Molecular analysis of the nuclear organellar genotype of somatic hybrid plants between tomato (Lycopersicon esculentum) and Lycopersicon chilense

Summary Somatic hybrid plants were recovered following fusion of leaf mesophyll protoplasts isolated from tomato (Lycopersicon esculentum) cultivar UC82 with protoplasts isolated from suspension cultured cells of L. chilense, LA 1959. Iodoacetate was used to select against the growth of unfused tomato protoplasts. Two somatic hybrids were recovered in a population of 16 regenerants. No tomato regenerants were recovered; all of the non-hybrid regenerants were L. chilense. The L. chilense protoplast regenerants were tetraploid. The hybrid nature of the plants was verified using species-specific restriction fragment length polymorphisms for the nuclear, chloroplast and mitochondrial genomes. The somatic hybrids had inherited the chloroplast DNA of the tomato parent, and portions of the mitochondrial DNA of the L. chilense parent. The somatic hybrids formed flowers and developed seedless fruit.     

Nuclear and cytoplasmic genome composition of Solanum bulbocastanum (+) S. tuberosum somatic hybrids.

Somatic hybrids between the wild incongruent species Solanum bulbocastanum (2n = 2x = 24) and S. tuberosum haploids (2n = 2x = 24) have been characterized for their nuclear and cytoplasmic genome composition. Cytologic observations revealed the recovery of 8 (near-)tetraploid and 3 hexaploid somatic hybrids. Multicolor genomic in situ hybridization (GISH) analysis was carried out to study the genomic dosage of the parental species in 5 somatic hybrids with different ploidy. The GISH procedure used was effective in discriminating parental genomes in the hybrids; most chromosomes were unambiguously colored. Two (near-)tetraploid somatic hybrids showed the expected 2:2 cultivated-to-wild genomic dosage; 2 hexaploids revealed a 4:2 cultivated-to-wild genomic dosage, and 1 hexaploid had a 2:4 cultivated-to-wild genomic dosage. Characterization of hybrid cytoplasmic genomes was performed using gene-specific primers that detected polymorphisms between the fusion parents in the intergenic regions. The analysis showed that most of the somatic hybrids inherited the plastidial and mitochondrial DNA of the cultivated parent. A few hybrids, with a rearranged mitochondrial genome (showing fragments derived from both parents), were also identified. These results confirmed the potential of somatic hybridization in producing new variability for genetic studies and breeding.     

Non-random inheritance of organellar genomes in symmetric and asymmetric somatic hybrids between Lycopersicon esculentum and L. pennellii

Summary The organization of the mitochondrial genome and the genotype of the chloroplast genome was characterized using restriction fragment length polymorphisms in a population (82 individuals) of symmetric and asymmetric somatic hybrids of tomato. The protoplast fusion products were regenerated following the fusion of leaf mesophyll protoplasts of Lycopersicon esculentum (tomato cv UC82) with suspension cell protoplasts of L. pennellii that had been irradiated with 5, 10, 15, 25, 50, or 100 kRads from a gamma source. The chloroplast genome in the somatic hybrids showed a random pattern of inheritance, i.e., either parental genome was present in equal numbers of regenerants, while in asymmetric somatic hybrids, the chloroplast genotype reflected the predominant nuclear genotype, i.e., tomato. The mitochondrial genome in the symmetric somatic hybrids showed a non-random pattern of inheritance, i.e., predominantly from the L. pennellii parent; asymmetric somatic hybrids had more tomato-specific mitochondrial sequences than symmetric somatic hybrids. The non-random inheritance of the chloroplast and mitochondrial DNA in these tomato protoplast fusion products appears to be influenced by the nuclear background of the regenerant.     

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。     

甜橙与酸橙体细胞杂种核质组成鉴定

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 amplifiedpolymorphic 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.     

Transmission of organelle genomes in citrus somatic hybrids

Restriction fragment length polymorphisms (RFLPs), were used to analyze the organelle composition of two-year-old trees, recovered from two different experiments: protoplasts from embryogenic cell suspensions of `Succari' sweet orange (C. sinensis L. Osbeck) were fused with leaf protoplasts of Citropsis gilletiana Swingle & M. Kell or to leaf protoplasts of Atalantia ceylanica(Arn.) Oliv. The somatic hybrids of both fusion combinations had the mitochondrial genome from the embryogenic partner. In some somatic hybrids, non-parental fragments were observed among the mitochondrial patterns. Somatic hybrids between `Succari' + Atalantia had plastid DNA from the embryogenic parent, while the somatic hybrids of `Succari' + Citropsis all had both parental chloroplast genomes. The relative abundance of organelle DNAs in the donor embryogenic and leaf cells may explain the consistent transmission of the embryogenic parent mitochondrial DNA and the inheritance of the chloroplast genome from either parent. This revised version was published online in June 2006 with corrections to the Cover Date.     

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     

Intergeneric somatic hybridization in Gramineae: somatic hybrid plants between tall fescue (Festuca arundinacea Schreb.) and Italian ryegrass (Lolium multiflorum Lam.)

Summary Tall fescue (Festuca arundinacea Schreb.) protoplasts, inactivated by iodoacetamide, and non-morphogenic Italian ryegrass (Lolium multiflorum Lam.) protoplasts, both derived from suspension cultures, were electrofused and putative somatic hybrid plants were recovered. Two different genotypic fusion combinations were carried out and several green plants were regenerated in one of them. With respect to plant habitus, leaf and inflorescence morphology, the regenerants had phenotypes intermediate between those of the parents. Southern hybridization analysis using a rice ribosomal DNA probe revealed that the regenerants contained both tall fescue- and Italian ryegrass-specific-DNA fragments. A cloned Italian ryegrass-specific interspersed DNA probe hybridized to total genomic DNA from Italian ryegrass and from the green regenerated somatic hybrid plants but not to tall fescue. Chromosome counts and zymograms of leaf esterases suggested nuclear genome instability of the somatic hybrid plants analyzed. Four mitochondrial probes and one chloroplast DNA probe were used in Southern hybridization experiments to analyze the organellar composition of the somatic hybrids obtained. The somatic hybrid plants analyzed showed tall fescue, additive or novel mtDNA patterns when hybridized with different mitochondrial gene-specific probes, while corresponding analysis using a chloroplast gene-specific probe revealed in all cases the tall fescue hybridization profile. Independently regenerated F. arundinacea (+) L. multiflorum somatic hybrid plants were successfully transferred to soil and grown to maturity, representing the first flowering intergeneric somatic hybrids recovered in Gramineae.     

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.     

Differential fate of plastid and mitochondrial genomes in Petunia somatic hybrids

Summary The chloroplast (cp) and mitochondrial (mt) DNAs of Petunia somatic hybrid plants, which were derived from the fusion of wild-type P. parodii protoplasts with albino P. inflata protoplasts, were analyzed by endonuclease restriction and Southern blot hybridization. Using ³²P-labelled probes that distinguished the two parental cpDNAs at a BamH1 site and at a HpaII site, only the P. parodii chloroplast genome was detected in the 10 somatic hybrid plants analyzed. To examine whether cytoplasmic mixing had resulted in rearrangement of the mitochondrial genome in the somatic hybrids, restriction patterns of purified somatic hybrid and parental mtDNAs were analyzed. Approximately 87% of those restriction fragments which distinguish the two parental genomes are P. inflata-specific. Restriction patterns of the somatic hybrid mtDNAs differ both from the parental patterns and from each other, suggesting that an interaction occurred between the parental mitochondrial genomes in the somatic fusion products which resulted in generation of the novel mtDNA patterns. Southern blot hybridization substantiates this conclusion. In addition, somatic hybrid lines derived from the same fusion product were observed to differ in mtDNA restriction pattern, reflecting a differential sorting-out of mitochondrial genomes at the time the plants were regenerated.     

2004 SIVB Congress Symposium Proceedings “Thinking Outside the Cell”: Applications of Somatic Hybridization and Cybridization in Crop Improvement,with Citrus as a Model

Summary Although somatic hybridization techniques are being ignored by variety improvement programs for most commodities, their contribution to citrus variety improvement continnes to expland and with increasing complexity. Citrus is, one of the few commodities where somatic hybridization is reaching its predicted potential, as somatic hybrids are now possible from most desirable parental combinations. Somatic hybrid citrus plants have been produced from more than 250 parental combinations, including more than 130 at the CREC. The CREC hybrids include 34 from sexually compatible intergeneric combinations, 16 from sexually incompatible combinations, and 81 interspecific combinations. The objective of this report is to demonstrate the impact of somatic hybridization on citrus improvement programs, and to discuss its potential with other commodities. For citrus scion improvement, several applications are aimed at the development of improved seedless fresh fruit varieties, and these include symmetric somatic hybridization, haploid+diploid fusion, targeted cybridization to transfer cytoplasmic male sterility (mtCMS) from Satsuma mandarin, and triploidy via interploid crosses using somatic hybrid allotetrapoid breeding parents. For rootstock improvement symmetric somatic hybridization provides an opportunity to hybridize complementary rootstocks without breaking up successful gene combinations. Rootstock somatic hybridization is providing opportunities for improving disease and inseet resistance, soil adaptation, and tree size control. Wide somatic hybridization provides an opportunity for gene transfer from related species, including some that are sexually incompatible. Extensive field research on citrus somatic hybrid rootstocks combined with emerging molecular analyses of citrus has allowed for the development of additional strategies for rootstock improvement. These include rootstock breeding and selection, at the tetraploid level using somatic hybrid parents, and the resynthesis of important rootstocks at the tetraploid level via fusion of selected superior parents. Ongoing examples of each strategy will be provided, along with ideas for extending the technology to other commodities.     

Analysis of the plastome and chondriome origin in plants regenerated after asymmetric Solanum ssp. protoplast fusions

Protoplasts from potato cultivars used as recipient parents were fused with irradiated protoplasts from wild Solanum donor species. Regenerated plants were analysed by RAPDs to identify hybrids. Irradiation of donor protoplasts with ionizing irradiation induced a broad range of donor nuclear DNA elimination in the asymmetric hybrids. Usage of chloroplast (cp)- and mitochondrial (mt)-specific PCR markers made it possible to trace the different origins of the cp genome in seven fusion combinations, as well as the mt genomes in two fusion combinations. Regenerated plants with recipient nucleus and plastome markers from the donors were found in six of the seven analysed fusion combinations. Protoplast fusion has generated novel mt genome combinations consisting of different portions of the mt genomes from the fusion partners. Selection of heterofusion products based on fluorescence markers is an efficient method to obtain asymmetric Solanum hybrids and cy- brids from most fusion combinations. Possible models for cybrid formation are discussed. Received: 7 October 1999 / Accepted: 3 December 1999     

Genome reorganization in Nicotiana asymmetric somatic hybrids analysed by in situ hybridization

In situ hybridization was used to examine genome reorganization in asymmetric somatic hybrids between Nicotiana plumbaginifolia and Nicotiana sylvestris obtained by fusion of gamma-irradiated protoplasts from one of the parents (donor) with non-irradiated protoplasts from the other (recipient). Probing with biotinylated total genomic DNA from either the donor or the recipient species unequivocally identified genetic material from both parents in 31 regenerant plants, each originating from a different nuclear hybrid colony. This method, termed genomic in situ hybridization (GISH), allowed intergenomic translocations containing chromosome segments from both species to be recognized in four regenerants. A probe homologous to the consensus sequence of the Arabidopsis thaliana telomeric repeat (5'-TTTAGGG-3')n, identified telomeres on all chromosomes, including 'mini-chromosomes' originating from the irradiated donor genome. Genomic in situ hybridization to plant chromosomes provides a rapid and reliable means of screening for recombinant genotypes in asymmetric somatic hybrids. Used in combination with other DNA probes, it also contributes to a greater understanding of the events responsible for genomic recovery and restabilization following genetic manipulation in vitro.     

Genome composition of asymmetric hybrids in relation to the phylogenetic distance between the parents. Nucleus-chloroplast interaction

Summary A series of fusion experiments were performed between protoplasts of a cytoplasmic albino mutant of tomato, Lycopersicon esculentum (ALRC), and gamma-irradiated protoplasts of L. hirsutum and the Solanum species S. commersonii, S. etuberosum and S. nigrum. These species were chosen for their different phylogenetic relationships to tomato. In all fusion combinations except from those between ALRC and S. nigrum, green calli were selected as putative fusion products and shoots regenerated from them. They were subsequently analyzed for their morphology, nuclear DNA composition and chloroplast DNA origin. The hybrids obtained between ALRC and L. hirsutum contained the chloroplasts of L. hirsutum and had the flower and leaf morphology of L. esculentum. After Southern blot analysis, using 13 restriction fragment length polymorphisms (RFLPs) randomly distributed over all chromosomes, all hybrids showed L. esculentum hybridization patterns. No chromosomes of L. hirsutum were found. These results indicate that these hybrids were true cybrids.The putative asymmetric hybrids, obtained with S. commersonii and S. etuberosum, showed phenotypic traits of both parents. After hybridization with species-specific repetitive nuclear DNA probes it was found that nuclear material of both parents was present in all plants. In the case of S. nigrum, which combination has the greatest phylogenetic distance between the fusion parents, no hybrid plants could be obtained. The chloroplast DNA of all hybrid plants was of the donor type suggesting that chloroplast transfer by asymmetric protoplast fusion can overcome problems associated with large phylogenetic distances between parental plants.     

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.     

Analysis of cytoplasmic genomes in somatic hybrids between navel orange (Citrus sinensis Osb.) and ‘Murcott’ tangor

Summary Somatic hybrid plants were produced by protoplast fusion of navel orange and Murcott tangor. Hybridity of the plants was confirmed by the restriction endonuclease analysis of nuclear ribosomal DNA. All of the plants (16 clones) were normal, uniform, and had the amphidiploid chromosome number of 36 (2n=2x=18 for each parent). The cpDNA analysis showed that each of the 16 somatic hybrids contained either one parental chloroplast genome or the other. In all cases, the mitochondrial genomes of the regenerated somatic hybrids were of the navel orange type.Contribution No. E-132 of the Fruit Tree Research Station     

苜蓿红豆草属间体细胞杂种的分子生物学鉴定

通过原生质体融合和培养获得苜蓿红豆草属间体细胞杂种植株。采用一种简便方法从杂种组织再生植株叶片、红豆草羟脯氨酸抗性系再生植株叶片和苜蓿根癌农杆菌702转化系愈伤组织提取DNA用于RAPD和Southern杂交分析。随机引物扩增结果显示两种亲本的RAPD多态具有明显差异。在所用20种随机引物中,6种产生较多的DNA片段。杂种组织具有两种亲本特有的DNA片段,但倾向于排除红豆草亲本的染色体,表明该杂种为非对称杂种,两种亲本染色体之间可能发生了重组。由于红豆草DNA的介入,杂种组织表现出较强的分化能力。分别利用RAPD扩增得到的OPA141000bp红豆草羟脯氨酸抗性系特异产物和OPA141600bp苜蓿根癌农杆菌702转化系特异产物为探针进行Southern分子杂交,证明杂种组织同时具有这两种DNA片段的同源序列。     

Comparison of Nicotiana tabacum and Nicotiana nesophila hybrids produced by ovule culture and protoplast fusion

Summary Somatic hybrids were produced between Nicotiana tabacum and N. nesophila, two species incapable of conventional sexual hybridization. Sexual hybrids, though, could be produced between these two species by using ovule culture only when N. nesophila was female. Clones of somatic hybrids were compared with sexual hybrids. Statistically significant variation was observed between clones, but not between sexual hybrids, for pollen viability, flower morphology, leaf morphology, and trichome density. As all clones of somatic hybrids have 96 chromosomes, the variability could not be explained by interclonal variation in chromosome number. Variation between somatic hybrids could be the result of cytoplasmic segregation or recombination, mitotic recombination or small chromosomal rearrangements prior to plant regeneration. Variation between clones could be exploited as these interspecies hybrids are now being used to incorporate disease resistance into cultivated tobacco.     

The fate of recombinant chromosomes and genome interaction in Nicotiana asymmetric somatic hybrids and their sexual progeny

Genomic in-situ hybridization (GISH) was used to monitor the behaviour of parental genomes, and the fate of intergenomic chromosome translocations, through meiosis of plants regenerated from asymmetric somatic hybrids between Nicotiana sylvestris and N. plumbaginifolia. Meiotic pairing in the regenerants was exclusively between chromosomes or chromosome segments derived from the same species. Translocation (recombinant) chromosomes contained chromosome segments from both parental species, and were detected at all stages of meiosis. They occasionally paired with respectively homologous segments of N. sylvestris or N. plumbaginifolia chromosomes. Within hybrid nuclei, the meiotic division of N. plumbaginifolia lagged behind that of N. sylvestris. However, normal and recombinant chromosomes were eventually incorporated into dyads and tetrads, and the regenerants were partially pollen fertile. Recombinant chromosomes were transmitted through either male or female gametes, and were detected by GISH in sexual progeny obtained on selfing or backcrossing the regenerants to N. sylvestris. A new recombinant chromosome in one plant of the first backcross generation provided evidence of further chromosome rearrangements occurring at, or following, meiosis in the original regenerants. This study demonstrates the stable incorporation of chromosome segments from one parental genome of an asymmetric somatic hybrid into another, via intergenomic translocation, and reveals their transmission to subsequent sexual progeny.     

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.