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.     

Induction of novel organelle DNA variation and transfer of resistance to frost and Verticillium wilt in Solanum tuberosum through somatic hybridization with 1EBN S. commersonii

Somatic hybridization can be used to induce genetic variability in plastidial and mitochondrial genomes, and transfer multiple uncloned genes across sexual barriers. Somatic hybrids were produced between a dihaploid clone of the common potato, S. tuberosum subsp. tuberosum, and the wild sexually incongruent diploid species S. commersonii. Fusion products were selected on the basis of callus growth and regeneration in vitro. Genome composition of putative somatic hybrids was determined by flow cytometric analysis of nuclear DNA content, RAPD analysis, and Southern analysis with probes specific to organellar DNA. All regenerated fusion products proved to be hybrids based on RAPD analysis. Seventy per cent of somatic hybrids were (near) tetraploids, 22% (near) hexaploids and 8%(near) octoploids. A high correlation was found between the nuclear DNA content determined by flow cytometry and the number of chloroplasts in stomata guard cell pairs. Somatic hybrids inherited the parental plastids in a random manner. On the contrary, they preferentially inherited the mitochondrial DNA fragments of S. tuberosum. The majority of them had a rearranged mitochondrial genome with fragments from both parents. Hybrids were highly vigorous and morphologically more similar to the cultivated than to the wild parent, produced tubers on long stolons under long photoperiod conditions, showed a high degree of flowering, but did not produce pollen. In addition, somatic hybrids were generally more resistant to frost and Verticillium wilt than the cultivated parent, indicating the introgression of relevant resistance genes from the wild species into the genetic background of S. tuberosum. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Spontaneous and induced loss of chromosomes in slow-growing somatic hybrid calli of Solanum tuberosum and Nicotiana plumbaginifolia

Summary Rate and extent of spontaneous and induced chromosome loss have been determined at the callus level of somatic hybrids of mutants of Solanum tuberosum and Nicotiana plumbaginifolia. AEC (amino ethyl cystein) resistance in potato and Nitrate-Reductase deficiency in N. plumbaginifolia have been used as genetic markers and chromosome morphology as a cytological marker. In this combination, development of hybrid callus was late and slow. Only a limited number of non-regenerable hybrid calli have become available. Chromosome loss could clearly be established in these hybrids from loss of markers and from chromosome cytology. Loss of markers occurred independantly.The best conditions to induce loss of chromosomal material in donor cells by irradiation were found by cytological investigations. A very drastic reduction in chromosome transfer by fusion could be effected by irradiation of plant tissue and subsequent preparation of protoplasts after a few days. Following fusion, hybrid callus was recovered with the potato genome drastically reduced. The amount of loss was deduced from the presence of a few fragments in metaphase cells or from interphase nuclei after in situ hybridization with a repetitive potato DNA probe.     

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.     

Assessment of genetic and phenotypic variation among intraspecific somatic hybrids of potato, Solanum tuberosum L.

Intraspecific somatic hybrids have been produced by protoplast fusion in eight combinations involving 10 dihaploids (2n= 2x= 24) in an attempt to provide new material for potato breeding. Cytological analysis revealed extensive variation in chromosome number, such as aneuploid, aneusomatic and mixoploid hybrids. Most of the hybrids represented the expected chromosome number of 48; however, the frequency of aneuploids reached 50% in some combinations. Some hybrids carried structurally rearranged chromosomes and exhibited a high frequency of aberrant anaphases. Isozyme and random amplified polymorphic DNA (RAPD) patterns of the hybrids from the same fusion combination were uniform. In the field, somatic hybrids showed wide phenotypic variation in 20 morphological characters. There was a significant correlation between certain leaf characters and the ploidy level, which may be used to distinguish the tetraploid hybrids from hexaploids and octoploids. Tuber yield and flowering intensity were highest in tetraploid hybrids (2n= 4x= 48). Eighteen of the 73 hybrids reached higher yields than the standard variety ‘Adretta’. Floral development and fertility were restored in hybrids derived from fusions between non-flowering or sterile dihaploids.     

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.     

Brassica napocampestris L. (2n=58). 1. Synthesis,cytology, fertility and general considerations

Summary The synthesis of leafy, forage forms of Brassica napocampestris (2n=58, aaaacc) by colchicine treatment of hybrids between B. napus (2n=38, aacc) and B. campestris (2n=20, aa) is described.The cytology of both triploid and hexaploid hybrids was studied. B. napocampestris proved fairly stable meiotically although multivalents were formed and some aneuploids detected in later generations. Seed fertility proved adequate for commercial production.The variation within the parental species and their easy crossability suggests that several distinct forms of B. napocampestris could be created, some of which may have agronomic potential. B. napocampestris may be used as a stepping stone in elaborating the range of variability within B. napus.     

Intergeneric symmetric and asymmetric somatic hybridization in Festuca and Lolium

Summary Intergeneric symmetric and asymmetric somatic hybrids have been obtained by fusion of metabolically inactivated protoplasts from embryogenic suspension cultures of tall fescue (Festuca arundinacea Schreb.) and unirradiated or 10–500 Gy-irradiated protoplasts from non-morphogenic cell suspensions of Italian ryegrass (Lolium multiflorum Lam.). Genotypically and phenotypically different somatic hybrid Festulolium mature flowering plants were regenerated.Species-specific sequences from F. arundinacea and L. multiflorum being dispersed and evenly-represented in the corresponding genomes were isolated and used for the molecular characterization of the nuclear make-up of the intergeneric, somatic Festulolium plants recovered. The irradiation of Italian ryegrass protoplasts with 250 Gy X-rays prior to fusogenic treatment favoured the unidirectional elimination of most or part of the donor chromosomes. Irradiation of L. multiflorum protoplasts with 500 Gy produced highly asymmetric (over 80% donor genome elimination) nuclear hybrids and clones showing a complete loss of donor chromosomes.The RFLP analysis of the organellar composition in symmetric and asymmetric tall fescue (+) Italian ryegrass regenerants confirmed their somatic hybrid character and revealed a bias towards recipient-type organelles when extensive donor nuclear genome elimination had occurred.Approaches aimed at improving persistence of ryegrasses based on asymmetric somatic hybridization with largely sexually-incompatible grass species (F. rubra and Alopecurus pratensis), and at transferring the cytoplasmic male sterility trait by intra- and inter-specific hybridization in L. multiflorum and L. perenne, have been undertaken.Abbreviations cpDNA chloroplast DNA - CMS cytoplasmic male sterility - 2,4-D 2,4-dichlorophenoxy-acetic acid - IOA iodoacetamide - mtDNA mitochondrial DNA - RFLP restriction fragment length polymorphism     

Analysis of chromosome, mtDNA and cpDNA patterns in five somatic hybrids between Brassica alboglabra Bailey and Brassica campestris L.

Summary Somatic hybrids between Brassica alboglabra (CC) and B. campestris (AA) were produced through protoplast fusion. Hybrids of this combination have already been produced through sexual crosses and among other traits in the offspring, a CMS of ogura type was discovered. The reason for generating somatic hybrids was to create new nuclear-cytoplasmic combinations and/or cytoplasmic variants, which could be exploited in combination with the sexual hybrids. Mitosis and meiosis were studied and all hybrids were found to be of the AACCCC genomic constitution. The fusion of somatic cells resulted in altered cytoplasmic genomes.     

Somatic hybridization as a tool for tomato breeding

Protoplast fusion can be used to produce somatic hybrids of species that cannot be obtained by sexual hybridization. The possibility to introgress genes from Solanum species into the cultivated tomato species Lycopersicon esculentum, and to obtain novel cytoplasm-nucleus combinations (cybrids) was considered as an important strategy to extend the genetic variation available for tomato breeding. Somatic hybrids between L. esculentum and other Lycopersicon species, as well as between L. esculentum and Solanum or Nicotiana species, have been produced. Specific mutants, genotypes with antibiotic resistances, and metabolic inhibition by iodoacetate or iodoacetamide and irradiation were used for the selection of hybrids. In addition, the improvement of protoplast culture techniques and the use of the favourable tissue culture traits derived from species such as L. peruvianum, which have been introduced into tomato by classical breeding, allowed the efficient recovery of somatic hybrids. However, the occurrence of somatic incongruity in fusion combinations of L. esculentum and Solanum and even more in L. esculentum and Nicotiana, did not allow the production of true cybrids and/or fertile hybrids, indicating the importance of both cytoplasm-nucleus and nucleus-nucleus interactions in somatic incongruity. Another problem with fusions between distantly related species is the strongly reduced fertility of the hybrids and the very limited homoeologous recombination between chromosomes of the parental species. Partial genome transfer from donor to recipient through microprotoplast (+) protoplast fusion, and the production of monosomic or disomic chromosome addition lines, light overcome some of these problems. In symmetric somatic hybrids between L. esculentum and S. tuberosum the occurrence of limited somatic and meiotic recombination was demonstrated. Fertile progeny plants could be obtained, though at a low frequency, when embryo rescue was performed on a large scale after backcrossing hexaploid somatic tomato (+) potato hybrids with a tetraploid potato genotype. The potential value of genomic in situ hybridization (GISH) and RFLPs for the analysis of the genome/chromosome composition of the hybrids has been demonstrated for intergeneric somatic hybrids between Lycopersicon and Solanum.Abbreviations cpDNA chloroplast DNA - mtDNA mitochondrial DNA     

A stable cytoplasmic male-sterile line of Brassica juncea carrying restructured organelle genomes from the somatic hybrid Trachystoma ballii+B. juncea

A cytoplasmic male-sterile (CMS) line of Brassica juncea has been developed by combining the cytoplasm originating from the somatic hybrid Trachystoma ballii+B. juncea, and the nucleus of B. juncea cv. Pusa Bold by repeated backcrossing. Male-sterile plants closely resembled the normal fertile B. juncea in general morphology, but had delayed flowering (5–7 days) when compared with fertile ‘Pusa Bold’ which flowered in 45 days. Stamens of the male-sterile line were transformed into petaloid structures. Pollen abortion occurred after tetrad formation. Female fertility of the male-sterile line was normal. Molecular analysis of organelle genomes indicated extensive mitochondrial DNA recombinations in the CMS line. Preliminary analysis of the chloroplast genome of the CMS line also indicated chloroplast DNA recombination.     

Fertile somatic hybrids of Solanum etuberosum (+) dihaploid Solanum tuberosum and their backcrossing progenies: relationships of genome dosage with tuber development and resistance to potato virus Y

The wild non-tuberous species Solanumetuberosum is resistant to biotic andabiotic stresses, but is very difficult tocross with cultivated potato. Therefore,interspecific somatic hybrids between adihaploid clone of potato S.tuberosum (2n=2x=24, AA genome) and thediploid species S. etuberosum(2n=2x=24, EE genome) were produced byprotoplast fusion. Among the 7 fertilefusion hybrids analysed by genomic insitu hybridisation (GISH), three groups ofplants were found with the genomicconstitution of AAEE, AAEEEE and AAAAEE.Four fusion hybrids had exactly theexpected chromosome composition, while eachof the three aneuploid hybrids had lost twochromosomes of S. etuberosum. Twobackcross progenies were developed, andGISH analysis was applied to analysetransmission of the parental chromosomesinto the sexual generations. BC₁hybrids derived from the crosses of thehexaploid somatic hybrids with tetraploidpotato were pentaploid with thetheoretically expected genomic compositionor with slight deviation from thisexpectation. In the three BC₂ hybridsanalysed by GISH seven to 12 chromosomes ofS. etuberosum were detected in thepredominant S. tuberosum background.No recombinant chromosomes in the hybridswere detected. Genome dosage affects tuberformation in hybrids and their progenies,but has less effect on resistance to potatovirus Y (PVY) in fusion hybrids. Severalgenotypes of the fusion hybrids andBC₁ progeny did not show viralinfection even in the graftingexperiments.     

Alteration of the genomic composition of Solanum nigrum (+) potato backcross derivatives by somatic hybridization: selection of fusion hybrids by DNA measurements and GISH

Fusion experiments were performed with a first (BC₁‐6738) and a second (BC₂‐9017) generation backcross hybrid of 6x Solarium nigrum (+) 2x potato somatic hybrids with potato cultivars. Because no progeny was obtained from the BC₂ genotypes, alternative approaches were sought to overcome the sexual crossing barrier. Five potato genotypes, one of which contains the hygromycin resistance gene, were used in the fusion experiments. All vigorous regenerants were used for the estimation of nuclear DNA content using flow cytometry. Plants with a DNA content higher than that of the BC₁‐6738 or BC₂ genotypes were considered potential somatic hybrids. Forty‐nine potential somatic hybrids resulted from fusion experiments with BC₁‐6738, from which 20 grew vigorously in the greenhouse and flowered. After pollination with several 4x potato cultivars, eight genotypes produced seeded berries and five genotypes gave seedless berries. In addition, 11 of these 13 somatic hybrids were selected for genomic in situ hybridization (GISH) analysis to determine their genomic composition. Nine had exactly or approximately the expected number of 36 S. nigrum and 60 potato chromosomes. In one genotype, only 22 instead of 36 S. nigrum chromosomes were found and one potato chromosome was possibly missing. Only five potential somatic hybrids were detected among the 79 regenerants from BC₂‐9017 (+) 2x potato fusion experiments that were analysed by flow cytometry. Two of these hybrids were rather vigorous and did flower, but pollinations with potato have not yet set any berries.     

Somatic Fusion for Breeding of Tetraploid Potatoes

From two tetraploid, one Transformed tetraploid, one triploid and 11 dihaploid clones of Solanum tuberosum somatic hybrids were produced by polyethylene glycol mediated somatic fusion. The inter-dihaploid clones comprised clones of agronomic value, homozygous doubled monohaploids, and in vitro selected clones resistant t0 Fusarium or Phytophthora toxins. Presumptive hybrids were enriched at the callus Stage in vitro by using differentiating media and by growth characteristics; further identification was performed by chromosome counting in vitro shoots and by isozyme analysis of in vitro plants. Final analysis was made from morphological characteristic of plant and tuber phenotypes. From 15 different combinations, 6009 plantlets have been regenerated. From five combinations, 310 reentrants were checked for hybrid nature by morphology and cytology and 88 by peroxidase and esterase isozyme analyses. Amongst these, from two combinations, a total of 17 different hybrids were confirmed by all methods. The procedures described are general enough to allow genome combination of interdihaploids resulting in tetraploids of practical breeding value.     

New interspecific rice genome constructions obtained by symmetric cell fusion

Newly released rice varieties are mostly progenies of common commercial cultivars and so most gene sources for rice breeding have closely related genotypes. Protoplast fusion technique is a method that can produce hybrid plants in one‐step within a desired cultivar. To introduce new genetic diversity into rice‐breeding materials, hybrids between Oryza sativa L. (AA) and Oryza punctata Kotschy ex Steud. (BBCC) were generated by symmetric protoplast fusion. The genomic distribution in interspecific somatic hybrids was investigated by multi‐colour genomic in situ hybridization. Differences in chromosome numbers were observed not only between different somatic hybrid plants but also within individual hybrid plants. Chromosomal reduction of the A genome was found in two somatic hybrids. These results suggest that it is possible to reconstruct new interspecific rice genomes. Chromosomal reduction in interspecific somatic hybrids may be used to generate breeding lines with novel chromosomal constitutions.     

Wide somatic hybrids of Citrus with its related genera and their potential in genetic improvement

Citrus wild relatives are an untapped germplasm reservoir, which possesses many elite resistance traits. Genetic introgression into Citrus by conventional methods has been greatly hindered by polyembryony, pollen / ovule sterility, sexual / graft incompatibility, long juvenility etc. Somatic hybridization via protoplast fusion may make it possible to transfer genes from wild relatives to Citrus. To date, more than sixty sexually compatible or incompatible intergeneric somatic hybrids between Citrus and its various related wild genera have been produced by PEG - orelectrically - induced fusion. These wide somatic hybrids were identified by morphology, cytology, isozyme, RAPD and RFLP analyses. Genetic variation or recombination was also revealed in some of them. Several sexually compatible combinations have flowered and set fruits. The agronomic performance of these wide somatic hybrids is diverse, and the current results are not conclusive. Somatic hybrids are being tested as rootstocks. The prospects of wide somatic hybridization of Citrus with its wild relatives are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Transfer of resistance to race 2 of Plasmodiophora brassicae from Brassica napus to cabbage (B. oleracea var. capitata). II. Meiosis in the interspecific hybrids between B. napus and 2x and 4x cabbage

Summary Meiosis in 14 interspecific F₁ hybrids with three chromosomal levels (triploid, tetraploid, hexaploid; 2n=28, 37 and 55) between Brassica napus L. and 2x and 4x cabbage (B. oleracea var. capitata L.) was studied. The oleracea genome from B. napus maintained close homology with the c genome of cabbage while the campestris genome of B. napus showed partial homology with the c genome contained in the hybrids. Genotypic influence on chromosome pairing was indicated. Structural chromosome differences and spontaneous chromosome breakage and reunion were suggested as causes for the abnormalities which related to the unbalance of the genotypes. The divergence of the genomes of B. napus and B. oleracea and the need for the qualification of the term secondary association were discussed.Contribution No. J. 673, Research Station, Agriculture Canada, St. Jean, Québec.     

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.     

Variability and inheritance of flowering time and duration in Actinidia chinensis (kiwifruit)

We investigated the variability and inheritance of flowering in a seedling population of diploid Actinidia chinensis Planch. We found considerable variation in time to reach 50% flowering (D50) and flowering duration (FD). D50 was highly heritable (h ² > 0.85) suggesting that this trait would respond well to selection in this population, whereas FD was greatly affected by the environment (h ² < 0.20). The results also showed that the flowering times of male vines were more sensitive to the environment than those of female vines. However, significant specific combining ability (SCA) effects could be achieved by targeting specific bi-parental combinations for breeding and selection of flowering time. The diversity in flowering traits among males will facilitate the selection of male pollinizers that coincide in flowering time with present or future female cultivars. The possibility of breeding female cultivars with a short flowering period to improve the consistency of fruit maturity at harvest is discussed.