fusion method (with X-irradiation of donor protoplasts) reported by Zelcer et al. (1978) for the production of cybrids. Using hygromycin-B for selection, a total of 332 hybrid calli were obtained. Regenerants, resistant or susceptible to both diseases, were selected. Many hybrids expressed resistance to only one pathogen. Dot blot experiments showed that the asymmetric hybrid plants contained varying amounts of the donor genomic DNA. Furthermore, a correlation was detected between the radiation dose and the degree of donor DNA elimination.     

Introgression of Resistance to Shattering in Brassica napus from Brassica juncea through Non-Homologous Recombination

Genetic information conffering non- shattering of siliques has been introgressed in rapeseed (Brassica napus) following; interspecific hybridization between Brassica juncea and B. napus. A reconstituted B. napus plant with complete non-dehiscence of its fruits was isolated in the BC-, generation. This plant had normal meiosis and formed 19 bivalents. Its seed fertility, however, was low (23 percent). It is suggested that the gene(s) for shattering-resistance were incorporated into a B. oleracea chromosome following allosyndetic; chromosome pairing and. segmental exchange between B. nigra and B. oleracea chromosomes in the initial interspecific AABC hybrid.     

Development of self-incompatible Brassica napus: (III) B. napus genotype effects on S-allele expression

Use of self‐incompatibility (SI) as a pollination control method for Brassica napus hybrid production requires the development of a sufficient number of S‐alleles that are expressed consistently in a range of B. napus lines. Self‐incompatibility (SI) alleles have been transferred from Brassica oleracea and Brassica rapa into B. napus var. oleifera. An understanding of expression of these alleles in B. napus is essential for their commercial use. Four SI B. napus doubled haploids containing the B. oleracea S‐alleles S2, S5, S13 and S24 were crossed to three B. napus cultivars to measure the B. napus genetic background effect on S‐allele expression. A line x tester analysis indicated that the largest source of variation in the expression rate of SI was the S‐allele itself. The B. napus genotypes tested contained modifier gene(s), some that enhanced SI expression and others that inhibited SI expression. The B. napus Canadian cultivar ‘Westar’ generally had a negative effect on SI expression while the European cultivar ‘Topas’ had a positive effect on the B. oleracea S‐allele expression. The B. oleracea S‐allele S24 was very similar in expression to the B. rapa allele W1. The application of these results for the use of B. oleracea S‐alleles for hybrid production in B. napus is discussed.     

Production and characterization of intergeneric somatic hybrids between Moricandia arvensis and Brassica oleracea

Somatic hybrids were produced between Moricandia arvensis (MaMa, 2n= 28) and Brassica oleracea (CC, 2n= 18) through cell fusion and then characterized by analysing their morphology, cytology, DNA constitution, leaf anatomy and seed fertility. Cell fusion was carried out between greenish protoplasts isolated from the mesophyll of M. arvensis and colourless ones from hypocotyls of B. oleracea. Three plants were generated from one shoot via cuttings and acclimatized in vivo. They closely resembled each other in morphology, exhibiting traits intermediate between the parental species. They were confirmed to be amphidiploids by mitotic and meiotic analyses, being 2n= 46 (MaMaCC), with pollen fertility of about 50%, which was enough to develop the subsequent progenies. Anatomical analysis of the for leaf tissue showed that the bundle sheath cells of the somatic hybrids contained some centripetally arranged organelles, like those of M. arvensis. The hybridity was also confirmed by random amplified polymorphic DNA analysis. Both chloroplast DNA and mitochondrial DNA of the somatic hybrids were estimated to be derived from M. arvensis. In leaf anatomy, the somatic hybrid showed the C₃‐C₄ intermediate trait as in M. arvensis. Many progenies resulted from backcrossing with parental species. The somatic hybrids are expected to be used as bridging plant material to introduce the C₃‐C₄ intermediate trait into Brassica crop species.     

Development of self-incompatible Brassica napus: (I) introgression of S-alleles from Brassica oleracea through interspecific hybridization

Cultivars in Brassica napus var. oleifera, a self‐pollinating, self‐compatible species, have traditionally been developed as open‐pollinated lines or populations. Significant yield gains in this species have been realized through the exploitation of heterosis. Commercial hybrid production has been possible as a result of the development of a number of pollination control systems. Self‐incompatibility was transferred from B. oleracea var. italica to B. napus var. oleifera through interspecific hybridization. The response to interspecific pollination, as measured by pod elongation and initial stages of ovule development, was genotype dependent, and two highly responsive B. napus genotypes were identified. Embryo rescue was used to produce the interspecific hybrids. Isoelectric focusing of stigma proteins was used to identify S‐alleles in the interspecific hybrids to facilitate backcrossing. Segregation of the S‐locus through a series of back‐crosses to B. napus was complicated by aneuploidy; however, the S‐locus was found to segregate as a single gene. Usefulness of B. oleracea as a source of S‐alleles for pollination control in B. napus is discussed.     

Transfer of powdery mildew resistance from Brassica carinata to Brassica oleracea through embryo rescue

Interspecific hybrid plants and backcross 1 (BC₁) progeny were produced through sexual crosses and embryo rescue between Brassica carinata accession PI 360883 and B. oleracea cvs Titleist’and‘Cecile’to transfer resistance to powdery mildew to B. oleracea. Four interspecific hybrids were obtained through application of embryo rescue from crosses with B. carinata as the maternal parent, and their interspecific nature confirmed through plant morphology and random amplified polymorphic DNA (RAPD) analysis. Twenty‐one BC₁ plants were obtained through sexual crosses and embryo rescue although embryo rescue was not necessary to produce first backcross generation plants between interspecific hybrids and B. oleracea. All interspecific hybrids and eight of the BC₁ plants were resistant to powdery mildew.     

A cytogenetic study of the progenies of hybrids between Brassica napus and Brassica oleracea, Brassica bourgeaui, Brassica cretica and Brassica montana

In this cytogenetic study the progeny of all crosses were investigated in F₁, F₂ and backcross (BC₁) hybrids. Brassica napus and F₁ hybrids between B. napus and B. oleracea, and between B. napus and three wild relatives of B. oleracea (B. bourgeaui, B. cretica and B. montana). Each of the wild relatives has 18 somatic chromosomes. Interspecific F₁ hybrids were obtained through ovary culture mean. These had 28 and 37 chromosomes and their mean pollen fertility was 10.7% and 93.0%, respectively. Many F₂ and BC₁ seeds were harvested from the F₁ hybrids with 37 chromosomes after self‐pollination and open pollination of the F₁ hybrids, and backcrossing with B. napus. Many aneuploids were obtained in the F₂ and BC₁ plants. It is evident from these investigations that the F₁ hybrids may serve as bridge plants to improve B. napus and other Brassica crops.     

Development of self-incompatible Brassica napus: (II) Brassica oleracea S-allele expression in B. napus

Brassica napus var. oleifera varieties have traditionally been developed as open‐pollinated varieties. The successful introduction of several high‐yielding hybrids based on cytoplasmic male sterility or transgenic pollination control systems has generated interest in the development of new hybrid systems. Self‐incompatibility could be an additional useful pollination control system for B. napus if a sufficient number of S‐alleles could be developed in this species. The S‐alleles, S2, S5, S13, S24 and S39, were identified in five hybrids of B. oleracea var. italica and subsequently transferred to B. napus. Doubled haploid lines were produced for the self‐incompatible (SI) lines in B. napus and intercrossed to produce SI heterozygotes in order to study allele interaction. There was a greater incidence of interallelic dominance in the stigmas and pollen of B. napus than was reported for the S‐alleles in B. oleracea. Allele S24 exhibited the greatest degree of dominance over the other alleles tested, while allele S2 was generally recessive or codominant with other alleles. Self‐incompatible expression was very similar in the SI homozygotes and heterozygotes, thus no weakening of the SI trait in the heterozygote was observed. The implications of S‐allele interaction for the use of SI in B. napus are discussed.     

Production and characterization of intergeneric hybrids between Brassica oleracea and a wild relative Moricandia arvensis

Intergeneric crosses were made between Brassica oleracea and Moricandia arvensis utilizing embryo rescue. Six F₁ hybrid plants were generated in the cross‐combination of B. oleracea × M. arvensis from 64 pods by the placenta‐embryo culture technique, whereas three plants were produced in the reciprocal cross from 40 pods by the ovary culture technique. The hybrid plants were ascertained to be amphihaploid with 2n = 23 chromosomes in mitosis and a meiotic chromosome association of (0–3)II + (17–23)I at metaphase I (M I). In the backcross with B. oleracea, some of these hybrids developed sesquidiploid BC₁ plants with 2n = 32 chromosomes that predominantly exhibited a meiotic configuration of (9II + 14I) in pollen mother cells. The following backcross of BC₂ plants to B. oleracea generated 48 BC₃ progeny with somatic chromosomes from 2n = 19 to 2n = 41. The 2n = 19 plants showed a chromosomal association type of (9II + 1I) and a chromosomal distribution type of (9¹/₂ + 9¹/₂) or (9 + 10) at M I and M II, respectively. These facts might suggest that they were monosomic addition lines (MALs) of B. oleracea carrying a single chromosome of M. arvensis that could offer potential for future genetic and breeding research, together with other novel hybrid progeny developed in this intergeneric hybridization.     

Utilization of Asymmetric Somatic Hybridization for the Transfer of Disease Resistance from Brassica nigra to Brassica napus

Asymmetric somatic hybrid calli were produced between Brassica napus and a transgenic (Hyg ^R) line of B. nigra using a donor recipient fusion method for the production of cybrids. The transgenic line of B. nigra used as a donor also possessed genetic resistance to the pathogenic fungi Phoma lingam and Plasmodiophora brassicae. Using hygromycin for selection, 332 hybrid calli were obtained from which 30 produced shoots (1—-20 per callus) which were rooted on a hormone-free culture medium. The rooted shoots were transferred into soil and cultivated in a growth chamber where the plants were tested for resistance against the two pathogens. Out of 129 hybrid plants tested for resistance against P. brassicae, 30 (23.3 %) plants proved to be resistant and from 78 plants tested for resistance against P. lingam, 41 (52.6 %) plants remained disease-free after infection.     

Oligonucleotide fingerprinting of resynthesized Brassica napus

Brassica napus plants, artificially synthesized through somatic hybridization of B. oleracea and B. campestris protoplasts, were analyzed by oligonucleotide fingerprinting. While the fingerprint patterns of the different hybrid plants looked very much alike, they did not simply represent a combination of the parental patterns. Instead, the absence of parental bands as well as the presence of new bands suggest that elimination and/or rearrangements occurred during or after the fusion of the two genomes. The fingerprints of individual F1 progeny plants of selfed hybrids did not detect major changes. Thus, once formed, the artificially resynthesized amphidiploid B. napus genome appears to be stable. Taken together, our experiments demonstrate the usefulness of oligonucleotide fingerprinting for the characterization of artificial hybrids in the genus Brassica.     

Introgression of genes into cultivated Brassica napus through resynthesis of B. napus via ovule culture and the accompanying change in fatty acid composition

Fifteen lines of Brassica napus were resynthesized via ovule culture through 24 interspecific crosses between four Brassica oleracea and three Brassica campestris accessions. The degree of success in the interspecific crosses was significantly influenced by maternal genotypes. The interspecific hybrid production rate (HPR) varied with combinations from 0 to 76.9%, with a mean HPR of 24.7% for the crosses with B. campestris as the female parent and 6.9% for the crosses with B. oleracea as female parent. Twenty‐four crosses between seven natural and six resynthesized B. napus gave, on average, 10.3 seeds per pod, and ranged from 1.2 to 22.0 seeds per pod, depending on genotypes of both parents. Resynthesized lines of B. napus showed high erucic acid content and variable content of linolenic acid, ranging from 3.4% to 9.9%. The fatty acid composition in hybrid seeds between natural and resynthesized B. napus was dominated by the embryo genotypes; an additive mode was shown for erucic acid and positive over‐dominance for linolenic acid content.     

Inheritance of the resistance to Leptosphaeria maculans of Brassica nigra and B. juncea in near-isogenic lines of B. napus

The inheritance of resistance to Leptosphaeria maculans was studied in near-isogenic lines derived from asymmetric somatic hybrids between Brassica napus+Brassica nigra and Brassica napus+Brassica juncea, respectively. The hybrids had been backcrossed to B. napus for seven generations before the genetic segregation of the blackleg resistance was determined. The results of the inheritance studies suggested that one single dominant allele controls the resistance in the Brassica napojuncea line, whereas two independent dominant loci were found in the Brassica naponigra line. Total leaf DNA from the near-isogenic lines was isolated and 89 loci were detected by hybridization to 66 restriction fragment length polymorphism (RFLP) markers previously mapped in the B. nigra genome. Out of the 89 loci, eight loci were detected in the B. naponigra line and six were found in the B. napojuncea line. RFLP markers co-segregating with blackleg resistance in adult leaves were also found. Two markers associated with linkage group 5 and 8, respectively, of the B genome were found in the B. naponigra line and one marker was associated with linkage group 2 in the B. napojuncea line.     

Assessment of transgene escape from Brassica rapa (B. campestris) into B. nigra or Sinapis arvensis

The possibility of gene transfer between Brassica rapa and the two weedy species B. nigra and Sinapis arvensis was evaluated with the special concern on transgene escape from B. rapa to these two weedy species. B. rapa cultivar Tobin was reciprocally crossed to five and four strains of B. nigra and S. arvensis, respectively, using controlled cross. A single interspecific hybrid was obtained from the cross B. rapa×B. nigra, but no other cross was successful. The fertility of this hybrid on open pollination, selfing and backcrosses was investigated. The data of the present study and the information available to date indicate that gene transfer between B. rapa and B. nigra is possible. The chance of transgene escape from B. rapa to B. nigra depends essentially on whether natural cross can occur between these two species. Gene transfer between B. rapa and S. arvensis is at the most difficult, whereas trans-gene escape directly from B. rapa to S. arvensis appears very unlikely.     

Interspecific hybridization between Brassica carinata and Brassica rapa

The crossability between Brassica carinata (BBCC, 2n=34) and Brassica rapa (AA, 2n=20), and the cytomorphology of their F₁ hybrids were studied. Hybrids between these two species were only obtained when B. carinata was used as the female parent. The hybrid plants exhibited intermediate leaf and flower morphology, and were found to be free from white rust and Alternaria blight diseases. One of the four F₁ plants was completely male sterile, while the remaining plants had 4.8, 8.6, and 10.9% stainable pollen, respectively. No seed was produced on hybrid plants under self pollination or in backcrosses; but seed was obtained from open pollination. The occurrence of the maximum of 11 bivalents as well as up to 44.8%) of cells with multivalent associations in the form of trivalents (0‐2) and a quadrivalent (0‐1) in the trigenomic triploid hybrid (ABC, 2n = 27) revealed intergenomic homoeology among the A, B and C genomes. Meiotic analysis of F₁ hybrids indicated that traits of economic importance, such as disease resistance, could be transferred from B. carinata to B. rapa through interspecific crosses.     

Resynthesis of rapeseed (Brassica napus L.): A comparison of sexual versus somatic hybridization

Sexual and somatic Brassica napus hybrids produced from the same parental plants were compared. Sexual crosses between a white-flowered, self-compatible broccoli selection (B. oleracea var. italica, cc genome) as the maternal parent and a flowering pak choi accession (B. chinensis, aa genome) yielded one unique spontaneous hybrid and four hybrids through embryo rescue. Thirty-nine somatic hybrids were recovered from a protoplast fusion experiment. Hybridity was confirmed by morphology, isozyme expression, flow cytometry, and DNA hybridization. Sexual and somatic hybrids exhibited differences in leaf morphology, flower colour, flowering habit, and organellar inheritance. Sexual hybrids were all fertile amphidiploids (2n = 38, aacc) following spontaneous chromosome doubling. All somatic hybrids had high nuclear DNA contents; most were probably hexaploids (aaaacc or aacccc) from the fusion of three portoplasts. Two initially sterile hexaploid (aaaacc) regenerates eventually set selfed seed after the loss of the putative extra aa genome following regrowth from axillary buds. A bias toward inheritance of B. chinensis chloroplasts was observed with somatic hybrids.     

Development of black rot resistant interspecific hybrids between Brassica oleracea L. cultivars and Brassica accession A 19182, using embryo rescue

Black rot is a bacterial disease of Brassica oleracea caused by Xanthomonas campestris pv. campestris. Resistance to the major black rot races 1 or 4 has been identified in related Brassica species including B. carinata and B. napus. In this study, two B. juncea accessions (A 19182 and A 19183) that are resistant to races 1 and 4 of Xcc were used as maternal and paternal parents to generate interspecific hybrids with B. oleracea cultivars. Interspecific hybrids were recovered using the embryo rescue technique and confirmed through inheritance of paternal molecular markers. Twenty-six interspecific hybrid plants were obtained between A 19182 and B. oleracea cultivars, but no interspecific hybrids were obtained using A 19183. Although interspecific hybrid plants were male sterile, they were used successfully as maternal parents to generate backcross plants using embryo rescue. All hybrid and BC₁ plants were resistant to black rot races 1 and 4.     

Efficiency of PCR-RF-SSCP marker production in Brassica oleracea using Brassica EST sequences

Efficiencies of SCAR, CAPS and PCR-RF-SSCP marker production were investigated using two combinations of breeding lines in Brassica oleracea. Published EST sequences of B. oleracea, Brassica rapa, Brassica napus, and Arabidopsis thaliana and newly determined nucleotide sequences of anther cDNA clones from B. oleracea were used for designing primer pairs to amplify genes. The percentage of primer pairs yielding DNA amplification of a single gene was higher in primer pairs of B. oleracea (91%) than those of B. rapa (56%) and A. thaliana (17%). Single DNA fragments amplified by 9% of the primer pairs showed polymorphism as SCAR markers between a broccoli line and a Chinese kale line by agarose-gel electrophoresis. CAPS analysis showed different band patterns in 32% of the same-sized DNA fragments, and PCR-RF-SSCP analysis revealed DNA polymorphism in 52% of those showing no DNA polymorphism by CAPS. In total, 71% of the single DNA fragments were converted to DNA markers. The frequency of DNA polymorphism between parental lines of a cabbage F₁ hybrid was lower, 5% by SCAR and 12% by CAPS. However PCR-RF-SSCP analysis revealed DNA polymorphism in 21% of the DNA fragments showing no polymorphism by CAPS. These results suggest that PCR-RF-SSCP analysis enables highly efficient DNA marker production for mapping of genes in Brassica using progeny, even progeny of closely related parents. Analysis of selfed seeds of broccoli F₁ cultivars using PCR-RF-SSCP markers indicated that PCR-RF-SSCP analysis is also applicable to seed purity tests.     

Characterization of Somatic Brassies napus Hybrids by Polyacrylamide Gel Electrophoresis

Three somatic hybrids obtained by fusion of protoplasts from Brassica oleracea and B. campestris were analyzed by gel electrophoresis and compared with their respective parental species. By comparing multiple forms of esterases and phosphorylases it could be demonstrated that in all cases the hybrid plants contained one or more enzymes from each parent.     

The transfer of triazine resistance from Brassica napus L. to B. oleracea L. III. First backcross to parental species

Summary Atrazine resistant Brassica napus × B. oleracea F₁ hybrids were backcrossed to both parental species. The backcrosses to B. napus produced seeds in both directions but results were much better when the F₁ hybrid was the pollen parent. Backcrosses to B. oleracea failed completely but BC₁s were rescued by embryo culture both from a tetraploid hybrid (2n = 4x = 37; A₁C₁CC) and sesquidiploid hybrids (2n = 3x = 8; A₁C₁C). Progeny of crosses between the tetraploid hybrid and B. oleracea had between 25 and 28 chromosomes. That of crosses between the sesquidiploid hybrid and B. oleracea had between 21 and 27. A few plants that had chromosome counts outside the expected range may have originated from either diploid parthenogenesis, unreduced gametes or spontaneous chromosome doubling during in vitro culture. Pollen stainability of the BC₁s ranged from 0% to 91.5%. All the BC₁s to B. oleracea were resistant to atrazine.