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.     

Transfer of resistance against Phoma lingam to Brassica napus by asymmetric somatic hybridization combined with toxin selection

Summary Irradiated mesophyll protoplasts from nine different accessions of B. juncea, B. nigra and B. carinata, all resistant to Phoma lingam, were used as gene donors in fusion experiments with hypocotyl protoplasts isolated from B. napus as the recipient. A toxin, sirodesmin PL, was used to select those fusion products in which the resistant gene(s) was present. In the fusion experiments different gene donors, various irradiation dosages and toxin treatments were combined. Symmetric and asymmetric hybrid plants were obtained from the cell cultures with and without toxin selection. Isozymes were used to verify hybrid characters in the symmetric hybrids, whereas two DNA probes were used to identify donor-DNA in the asymmetric hybrids. Resistance to P. lingam was expressed in all symmetric hybrids, and in 19 of 24 toxin-selected asymmetric hybrids, while all the unselected asymmetric hybrids were susceptible.     

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.     

Production of intertribal somatic hybrids between Brassica napus L. and Lesquerella fendleri (Gray) Wats

Intertribal Brassica napus (+) Lesquerella fendleri hybrids have been produced by polyethylene glycol-induced fusions of B. napus hypocotyl and L. fendleri mesophyll protoplasts. Two series of experiments were performed. In the first, symmetric fusion experiments, protoplasts from the two materials were fused without any pretreatments. In the second, asymmetric fusion experiments, X-ray irradiation at doses of 180 and 200 Gy were used to limit the transfer of the L. fendleri genome to the hybrids. X-ray irradiation of L. fendleri mesophyll protoplasts did not suppress the proliferation rate and callus formation of the fusion products but did significantly decrease growth and differentiation of non-fused L. fendleri protoplasts. In total, 128 regenerated plants were identified as intertribal somatic hybrids on the basis of morphological criteria. Nuclear DNA analysis performed on 80 plants, using species specific sequences, demonstrated that 33 plants from the symmetric fusions and 43 plants from the asymmetric fusions were hybrids. Chloroplast and mitochondrial DNA analysis revealed a biased segregation that favoured B. napus organelles in the hybrids from the symmetric fusion experiments. The bias was even stronger in the hybrids from the asymmetric fusion experiments where no hybrids with L. fendleri organelles were found. X-ray irradiation of L. fendleri protoplasts increased the possibility of obtaining mature somatic hybrid plants with improved fertility. Five plants from the symmetric and 24 plants from the asymmetric fusion experiments were established in the greenhouse. From the symmetric fusions 2 plants could be fertilised and set seeds after cross-pollination with B. napus. From the asymmetric fusions 9 plants could be selfed as well as fertilised when backcrossed with B. napus. Chromosome analysis was performed on all of the plants but 1 that were transferred to the greenhouse. Three plants from the symmetric fusions contained 50 chromosomes, which corresponded to the sum of the parental genomes. From the asymmetric fusions, 11 hybrids contained 38 chromosomes. Among the other asymmetric hybrids, plants with 50 chromosomes and with chromosome numbers higher than the sum of the parental chromosomes were found. When different root squashes of the same plant were analysed, a total of 6 plants were found that had different chromosome numbers.     

Somatic hybrids with substitution type genomic configuration TCBB for the transfer of nuclear and organelle genes from Brassica tournefortii TT to allotetraploid oilseed crop B. carinata BBCC

Oilseed crop Brassica carinata BBCC is a natural allotetraploid of diploid species B. nigra BB and B. oleracea CC. To transfer the nuclear and organelle genes in a concerted manner from an alien species, B. tournefortii TT, to B. carinata, we produced somatic hybrids with genomic configuration TCBB using B. nigra and B. oleracea stocks that carried selectable marker genes. B. tournefortii TT was sexually crossed with hygromycin-resistant B. oleracea CC. Protoplasts isolated from shoot cultures of hygromycin-resistant F₁ hybrids of B. tournefortiixB. oleracea TC were fused with protoplasts of kanamycin-resistant B. nigra BB. In two different fusion experiments 80 colonies were obtained through selection on media containing both hygromycin and kanamycin. Of these, 39 colonies regenerated into plants. Analysis of 15 regenerants by random amplified polymorphic DNA (RAPD) markers showed the presence of all three genomes, thereby confirming these to be true hybrids. Restriction fragment length polymorphism (RFLP) analysis of organelle genomes with heterologous chloroplast (cp)and mitochondrial (mt) DNA probes showed that the chloroplast genome was inherited from either of the two parents while mitochondrial genomes predominantly showed novel configurations due to either rearrangements or intergenomic recombinations. We anticipate that the TCBB genomic configuration will provide a more conducive situation for recombination between the T and C genomes during meiosis than the TTCCBB or TCCBB type configurations that are usually produced for alien gene transfer. The agronomic aim of producing TCBB hybrids is to transfer mitochondrial genes conferring cytoplasmic male sterility and nuclear genes for fertility restoration from B. tournefortii to B. carinata.     

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.     

Effect of radiation dose on the production of and the extent of asymmetry in tomato asymmetric somatic hybrids

Summary Asymmetric somatic hybrids were recovered following fusion of tomato leaf mesophyll protoplasts with irradiated protoplasts isolated from Lycopersicon pennellii suspension cells. The asymmetry was determined by scoring the regenerants at between 20 and 24 loci using isozymes and restriction fragment length polymorphisms. In addition, three quantitative traits, fruit size, leaf shape, and stigma exsertion, were measured in the regenerants. The recovery of asymmetric somatic hybrids was as high as 50% of the regenerants, and there was no requirement for the transfer of a selectable marker gene from the irradiated partner. The amount of nuclear DNA transferred from the irradiated protoplast fusion partner was found to be inversely proportional to the radiation dose. It was possible to recover tomato asymmetric somatic hybrids which were self-fertile and contained limited amounts of genetic information from L. pennelli.     

Asymmetric somatic hybridization between tall fescue (Festuca arundinacea Schreb.) and irradiated Italian ryegrass (Lolium multiflorum Lam.) protoplasts

Intergeneric asymmetric somatic hybrids have been obtained by the fusion of metabolically inactivated protoplasts from embryogenic suspension cultures ofFestuca arundinacea (recipient) and protoplasts from a non-morphogenic cell suspension ofLolium multiflorum (donor) irradiated with 10, 25, 50, 100, 250 and 500 Gy of X-rays. Regenerating calli led to the recovery of genotypically and phenotypically different asymmetric somatic hybridFestulolium plants. The genome composition of the asymmetric somatic hybrid clones was characterized by quantitative dot-blot hybridizations using dispersed repetitive DNA sequences specific to tall fescue and Italian ryegrass. Data from dot-blot hybridizations using two cloned Italian ryegrass-specific sequences as probes showed that irradiation favoured a unidirectional elimination of most or part of the donor chromosomes in asymmetric somatic hybrid clones obtained from fusion experiments using donor protoplasts irradiated at doses 250 Gy. Irradiation of cells of the donor parent with 500 Gy prior to protoplast fusion produced highly asymmetric nuclear hybrids with over 80% elimination of the donor genome as well as clones showing a complete loss of donor chromosomes. Further information on the degree of asymmetry in regenerated hybrid plants was obtained from chromosomal analysis including in situ hybridizations withL. multiflorum-specific repetitive sequences. A Southern blot hybridization analysis using one chloroplast and six mitochondrial-specific probes revealed preferentially recipient-type organelles in asymmetric somatic hybrid clones obtained from fusion experiments with donor protoplasts irradiated with doses higher than 100 Gy. It is concluded that the irradiation of donor cells before fusion at different doses can be used for producing both nuclear hybrids with limited donor DNA elimination or highly asymmetric nuclear hybrid plants in an intergeneric graminaceous combination. For a wide range of radiation doses tested (25–250Gy), the degree of the species-specific genome elimination from the irradiated partner seems not to be dose dependent. A bias towards recipient-type organelles was apparent when extensive donor nuclear genome elimination occurred.Abbreviations cpDNA Chloroplast DNA - 2, 4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - IOA iodoacetamide - mtDNA mitochondrial DNA - RFLP restriction fragment length polymorphism     

Comparative study of symmetric and asymmetric somatic hybridization between common wheat andHaynaldia villosa

Symmetric and asymmetric protoplast fusion between long term cell suspension-derived protoplasts ofTriticum aestivum (cv. Jinan 177) and protoplasts ofHaynaldia villosa prepared from one-year-old embryogeneric calli was performed by PEG method. In asymmetric fusion, donor calli were treated with gamma ray at a dose of 40, 60, 80 Gy (1.3 Gy/min) respectively and then used to isolate protoplasts. Results of morphological, cytological, biochemical (isozyme) and 5S rDNA spacer sequence analysis revealed that we obtained somatic hybrid lines at high frequency from both symmetric and asymmetric fusion. Hybrid plants were recovered from symmetric and low dose γ-fusion combinations. GISH (genomicin situ hybridization) analysis proved exactly the existence of both parental chromosomes and the common occurrence of several kinds of translocation between them in the hybrid clones regenerated from symmetric and asymmetric fusion. And the elimination of donor DNA in hybrid clones regenerated from asymmetric fusion combinations was found to increase with the increasing gamma doses. It is concluded that transference and recombination of nuclear DNA can be achieved effectively by symmetric and asymmetric fusion, hybrids with small fragment translocation which are valuable in plant breeding can be obtained directly by asymmetric fusion.     

Transfer of resistance to the beet cyst nematode (Heterodera Schachtii Schm.) from Sinapis alba L. (white mustard) to the Brassica napus L. gene pool by means of sexual and somatic hybridization

Summary Sexual and somatic hybrid plants have been produced between Sinapis alba L. (white mustard) and Brassica napus L. (oil-seed rape), with the aim to transfer resistance to the beet cyst nematode Heterodera schachtii Schm. (BCN) from white mustard into the oil-seed rape gene pool. Only crosses between diploid accessions of S. alba (2n = 24, S_a1S_a1) as the pistillate parent and several B. napus accessions (2n = 38, AACC) yielded hybrid plants with 31 chromosomes. Crosses between tetraploid accessions of S. alba (2n = 48, S_a1S_a1S_a1S_a1) and B. napus were unsuccessful. Somatic hybrid plants were also obtained between a diploid accession of S. alba and B. napus. These hybrids were mitotically unstable, the number of chromosomes ranging from 56 to more than 90. Analysis of total DNA using a pea rDNA probe confirmed the hybrid nature of the sexual hybrids, whereas for the somatic hybrids a pattern identical to that of B. napus was obtained. Using chloroplast (cp) and mitochondrial (mt) DNA sequences, we found that all of the sexual F₁ hybrids and somatic hybrids contained cpDNA and mtDNA of the S. alba parent. No recombinant mtDNA or cpDNA pattern was observed. Three BC₁ plants were obtained when sexual hybrids were back-crossed with B. napus. Backcrossing of somatic hybrids with B. napus was not successful. Three sexual hybrids and one BC₁ plant, the latter obtained from a cross between a sexual hybrid and B. napus, were found to show a high level of BCN resistance. The level of BCN resistance of the somatic hybrids was in general high, but varied between cuttings from the same plant. Results from cytological studies of chromosome association at meiotic metaphase I in the sexual hybrids suggest partial homology between chromosomes of the AC and S_a1 genomes and thus their potential for gene exchange.     

Production of interspecific somatic hybrids between tuber mustard (<Emphasis Type="Italic">Brassica</Emphasis><Emphasis Type="Italic">juncea</Emphasis>) and red cabbage (<Emphasis Type="Italic">Brassica oleracea</Emphasis>)

In the present investigation, the interspecific somatic hybridization between tuber mustard and red cabbage was established in order to introduce valuable genes from red cabbage (Brassica oleracea) into Brassica juncea. Prior to fusion treatment, protoplasts of red cabbage were inactivated with 2 mM iodoacetamide to inhibit cell division. Micro-calluses were obtained at a frequency of 10.3% after approximately 5 weeks culture following protoplast fusion. Some of the fusion-derived calluses possessed red pigmented cells after being transferred to proliferation medium, and they were presumably considered to be somatic hybrid cell lines. Plantlets were regenerated from 12 cell lines, of which nine plantlets exhibited characteristics intermediate of both parents in terms of plant morphology. With the exception of common protein bands featured by two parents, there were unique banding patterns produced in the hybrids by using SDS-PAGE analysis. By chromosome countings, it was showed that they ranged approximately from 2n=30 to 42 in chromosome numbers. Their hybridity were further confirmed by RAPD analysis revealing that genes of both parents were partially incorporated into the hybrids. Positively, all these hybrids were capable of seed-setting. The pod-setting was 4.2 in somatic hybrid H₇ when backcrossed with tuber mustard.     

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.     

Streptomycin and lincomycin resistances are selective plastid markers in cultured Nicotiana cells

Summary Resistance to streptomycin and lincomycin in plant cell culture is used as a color marker: resistant cells are green whereas sensitive cells are white on the selective medium. Streptomycin and lincomycin at appropriate concentrations do not kill sensitive Nicotiana cells. The selective value of plastid ribosomal DNA mutations, conferring resistance to streptomycin and lincomycin, was investigated by growing heteroplastidic cells on a selective medium. The heteroplastidic cells were obtained by protoplast fusion, and contained a mixed population of streptomycin resistant plastids from the N. tabacum line Nt-SR1-Kan2, and lincomycin resistant plastids from the N. plumbaginifolia line Np-LR400-Hyg1. Clones derived from protoplast fusion were selected by kanamycin and hygromycin resistance, transgenic nuclear markers. Somatic hybrids were then grown on a selective streptomycin or lincomycin medium, or in the absence of either drug to a 50 to 100 mg size callus. Southern analysis of a polymorphic region of plastid DNA (ptDNA) revealed that somatic hybrids grown on streptomycin contained almost exclusively ptDNA from the streptomycin resistant parent, somatic hybrids grown on lincomycin contained almost exclusively ptDNA from the lincomycin resistant parent whereas somatic hybrids grown in the absence of either drug contained mixed parental plastids. Sensitive ptDNA was below detection level in most clones on selective medium, but could be recovered upon subsequent culture in the presence of the appropriate drug. The drugs streptomycin and lincomycin provide a powerful selection pressure that should facilitate recovery of plastid transformants.     

Synthesis of Ogura male sterile rapeseed (Brassica napus L.) with cold tolerance by protoplast fusion and effects of atrazine resistance on seed yield

Twenty-one cold-tolerant, male sterile Brassica napus somatic hybrids were produced by protoplast fusion. The fusion partners were a coldsensitive, Ogura cytoplasmic male sterile cauliflower inbred (B. oleracea var. botrytis inbred NY7642A) and a cold-tolerant, fertile canola-type B. rapa cv. Candle. Hybridity was confirmed by morphology, isozyme expression, flow cytometry, and DNA hybridization. Organellar analyses revealed a very strong bias for Brassica over Raphanus chloroplasts. Cold tolerance was confirmed by cold chamber studies and chloroplast DNA analyses. Good female fertility with 21.4 ± 3.1 seeds/pod was observed in the field using natural pollination vectors. Total seed yield was significantly greater for the atrazine-sensitive somatic hybrids produced in this study than for atrazine-resistant isolines.Abbreviations CMS cytoplasmic male sterility - IA iodoacetate - cpDNA chloroplast DNA     

Somatic hybridization between Brassica juncea and Moricandia arvensis by protoplast fusion

Intergeneric somatic hybrids have been produced between Brassica juncea (2n=36, AABB) cv. RLM-198 and Moricandia arvensis (2n=28, MM) by protoplast fusion. Hypocotyl protoplasts of B. juncea were fused with mesophyll protoplasts of M. arvensis using polyethylene glycol. Fusion frequency, estimated on the basis of differential morphological characterstics of parental protoplasts was about 5%. Of the 156 calli obtained, four calli produced shoots intermediate in morphology between the parents. Hybrid nature of the plants was confirmed using wheat nuclear rDNA probe. Hybridization of total DNA with a mitochondrial DNA probe carrying 5s–18s rRNA genes of maize showed that the mitochondria of the somatic hybrids were derived from the wild species M. arvensis. Meiosis in the only hybrid that produced normal flowers revealed the occurrence of 64 chromosomes, the sum of chromosomes of parental species. Inspite of complete pollen sterility, siliquas were produced in this hybrid by back-crossing with B. juncea. These siliquas on in vitro culture produced 12 seeds.     

Synthesis of high erucic acid rapeseed (Brassica napus L.) somatic hybrids with improved agronomic characters

Novel Brassica napus somatic hybrids have been created through protoplast fusion of B. oleracea var. botrytis and B. rapa var. oleifera genotypes selected for high erucic acid (22:1) content in the seed oil. Fifty amphidiploids (aacc) and one putative hexaploid (aacccc) hybrid were recovered in one fusion experiment. Conversely, only one amphidiploid and numerous regenerates with higher DNA contents were produced in a similar fusion using a different B. rapa partner. Hybridity was confirmed by morphology, isozyme expression, flow cytometry, and DNA hybridization. Analysis of organellar DNA revealed a distinct bias toward the inheritance of chloroplasts from the B. rapa (aa) genome. All amphidiploids set self-pollinated seed. A erucic acid content as high as 57.4% was found in the seed oil of one regenerated plant. Fatty acid composition was stable in the R₁ generation and was coupled with increased female fertility. Other novel agronomic characters in the hybrids recovered include large seed size, lodging resistance, and non-shattering seed pods.     

Comparison of UV light, X-ray and restriction enzyme treatment as tools in production of asymmetric somatic hybrids between Brassica napus and Arabidopsis thaliana

 Asymmetric somatic hybrids between Brassica napus (receptor) and Arabidopsis thaliana (donor) have been produced by three different methods supposed to induce asymmetry. The donor protoplasts were either UV- or X-irradiated, or the mixture of protoplasts was treated with the restriction enzyme PvuII immediately before fusion. The genome composition of the hybrids was analysed with Southern blot hybridisations using 15 different mapped A. thaliana RFLP markers as probes. Both UV- and X-irradiation were found to be efficient treatments for induction of asymmetry in somatic hybrids in a dose-dependent manner. The addition of a restriction enzyme to the protoplast mixture did not have any effect on the frequency of asymmetric hybrids or on the degree of asymmetry in the hybrids produced. UV- and X-irradiation resulted in higher fertility in the hybrids, while PvuII treatment did not have any effect on seed set. A significant positive correlation between degree of asymmetry in different plants and seed set after selfing was detected. Received: 18 December 1997 / Accepted: 7 January 1998     

Production and characterization of asymmetric somatic hybrids between Arabidopsis thaliana and Brassica napus

Summary Cell suspension-derived protoplasts of a chlorsulfuron-resistant (GH50) strain of Arabidopsis thaliana cv Columbia were X-irradiated at 60 or 90 krad, to facilitate the elimination of GH50 donor chromosomes in fusion products. Irradiated GH50 protoplasts were fused, with polyethylene glycol, to protoplasts derived from stem epidermal strips of Brassica napus cv Westar. Chlorsulfuron-resistant colonies were selected in vitro and then transferred to shoot and root regeneration medium. Seventeen hybrid lines were regenerated in vitro, and eight were successfully established in the greenhouse, where they flowered. These eight asymmetric hybrids were intermediate in vegetative morphology between Arabidopsis and Brassica. The flowers from these hybrids were male-sterile with abnormal petal and pistil structures. Zymograms for phosphoglucomutase, esterase, and peroxidase showed the presence of all parental isozymes in each of the hybrids tested. Nuclear hybridity was also confirmed for the ribosomal RNA genes using a wheat rDNA probe; however, the chloroplast genome in each of the hybrids was derived solely from the Brassica parent. All selected somatic hybrids were capable of rooting at levels of chlorsulfuron which were inhibitory to unfused Brassica plantlets. The degree of herbicide resistance in the hybrid shoots is presently being evaluated.Contribution No. 1428, Plant Research Centre, Agriculture Canada     

Plant regeneration capacity of mesophyll protoplasts from Brassica napus and related species

Protoplasts from a total of thirty-six genotypes of Brassica species – B. napus, B. campestris (syn. B. rapa), B. juncea, and three distant relatives, Orychophragmus violaceus, Isatis indigotica and Xinjiang wild rape – were analysed for shoot regeneration using a feeder culture system. With the exception of B. campestris and Xinjiang wild rape, some genotypes of all the species could regenerate plants with high efficiency (above 20% of isolated calli initiating shoots). Several genotypes with high regeneration ability were elite breeding lines. Culture conditions as well as genotype had a significant impact on shoot regeneration frequency. In particular, silver nitrate added to the regeneration medium at doses of 6 and 30 μM improved shoot regeneration frequency to 25.4% and 52.2% of isolated calli, respectively, compared to 7.3% percent shoot regeneration without silver nitrate in seven responsive genotypes. Addition of silver nitrate to the regeneration medium also induced shoot regeneration in non-responsive genotypes. Intact plants could be obtained within three months from protoplast isolation in the regenerative genotypes using the current culture system. Advantages of mesophyll protoplasts as compared to protoplasts isolated from hypocotyls for genetic manipulation in Brassica species are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Genetic characterization of asymmetric somatic hybrids between Bupleurum scorzonerifolium Willd and Triticum aestivum L.: potential application to the study of the wheat genome

In this paper, we describe how Bupleurum scorzonerifolium/Triticum aestivum asymmetric somatic hybrids can be exploited to study the wheat genome. Protoplasts of B. scorzonerifolium Willd were irradiated with ultraviolet light (UV) and fused with protoplasts of common wheat (T. aestivum L.). All cell clones were similar in appearance to those of B. scorzonerifolium, while the regenerated plantlets were either intermediate or B. scorzonerifolium-like. Genotypic screening using isozymes showed that 39.3% of cell clones formed were hybrid. Some of the hybrid cell clones grew vigorously, and differentiated green leaves, shoots or plantlets. DNA marker analysis of the hybrids demonstrated that wheat DNA was integrated into the nuclear genomes of B. scorzonerifolium and in situ karyotyping cells revealed that a few wheat chromosome fragments had been introgressed into B. scorzonerifolium. The average wheat SSR retention frequency of the RH panel was 20.50%, but was only 6.67% in fusions with a non-irradiated donor. B. scorzonerifolium chromosomes and wheat SSR fragments in most asymmetric hybrid cell lines remained stable over a period of 2.5–3.5 years. We suggest the UV-induced asymmetric somatic hybrids between B. scorzonerifolium Willd and T. aestivum L. have the potential for use in the construction of an RH map of the wheat genome.