Different Behavior of Brassica juncea and B. carinata as Sources of Phoma lingam Resistance in Experiments of Interspecific Transfer to B. napus

With the aim to transfer Phoma lingam resistance into rape, successful interspecific crosses were made between three oilseed rape varieties (Brassica napus) and the resistant species B. carinata and B. carinata. Although both hybrid types B. napus×B. juncea and B. napus×B. carinata showed the same high level of resistance as the respective resistant parent, the resistance could be only transferred from juncea crosses. After three backcross generations, lines morphologically undistinguishable from rape, fertile, and with a high degree of resistance were obtained. The resistance of B. carinata was practically lost in the first backcross. A possible explanation of this different behavior could be a higher recombination between the genomes B and C (juncea crosses) than between B and A (carinata crosses). The: applied embryo culture increased the yield of hybrids and first backcross plants and reduced considerably the generation time.     

Studies of pollen grain germination,pollen tube growth,micropylar penetration and seed set in intraspecific and intergeneric crosses within three Cruciferae species

Summary Pollen grain germination, pollen tube growth and micropylar penetration were investigated in intraspecific and intergeneric crosses involving Brassica napus L. ssp. oleifera (Metzg.) Sinsk. (oil-seed rape or fodder rape), xBrassicoraphanus Sageret (Raparadish) and diploid (2x) and tetraploid (4x) accessions of Sinapis alba L.(white mustard). For the reciprocal intergeneric crosses between B. napus and xBrassicoraphanus no effective barriers to pollen tube growth on stigmata or in styles were observed. The resulting low frequency of hybrid plants was mainly associated with a low rate of ovules with micropylar penetration per siliqua or with embryo abortion. Hybrid plants could be obtained without use of embryo rescue. In reciprocal crosses between B. napus and S. alba 2x or 4x incongruity barriers were observed on the stigma, in the style, and in the ovary resulting in a low frequency of ovules with micropylar penetration per siliqua. Open flower-pollination compared to bud-pollination generally was the more favourable procedure for pollen grain germination and pollen tube growth in crosses involving S. alba, but for micropylar penetration and seed set no differences were observed. Crosses between S. alba 2x () and B.napus () were found to result in a higher frequency of ovules with micropylar penetration as compared to reciprocal crosses or crosses with S. alba 4x. All reciprocal crosses between B. napus and S. alba 2x or 4x were unsuccessful when no embryo rescue was applied. Embryo rescue shortly after pollination, i.e. 2 to 5 days, however, resulted in hybrid seeds and plants, but only when applied to crosses between S. alba 2x () and B. napus (). The possible effects of the genome constitution, taxonomic distance and the parthenogenetic and parthenogenesis inducing ability of the parental genotypes on the observed malfunctions at the pre-and/or post-zygotic stage of the pollen grain-pistil interactions are discussed.Abbreviations DAP Days After Pollination - IAA Indole-3-Acetic Acid - kin Kinetin     

Characterization of Brassica nigra chromosomes and of blackleg resistance in B. napus–B. nigra addition lines

Brassica napus-B. nigra addition lines were previously created using the variety ‘Darmor’ as the oilseed rape genetic background. Two isozyme loci and 46 RAPD markers were added on five different B. nigra chromosomes. The oilseed rape variety used was highly susceptible to blackleg at the cotyledon stage and only the addition of chromosome 4 gave the same level of blackleg resistance as B. nigra. This resistance was efficient whatever the isolates used. A significant effect on the development of stem canker under field conditions was observed only for the line carrying chromosome 4 which was more resistant than the susceptible control. The potential effects of two other chromosomes have to be confirmed. F1 hybrids obtained by crosses between two highly susceptible lines and the monosomic addition line carrying chromosome 4 were examined under field conditions. No effect of the oilseed rape genetic background on the expression of resistance was detected. The introduction of this resistance and mapping of the gene(s) into oilseed rape varieties are discussed.     

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.     

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.     

Identification of Maintainer Genes in Brassica napus L. for the Male-Sterility-Inducing Cytoplasm of Diplotaxis muralis L.

Male fertility of F¹ interspecific hybrid plants derived from crosses between cytoplasmic male-sterile Brassica campestris in Diplotaxis muralis cytoplasm and 147 B. napus cultivars was Investigated. F¹, plants obtained, from crosses with the B. napus cultivars‘Mangum’and‘Hinchu’were male-sterile while F¹ plants derived from all other crosses were male-fertile. This indicated that these two cultivars carried maintainer genes far the male-sterility-inducing cytoplasm of D. muralis. Sterility was stable In plants derived from backcrosses of male-sterile F; plants with‘Mangun and‘Hinchu’but the seed set of backcross plants was low. With restorer genes readily available in B. napus, these findings could lead to the development of a new cytoplasmic male sterility system for the breeding of B. napus hybrid cultivars.     

An evaluation of the potential of intergeneric gene transfer between Brassica napus and Sinapis arvensis

The possibility of gene transfer between Brassica napus and Sinapis arvensis was evaluated. Six spring-type cultivars of B. napus and four strains of S. arvensis were reciprocally crossed through controlled crosses. No hybrid was yielded from any cross. However, one hybrid with 28 chromosomes was obtained from B. napus×S. arvensis through ovule culture. The hybrid plant was highly sterile and set no seed on open pollination. Two F₂ plants, with 35 and 36 chromosomes respectively, were obtained through self-pollination by hand. Backcross of B. napus produced 23 plants carrying some characteristics of S. arvensis, but backcross to S. arvensis failed to produce a plant. The chromosome counts of the BC₁F₁ plants indicated that gametes with more than nine chromosomes were favoured during the meiosis. The data demonstrated that gene transfer from S. arvensis to B. napus was very difficult under controlled cross and backcross, while to transfer genes from B. napus to S. arvensis would be extremely remote even under the most favorable conditions.     

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.     

Hybridization of radish (Raphanus sativus L.) and oilseed rape (Brassica napus L.) through a flower-culture method

Summary Hybridization between radish and oilseed rape has been cumbersome, requiring elaborate embryo rescue techniques. With a modified flower culture method, we have achieved successful hybridization between radish and (transgenic) oilseed rape without the laborious and technically demandingin vitro ovule or embryo rescue techniques.The hybrid nature of the intergeneric hybrids was demonstrated using morphological traits, and DNA analyses. The described method will facilitate the generation ofRaphanobrassica hybrids useful for biosafety studies of the potential for transgenes to spread in weedyCruciferae as well as for breeding programs aimed at introducing useful radish genes, e.g. nematode resistance genes, into oilseed rape.     

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.     

Cytogenetical and molecular investigations on somatic hybrids of Sinapis alba and Brassica juncea and their backcross progeny

Somatic hybrids of Sinapis alba+Brassica juncea (Sal Sal AABB) were synthesized by protoplast electrofusion. They were true genomic allopolyploids since they possessed 60 chromosomes, i.e. the sum of S. alba (2n= 24) and B. juncea (2n= 36) chromosomes. Chromosome pairing was predominantly bivalent with the occasional occurrence of multivalents in the pollen mother cells at diakinesis and metaphase I. Hybrids were completely pollen-sterile, but produced seeds on back-crossing with B. juncea and B. campestris. A total of 37 BC₁ plants were raised from two somatic hybrids (JS-1 and JS-2) and 24 of these were analysed cytologically. The 22 plants originating from the pollinations of somatic hybrids with B. juncea showed a chromosome configuration of 18II+12I and had 42–86% pollen fertility. Two plants from the backcrosses of the somatic hybrid with B. campestris formed 10II +20I, and had 0–4% fertile pollen. Total DNA analysis by probing with pTA71 carrying a full-length 18S–25S rDNA fragment of the wheat nuclear genome revealed that the two somatic hybrids possessed all the characteristic bands of both the species, confirming their hybridity. Probing with the mitochondrial coxI and atp9 genes indicated mitochondrial genome recombination in the hybrids. Hybridization with chloroplast-specific psbD indicated that both the somatic hybrids possessed the cp genome of S. alba origin.     

Components of a Cytoplasmic Male Sterility System in Resynthesized and Cultivated Forms of Oilseed Rape (Brassies napus L.)

Male sterile Brassica napus L. plants were found in breeding material which was used for the development of yellow-seeded oilseed rape. The genetic studies indicated that the male sterility was conditioned by the presence of maintamer genes in the nuclear backgrounds of two newly resynthesized B. napus lines, No7076 and No7406, in combination with a male sterility-inducing cytoplasm (S) which is frequently found in cultivated forms of B. napus. Test crosses with nap maintainer and restorer lines support the conclusion that the observed male sterility is of nap type. Furthermore, the Eco RI restriction pattern of mitochondrial DNA of the (S) cytoplasm was identical to that of the nap cytoplasm. Hence, we conclude that we have uncovered a new source of maintainer lines for the nap system which could potentially lead to the production of a better maintainer/restorer system for use in hybrid oilseed rape breeding programmes. However, more work is needed to reduce the glucosinolate content of the maintainer lines and to determine the factors controlling the phenotypic expression of the system.     

Inheritance and Agronomic Performance of the Waxless Character in Brassica napus L.

A mutant of oilseed rape (B. napus L.) with waxless leaves was discovered in 1988. Three pairs of reciprocal crosses were made between the mutant (Wl) and 3 normal wax layered lines (Nwl). By the summer planting in Kunming and Wenjiang and the autumn planting in Wenjiang, it was confirmed that the Wl character is completely dominant to the normal opposite Nwl. Chi-square tests revealed that the segregation ratios of Wl to Nwl among B₁ and F₂ plants fit to 1:1 and 3:1, respectively, i.e. the assumption of one gene pair. The yield of 12 hybrids, which were derived fom Wl and some Nwl lines, exceeded the check control ‘Zhong You 821’, a best cultivar in Chinese production by 19.31 % on the average. When the seedling had 3–4 leaves, both phenotypes can be accurately determined visually on the field. This material possesses potential value for studies of the genetics and breeding, as a prominent genetic marker in B. napus.     

Genetic relationships among resynthesized,semi-resynthesized and natural <Emphasis Type="Italic">Brassica napus</Emphasis> L. genotypes

The allopolyploidization event that created cultivated oilseed rape Brassica napus L, followed by intense breeding, reduced its genetic diversity. Resynthesized (RS) B. napus L. obtained by interspecific hybridization between genotypes of B. rapa L. and B. oleracea L. can be a valuable source for broadening genetic diversity in cultivated oilseed rape. In this study, we determined the extent of DNA polymorphism among natural accessions of oilseed rape, resynthesized B. napus, their parental species and double-low quality semi-RS lines carrying the Rfo gene. Using 10 selected primer combinations, 522 polymorphic AFLP markers were scored in the complete set of 100 Brassica sp. To detect relationships between these genotypes, a cluster analysis was performed using the Jaccard’s distance. Resynthesized allopolyploids clustered directly between their diploid parents. Cultivated accessions of oilseed rape created a compact group away from resynthesized allopolyploids as well as semi-RS lines. The natural oilseed rape group, which consists of 49 cultivars and breeding lines of oilseed rape, is characterized by lower genetic diversity than the group of 33 accessions of resynthesized oilseed rape, and the analysis showed that the double-low quality semi-RS lines represent a specific genetic variation of B. napus. The de novo resynthesized B. napus lines and the semi-RS lines of double-low quality generated from them, provide a significant opportunity for enrichment the gene pool of oilseed rape.     

The Effect of A Genome Substitution on the Resistance of Brassica napus to Infection by Leptosphaeria maculans

Six accessions belonging to four subspecies of Brassica rapa, including three accessions of B. rapa subsp. sylvestris, were crossed with B. oleracea subsp. alboglabra in order to develop a series of synthetic B. napus lines with a common C genome but contrasting A genomes. Different A genomes had significant effects on the efficiency of B. napus resynthesis and the sexual compatibility of the synthetic lines with oilseed rape cultivars. The synthetic lines were used to investigate the effect of A genome substitution on the resistance of B. napus to infection by Leptosphaeria maculans, and to explore the potential for the use of wild forms of B. rapa in oilseed rape breeding programmes. Synthetic lines derived from two wild accessions of B. rapa, and their F₁ hybrids with oilseed rape cultivars, expressed high levels of resistance to L. maculans in glasshouse experiments. One of these lines also expressed high levels of resistance in field experiments in England and Australia when exposed to a genetically diverse pathogen population. All other synthetic lines and cultivars were highly susceptible in both glasshouse and field experiments. F₁ hybrids between oilseed rape cultivars and synthetic lines derived from B. rapa subsp. chinensis were significantly more susceptible than either 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.     

Development of breeding populations from interspecific hybrids between Trifolium repens L. and T. occidentale Coombe

Trifolium occidentale is a diploid wild relative of T. repens (white clover) with adaptation to dry, saline coastal habitats. Transfer of drought and salt‐tolerant adaptive traits to white clover could be potentially valuable if interspecific hybridization can be achieved efficiently and leads to fertile hybrid populations. To achieve hybridization, 4x plants of T. occidentale were generated. Efficient techniques for generation of 4x plants and their identification using dry pollen shape are described. Interspecific 4x F₁ plants were achieved without embryo rescue. F₂ populations and first backcross hybrids to white clover were also efficiently achieved. Although male and female fertility were lower than in white clover, they were adequate to produce large amounts of seed from small numbers of inflorescences. Thus, early generation interspecific hybrid prebreeding populations can be readily developed, opening the way for transfer of traits from T. occidentale to white clover.     

Creating favourable morphological and yield variations for rapeseed by interspecific crosses between Brassica rapa and Brassica oleracea

Brassica napus is a most important oilseed grown worldwide with a limited genetic background, due to the short history of speciation, domestication and cultivation. To create novel germplasm for rapeseed breeding, we made interspecific crosses followed with chromosome doubling between B. rapa and B. oleracea to generate novel B. napus with favourable agronomic traits. The resynthesized (S0) hybrids were confirmed by SSR and cytogenetic analysis, and the fertility was increased from 32.7% in S0 generation to ~97.31% in S1 generation. The plant shapes of the progeny were dramatically improved compared to the diploid parents and B. napus cv. ‘Yangyou 6’, especially for the branch initiation height, branch number and pod number. The single‐plant yield was significantly improved in S1 progeny for the variations in branching sites and number. Significant improvement in plant shape and yield was observed on S2 generation compared to the local elite commercial open‐pollinated cultivar, which would be further fixed by intensive selection and pyramiding breeding. Such variation is of great value for breeding rapeseed with improved plant architecture and harvest index.     

Changes in the meiotic pairing behaviour of a winter wheat-winter barley hybrid maintained for a long term in tissue culture, and tracing the barley chromatin in the progeny using GISH and SSR markers

The aim of the present study was to produce backcross progenies in a new winter wheat (‘Asakaze komugi’) × winter barley (‘Manas’) hybrid produced in Martonvasar. As no backcross seeds were obtained from the initial hybrids, young inflorescences of the hybrids were used for in vitro multiplication in three consecutive cycles until a backcross progeny was developed. The chromosome constitution of the regenerated hybrids was analysed using genomic in situ hybridization (GISH) after each in vitro multiplication cycle. The seven barley chromosomes were present even after the third in vitro multiplication cycle but abnormalities were observed. Sixteen BC; plants containing, according to GfSH analysis, one to three complete barley chromosomes, two deletion barley chromosomes and a dicentric wheat‐barley translocation were grown to maturity from the single backcross progeny. The barley chromatin was identified using 20 chromosome‐specific barley SSR markers. All seven barley chromosomes were represented in the BC: plants. A deletion breakpoint at FL ±0,3 on the 5HL chromosome arm facilitated the physical localization of microsatellite markers.     

Efficient hybridization between Lycopersicon esculentum and L. peruvianum via 'embryo rescue' and in vitro propagation

For the transfer of valuable traits from wild species into the cultivated tomato, excised globular-stage embryos 13 and 15 days after pollination (DAP) were cultured in vitro. Plants were regenerated from interspecific crosses of Lycopersicon esculentum cv. ‘Early pink’×L. peruvianum PI270435, and backcrosses of L. esculentum‘Giban (JF) No. 1’× (‘Early pink’× PI270435). Somatic embryos and single cotyledons emerged on hypocotyl sections of the embryos. Five to nine plantlets per embryo were obtained by clonal propagation. The hybrid nature of the plants is confirmed by comparing hybrids and parents in their ability to regenerate shoots from leaf segments in vitro, by comparing plant morphology and characteristics and by chromosome number. This study describes an efficient ‘embryo rescue’ method, as well as somatic embryogenesis by clonal propagation. A novel and simple method for the characterization of the interspecific hybrids is also reported.