Somatic hybrids between<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis> and cytoplasmic male-sterile radish (<Emphasis Type="Italic">Raphanus sativus</Emphasis>)

Somatic hybrids were produced by protoplast fusion between Arabidopsis thaliana ecotype Columbia and a male-sterile radish line MS-Gensuke (Raphanus sativus) with the Ogura cytoplasm. Forty-one shoots were differentiated from the regenerated calli and established as shoot cultures in vitro. About 20 of these shoots were judged to be hybrids based on growth characteristics and morphology. Molecular analyses of 11 shoots were performed, confirming the hybrid features. Of these 11 shoots, eight were established as rooted plants in the greenhouse. Polymerase chain reaction and randomly amplified polymorphic DNA analyses of the nuclear genomes of all analyzed shoots and plants confirmed that they contained hybrid DNA patterns. Their chromosome numbers also supported the hybrid nature of the plants. Investigations of the organelles in the hybrids revealed that the chloroplast (cp) genome was exclusively represented by radish cpDNA, while the mitochondrial DNA configuration showed a combination of both parental genomes as well as fragments unique to the hybrids. Hybrid plants that flowered were male-sterile independent of the presence of the Ogura CMS-gene orf138.Abbreviations CMS Cytoplasmic male sterilityCommunicated by M.R. Davey     

通过不对称体细胞杂交向小麦转移青苗碱谷DNA

普通小麦"济南177"(Triticum aestivum L.cv.Jinan177)经继代培养和选择,形成了性质不同的愈伤组织,一种生长迅速,易于形成悬浮系,游离的原生质具有旺盛的分裂能力,但不能分化,称为Cha9;另一种具有一定能力,但游离原生质体分裂能力低,称为176.二者之一来源的原生质体与紫外线照射的青苗碱谷原生质体融合均不能获得再生植株.而将源于Cha9和176的两种原生质体混合,与经紫外线照射的青苗碱谷原生质体在PEG诱导下融合,融合产物再生了大量植株.再生的愈伤组织及植株经表现型、细胞学、有工酶、RAPD分析,证明了其杂种性质,用不麦叶绿体特异的简单得复序列(SSR)引物分析了再生杂种的叶绿体遗传组成情况.在不同的杂种克隆中,同时带有Cha9和176的遗传物质并含有供体核及胞质基因组的克隆4具有较高的分化能力,再生了大量生长旺盛的完整植株.     

Genotyping of somatic hybrids between <Emphasis Type="Italic">Festuca arundinacea</Emphasis> Schreb. and <Emphasis Type="Italic">Triticum aestivum</Emphasis> L.

In order to genotype hybrid genomes of distant asymmetric somatic hybrids, we synthesized hybrid calli and plants via PEG-mediated protoplast fusion between recipient tall fescue (Festuca. arundinacea Schreb.) and donor wheat (Triticum aestivum L.). Seventeen and 25 putative hybrid clones were produced from the fusion combinations I and II, each with the donor wheat protoplast treated by UV light for 30 s and 1 min, respectively. Isozyme and RAPD profiles confirmed that ten hybrid clones were obtained from combination I and 19 from combination II. Out of the 29 hybrids, 12 regenerated hybrid plants with tall fescue phenotype. Composition and methylation-variation of the nuclear and cytoplasmic genomes of some hybrids, either with or without regenerative ability, were compared by genomic in situ hybridization, restriction fragment length polymorphism, and DNA methylation-sensitive amplification polymorphism. Our results indicated that these selected hybrids all contained introgressed nuclear and cytoplasmic DNA as well as obvious methylation variations compared to both parents. However, there were no differences either in nuclear/cytoplasmic DNA or methylation degree between the regenerable and non-regenerable hybrid clones. We conclude that both regeneration complementation and genetic material balance are crucial for hybrid plant regeneration.     

Construction of a primary RH panel of Italian ryegrass genome via UV-induced protoplast fusion

Symmetric and asymmetric somatic hybrids were produced via protoplast fusion between common wheat ( TRITICUM AESTIVUM L.) cv. "Jinan 177" and Italian ryegrass ( LOLIUM MULTIFLORUM Lam.). The ryegrass without or with UV irradiation was used as a donor, providing a small amount of chromatin. In these somatic hybrids, most ryegrass chromosomes have been confirmed preferential elimination and the somatic hybrid calli and plants showed wheat-like morphology. Some of the hybrid lines were used for the analysis of distribution and heredity of donor DNA in the hybrid genome and the possibility of establishing a radiation hybrid (RH) panel of the ryegrass in the present experiment. These hybrids, subcultured for two and three years, retained the ryegrass DNA examined by RFLP and GISH analysis, respectively. Distribution of the ryegrass DNA in the wheat genomes of 20 single-cell individuals, randomly selected from hybrid cell lines produced, were analyzed by 21 ryegrass genome specific SSR markers. The average frequencies of molecular marker retention in symmetric hybrid lines (UV 0), as well as asymmetric hybrid lines from UV 30 s and 1 min were 10.88, 15.48 and 33.86, respectively. It was suggested that the UV dose increased the introgression of donor DNA into wheat genome. The ryegrass SSR fragments in most asymmetric hybrid cell lines remained stable over a period of 2 approximately 3 years. This revealed that those asymmetric somatic hybrids are suitable for the introgression of ryegrass DNA into wheat, and for RH panel and RH mapping.     

通过不对称体细胞杂交向小麦转移青苗碱谷DNA(英文)

普通小麦“济南177”(Triticum aestzvum L.cv.Jinan 177)经继代培养和选择,形成了两种性质不同的愈伤组织,一种生长迅速,易于形成悬浮系,游离的原生质体具有旺盛的分裂能力,但不能分化,称为Cha 9;另一种具有一定分化能力,但游离的原生质体分裂能力低,称为176。二者之一来源的原生质体与紫外线照射的青苗碱谷原生质体融合均不能获得再生植株。而将来源于Cha 9和176的两种原生质体混合,与经紫外线照射的青苗碱谷原生质体在PEG诱导下融合,融合产物再生了大量植株。再生的愈伤组织及植株经表型、细胞学、同工酶、RAPD分析,证明了其杂种性质,用小麦叶绿体特异的简单重复序列(SSR)引物分析了再生杂种的叶绿体遗传组成情况。在不同的杂种克隆中,同时带有Cha 9和176的遗传物质并含有供体核及胞质基因组的克隆4具有较高的分化能力,再生了大量生长旺盛的完整植株。     

Production and characterization of somatic hybrid plants between leek (Allium ampeloprasum L.) and onion (Allium cepa L.)

 Results are reported on the production and characterization of somatic hybrids between Allium ampeloprasum and A. cepa. Both symmetric and asymmetric protoplast fusions were carried out using a polyethylene-based mass fusion protocol. Asymmetric fusions were performed using gamma ray-treated donor protoplasts of A. cepa and iodoacetamide-treated A. ampeloprasum protoplasts. However, the use of gamma irradiation to eliminate or inactivate the donor DNA of A. cepa proved to be detrimental to the development of fusion calli, and thus it was not possible to obtain hybrids from asymmetric fusions. The symmetric fusions yielded a high number of hybrid calli and regenerated plants. The analysis of the nuclear DNA composition using interspecific variation of rDNA revealed that most of the regenerated plants were hybrids. Flow cytometric analysis of nuclear DNA showed that these hybrid plants contained a lower DNA content than the sum of the DNA amounts of the parental species, suggesting that they were aneuploid. A shortage of chromosomes in the hybrids was confirmed by genomic in situ hybridization. Chromosome counts in metaphase cells of six hybrids revealed that these plants lacked 2–7 leek chromosomes. One hybrid showed also the loss of onion chromosomes. The hybrids had an intermediate phenotype in leaf morphology. The application of these somatic hybrids in breeding is discussed. Received: 7 April 1997 / Accepted: 10 September 1997     

Morphological and molecular characterization of fertile tetraploid somatic hybrids produced by protoplast electrofusion and PEG-induced fusion between Lycopersicon esculentum Mill. and Lycopersicon peruvianum Mill.

Summary Mesophyl protoplasts of two genotypes of cultivated tomato (Lycopersicon esculentum Mill.) and one of its wild relative species (Lycopersicon peruvianum Mill.) were fused by using electrofusion and polyethyleneglycol-induced fusion. Forty-three fertile tetraploid somatic hybrid plants, each deriving from separate calli, were recovered from both fusion procedures. Electrofusion appeared more efficient than chemical fusion for the production of somatic hybrids. These plants appeared morphologically similar, whatever the fusion procedure and tomato genotype. They had intermediate leaf, inflorescence, and flower morphology. After self-pollination, the hybrids set fruit of intermediate size and color. The hybrid nature of these plants was confirmed by isoelectric focusing of the Rubisco small subunits used as nuclear markers. L. esculentum and L. peruvianum were distinguished by means of two chloroplast markers: CF1-ATPase subunit as analyzed by isoelectro-focusing and ct DNA restriction patterns. All hybrids displayed both ct markers of only one parent with no biased transmission. Mitochondrial (mt) DNAs were prepared from flower buds by using miniaturized CsCl gradients. Preliminary analysis indicated that mt genomes from the hybrids all differed from those of both parents. mt DNA Sall restriction enzyme analysis revealed that all but two hybrids contained one novel fragment of 13.5 kb. Gene mapping experiments showed that the mt apocytochrome b and ATPase subunit 9 homologies in the somatic hybrid mt DNA resembled L. esculentum and L. peruvianum, respectively; the mt nad5 probe distinguished at least four distinct patterns in the hybrids. These results indicated that mt DNA rearrangements involving intergenomic recombinations occurred through protoplast fusion. A greater mt DNA polymorphism was induced with chemical fusion than with electrofusion.     

Interspecific hybridization between Brassica juncea and B. spinescens through protoplast fusion

Hypocotyl derived protoplasts of B. juncea cv. RLM-198 were fused with mesophyll protoplasts of B. spinescens using polyethylene glycol to produce interspecific hybrids. Fusion products could be microscopically identified by characteristics of the protoplasts of both parents in the hybrid cells; they are colourless and vacuolated like the hypocotyl protoplasts and possess chloroplasts of the mesophyll protoplasts. The heterokaryotic fusion frequency was around 5%. However, the frequency of calli regenerating hybrid shoots was more than 10% of the regenerating calli. Putative somatic hybrids had morphological features characteristic of both the parents. Twelve plants analysed cytologically, possessed 52 chromosomes (26_II) at meiosis representing the complete genomes of B. juncea (18_II) and B. spinescens (8_II). For esterase isozymes, the hybrids had bands of Doth the parents. Hybrid nature of some of the plants was confirmed by their close resemblance to B. juncea, chromosome number and isozyme bands of B. spinescens as in Rsp-19. Somatic hybrids had rudimentary, non-dehiscent anthers and completely sterile pollen. However, on back crossing with B. juncea, 10 out of 12 plants produced seeds and about 100 plants were realized.Abbreviations PEG Polyethylene glycol     

Nuclear genomic composition of asymmetric fusion products between irradiated transgenic Solanum brevidens and S. tuberosum: limited elimination of donor chromosomes and polyploidization of the recipient genome

Summary The production of asymmetric somatic hybrid calli after fusion between gamma-irradiated protoplasts from transgenic Solanum brevidens and protoplasts from S. tuberosum are reported. Transgenic (kanamycin-resistant, GUS-positive) S. brevidens plants and hairy root clones were obtained after transformation with Agrobacterium tumefaciens LBA 1060 (pRi1855) (pBI121) and LBA 4404 (pRAL4404) (pBI121), and A. rhizogenes LBA 9402 (pRi1855) (pBI121), respectively. Leaf protoplasts isolated from the transgenic plants or root protoplasts from the hairy root clones were fused with S. tuberosum leaf protoplasts, and several calli were selected on kanamycin-containing medium. The relative nuclear DNA content of the hybrid calli was measured by flow cytometry (FCM), and the percentages of DNA of the S. brevidens and S. tuberosum genomes in the calli were determined by dot blot analysis using species-specific DNA probes. Chromosome-specific restriction fragment length polymorphism (RFLP) markers were used to investigate the elimination of specific S. brevidens chromosomes in the hybrids. The combined data on FCM, dot blot and RFLP analysis revealed that 18–62% of the S. brevidens DNA was eliminated in the hybrid calli and that the RFLP marker for chromosome 7 was absent in seven out of ten calli. The absence of RFLP markers for chromosomes 5 and 11 hardly ever occurred. In most of the hybrids the ploidy level of the S. tuberosum genome had increased considerably.     

Callus Formation from Somatic Hybridization of Wheat (Triticum aestivum L.) and Ryegrass (Lolium perenne L.) by Electrofusion

Somatic hybrid calli were recovered following electrofusion of protoplasts from a chloroplast-containing cell suspension culture of wheat (Triticum aestivum L.) and a cell suspension culture of ryegrass (Lolium perenne L.). The protoplasts of wheat were inactivated by iodoacetamide; in addition morphology and colour were used as markers to aid selection of putative hybrid calli. For isozyme analysis of putative hybrids, nine isozymes were tested for differences in bands between the parental lines. Of these, three showed differences (ADH, GOT, SDH). Analysis of ADH bands of calli indicated that six lines were hybrids. These lines were analysed with the ,ther isozymes, and at the DNA level by Southern hybridisation with a wheat ribosomal DNA probe. The overall results indicated that one line was an almost complete combination of the genomes of the parental lines, but the other 5 lines were probably partial hybrids. In the latter, some loss of the wheat genome had probably occurred.     

Production of somatic hybrids between Diospyros glandulosa and D. kaki by protoplast fusion

Interspecific somatic hybrids between Diospyros glandulosa (2n=2x=30) and D. kaki cv. Jiro (2n=6x=90) were produced by electrofusion of protoplasts. Protoplasts were isolated from calli derived from leaf primordia, fused electrically, and cultured by agarose-bead culture using a modified KM8p medium. Flow cytometry revealed that the nuclear DNA content was the sum of those of D. glandulosa and D. kaki cv. Jiro in 149 of the 166 calli obtained. RAPD analysis showed that the 149 callus lines yielded specific bands for both D. glandulosa and D. kaki cv. Jiro and further confirmed that they were interspecific somatic hybrid calluses. Shoots were regenerated from 63 of the 149 interspecific hybrid calluses. Chloroplast DNA analysis by PCR-RFLP, flow cytometric determination of nuclear DNA content, and RAPD analysis revealed that the 63 interspecific hybrid shoot lines contained the nuclear genomes from both parents but only the chloroplast genome from D. glandulosa. Microscopic observation of root tip cells demonstrated that somatic chromosome number of the interspecific hybrids was 2n=8x=120. This revised version was published online in June 2006 with corrections to the Cover Date.     

Organelle analysis of symmetric and asymmetric hybrids between Lycopersicon peruvianum and Lycopersicon esculentum

Summary The organelles of somatic hybrids obtained from symmetric and asymmetric fusions between the Lycopersicon species L. peruvianum and L. esculentum were analyzed by DNA hybridization methods. In the asymmetric fusions the L. peruvianum protoplasts were gamma-irradiated at a dose of 50, 300 and 1,000 Gy. The organelles were characterized using the Petunia chloroplast probe pPCY64 and the mitochondrial EcoRI-SalI fragment of the Pcf gene. In all symmetric and asymmetric hybrid plants, a total of 73 being analyzed, only one of the parental chloroplast genomes was present, except for one hybrid plant which harbored both parental chloroplast genomes. No recombination and/or rearrangement in the chloroplast genome could be identified with the pPCY64 probe. Irradiation of the L. peruvianum protoplasts did not significantly reduce the fraction of asymmetric hybrids with L. peruvianum chloroplasts. A novel mitochondrial restriction pattern was present in 5 out of 24 hybrids tested. In 9 hybrids novel combinations of chloroplasts and mitochondria were found, indicating that both organelle types sorted out independently.     

Construction of whole genome radiation hybrid panels and map of chromosome 5A of wheat using asymmetric somatic hybridization

To explore the feasibility of constructing a whole genome radiation hybrid (WGRH) map in plant species with large genomes, asymmetric somatic hybridization between wheat (Triticum aestivum L.) and Bupleurum scorzonerifolium Willd. was performed. The protoplasts of wheat were irradiated with ultraviolet light (UV) and gamma-ray and rescued by protoplast fusion using B. scorzonerifolium as the recipient. Assessment of SSR markers showed that the radiation hybrids have the average marker retention frequency of 15.5%. Two RH panels (RHPWI and RHPWII) that contained 92 and 184 radiation hybrids, respectively, were developed and used for mapping of 68 SSR markers in chromosome 5A of wheat. A total of 1557 and 2034 breaks were detected in each panel. The RH map of chromosome 5A based on RHPWII was constructed. The distance of the comprehensive map was 2103 cR and the approximate resolution was estimated to be ～501.6 kb/break. The RH panels evaluated in this study enabled us to order the ESTs in a single deletion bin or in the multiple bins cross the chromosome. These results demonstrated that RH mapping via protoplast fusion is feasible at the whole genome level for mapping purposes in wheat and the potential value of this mapping approach for the plant species with large genomes.     

小麦与多年生黑麦草原生质体的电融合及杂种愈伤组织的形成

多年生黑麦草(Lolium perenne L.)悬浮培养细胞来源的原生质体和小麦(Triticumaestivum L.)含叶绿体的悬浮培养细胞来源的原生质体间,用直流方波脉冲进行电融合,获得了体细胞杂种愈伤组织。小麦的原生质体经过碘乙酰胺失活。愈伤组织的形态和颜色被用作识别预期杂种的标记。为了对杂种愈伤组织进行同工酶分析,观察了亲本的9种同工酶谱,其中3种在亲本间表现出差异(ADH、GOT 和 SDH)。酒精脱氢酶(ADH)的分析结果表明,有6个细胞系表现出杂种带。这些细胞系经过其他两种同工酶分析和 rDNA 探针杂交试验表明,一个细胞系表现出基本完全的亲本基因组间的组合,其余5个细胞系是部分杂种。     

Independent segregation of the plastid genome and cytoplasmic male sterility in Petunia somatic hybrids

Summary The chloroplast genomes of three sets of Petunia somatic hybrids were analyzed to examine the relationship between chloroplast DNA (cpDNA) composition and cytoplasmic male sterility (CMS). Chloroplast genomes of somatic hybrid plants were identified either by restriction and electrophoresis of purified cpDNAs or by hybridization of total DNA digests with cloned cpDNA probes that distinguish the parental genomes.The chloroplast genomes of a set of seven somatic hybrids derived from the fusion of Petunia CMS line 2423 and fertile line 3699 were analyzed. All seven plants were fertile, and all exhibited the cpDNA restriction pattern of the sterile cytoplasm. Similarly, four fertile somatic hybrids derived from the fusion of CMS line 3688 and fertile line 3677 were found to contain the CMS chloroplast genome. The cpDNA compositions of four fertile and two sterile somatic hybrids derived from the fusion of CMS line 3688 and fertile line 3704 were determined by restriction analysis of purified cpDNAs; all six plants exhibited the cpDNA restriction pattern of line 3704. Thus the CMS phenotype segregates independently of the chloroplast genome in Petunia somatic hybrids, indicating that CMS in Petunia is not specified by the chloroplast genome.     

Novel flowering and fatty acid characters in rapid cycling Brassica napus L. resynthesized by protoplast fusion

Novel rapid cycling Brassica napus lines have been produced by protoplast fusion between rapid cycling B. oleracea and rapid cycling B. rapa. Fusion products were selected based on iodoacetate inactivation and regeneration ability. A total of 36 plants was recovered from 3 regenerating calli. All were confirmed as somatic hybrids by morphological features, flow cytometric estimation of nuclear DNA content, RAPD analysis and/or DNA hybridization. Plants from two of the calli contained chloroplasts from B. rapa, and plants from the third contained B. oleracea chloroplasts. Some plants flowered in vitro, but on average flowering was initiated 22 days after transfer to soil. Although seed set was fairly low after self pollination, more seeds were obtained from pollination of open flowers than from pollination of buds. Seeds of the somatic hybrid B. napus showed novel fatty acid compositions, different from the mean of the two parental lines. Flowering was monitored in plants grown from seeds of the somatic hybrids, rapid cycling B. napus (CrGC 5-1) and the two diploid parental genotypes. Progeny of the somatic hybrids flowered faster and were more vigorous than rapid cycling B. napus (CrGC 5-1). The improved lines contain chloroplasts from B. rapa, unlike rapid cycling B. napus (CrGC 5-1), which has B. oleracea chloroplasts. The somatic hybrid lines produced may be useful for genetic studies or further in vitro manipulations.Abbreviations CrGC Crucifer Genetics Cooperative, University of Wisconsin-Madison - MES 1-morpholino-ethane sulfonate - MS-3,0 Murashige and Skoog medium containing 3% sucrose and no growth regulators - RAPD random amplified polymorphic DNA - RC rapid cycling - RFLP restriction fragment length polymorphism - std standard deviation - TE 10mM Tris, 1 mM EDTA, pH 8     

Production of asymmetric hybrids between Arabidopsis thaliana and Brassica napus utilizing an efficient protoplast culture system

Application of the protoplast culture method developed for Brassica protoplasts to protoplasts of Arabidopsis thaliana has increased the opportunities for interspecific hybridizations involving Arabidopsis. A more-efficient and much-simpler method was established compared to the earlier-reported protocol developed for A. thaliana protoplasts in which alginate beads were utilized. Mesophyll protoplasts of A. thaliana (ecotypes 'Landsberg erecta' and 'Wassilewskija') were cultured in the modified 8p liquid medium, which had been developed for Brassica protoplasts. For comparison, protoplasts were cultured in sodium alginate beads supplied with B5 medium according to the protocol for A. thaliana. The protoplasts divided with high frequencies in the 8p medium, and calli proliferated more rapidly than in the sodium alginate beads. High frequencies of shoot differentiation and regeneration were observed in calli of both ecotypes, from about 30% in the ecotype 'Wassilewskija' to about 60% for 'Landsberg erecta'. The more-rapidly the calli developed, the higher the regeneration frequencies were. Asymmetric hybrids between A. thaliana and Brassica napus were obtained by treating the protoplasts of A. thaliana with iodoacetamide (IOA) and B. napus protoplasts with UV-irradiation before fusion with polyethylene glycol (PEG). By using the culture procedure developed for Brassica protoplasts, calli developed and plants were regenerated. Although most of the plants regenerated after cell fusion were A. thaliana-like and were judged to be escapes from IOA treatment, more than ten plants showed hybrid features of both morphological and molecular characters. Among the hybrids that have flowered so far, both male-fertile and male-sterile plants have been obtained. Back-crossings to A. thaliana are now in progress as is morphological and molecular characterization of the plants.     

甜橙与酸橙体细胞杂种核质组成鉴定(英文)

采用流式细胞术(flow cytometry, FCM)、简单重复序列(simple sequence repeat, SSR)和酶切扩增多型性序列(cleaved amplified polymorphic sequence, CAPS)等技术分析酸橙(Citrus aurantium L. )叶肉原生质体和甜橙(C. sinenis Osbeck cv. Shamouti)胚性愈伤组织原生质体电融合再生的体细胞杂种。FCM研究结果表明,所有的体细胞杂种植株荧光强度是二倍体对照的2倍,说明所分析的植株为四倍体。用SSR和CAPS分析了体细胞杂种的核质遗传组成,在试验的4对SSR引物中,有2对能区分开融合亲本。在2对引物中,体细胞杂种植株包含双亲的全部特异带,表明它们为异核杂种。通用引物扩增结合限制性内切酶酶切能鉴别融合亲本,在具有多型性的引物/酶组合中,所有体细胞杂种的线粒体和叶绿体DNA带型与胚性亲本(甜橙)完全一样。结果表明体细胞杂种核基因组来自双亲,而胞质基因组来自悬浮系亲本。讨论了所用技术的特点、柑橘四倍体体细胞杂种核质遗传规律及本组合体细胞杂种的应用。     

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.     

Fertility and chromosome stability in Brassica napus resynthesised by protoplast fusion

Summary Fertile somatic hybrids between Brassica campestris and B. oleracea have been produced by protoplast fusion. Fusion products were identified by their intermediate protoplast morphology. Heterokaryons were isolated either with micropipettes using a micromanipulator or by flow sorting. About 2% of the obtained calli differentiated to shoots. Of the shoots obtained from manually selected heterokaryons, 100% were true hybrids as confirmed by isozyme analysis while 87% of the flow sorted ones showed a hybrid pattern. Ploidy level of the hybrid plants was determined by chromosome counting and relative DNA-content analysis. The sum of the chromosome number (38) from the two fusion partners were found in 30% of the hybrids; 9% had fewer and 61% had more chromosomes. Pollen viability and seed set varied with ploidy level. Compared to natural B. napus, a pollen viability of 52%–93% and a fertility of 1%–40% was found for the somatic hybrids with normal chromosome number. Restriction enzyme analysis of chloroplast-DNA showed that either B. campestris or B. oleracea chloroplasts were present in the somatic hybrid plants. Of 11 hybrid plants 5 had the campestris and 6 had the oleracea type (11 ratio).