The production of fertile,triploid somatic hybrid plants (Nicotiana glutinosa (n) + N. tabacum (2n)) via gametic: somatic protoplast fusion

Summary The fusion of gametic protoplasts with somatic protoplasts giving rise to gametosomatic hybrid plants was investigated. Gametosomatic hybrid plants were regenerated following the fusion of nitrate reductase deficient (Nr^–) Nicotiana tabacum Nia-130 leaf mesophyll protoplasts with N. glutinosa tetrad protoplasts. The resulting plants were confirmed as hybrids, based on leaf and floral morphology, chromosome number, leaf esterase and leaf callus peroxidase zymograms and Fraction-1-protein analysis. The five gametosomatic hybrid plants had the expected pentaploid, but functionally triploid chromosome number of 3n=5x=60. The relevance of triploid gametosomatic hybrids in facilitating limited gene transfer, is discussed. The utilisation of tetrads as a generally available source of haploid protoplasts for fusion studies is proposed.     

Fusion-mediated transfer of triazine-resistant chloroplasts: Characterization of Nicotiana tabacum cybrid plants

Summary Terbutryn-resistant plastids of the Nicotiana plumbaginifolia TBR2 mutant were introduced into N. tabacum plants by protoplast fusion following X-irradiation of TBR2 protoplasts. The N. tabacum chloroplast recipient line, SR1-A15, carried mutant (albino) plastids. Following protoplast fusion, potential cybrid cell lines with an N. tabacum (SR1-A15) nucleus and N. plumbaginifolia (TBR2) chloroplasts were identified by their green color. The presence of TBR2 plastids in regenerated green N. tabacum plants was confirmed by hybridization with a chloroplast DNA probe and by the modified chloroplast fluorescence transients characteristic of the TBR2 mutant. Cybrid plants were resistant to high levels of atrazine (10 kg/ha). The protruding stigma and shorter than normal filaments of the cybrids resulted in male sterility. In the cybrids atrazine resistance was associated with reduced vigour, suggesting a causal relationship.     

Asymmetric hybridization in Nicotiana by fusion of irradiated protoplasts

Summary Mesophyll protoplasts of a kanamycin-resistant, nopaline-positive Nicotiana plumbaginifolia seed line were inactivated by -irradiation and electrically fused with unirradiated mesophyll protoplasts of N. tabacum. Hybrids were selected on kanamycin and regenerated. Genetic material from N. plumbaginifolia was detected in these plants by the following criteria: (1) morphology, (2) esterase isozyme profiles, and (3) the presence of nopaline in leaf extracts. All of the plants regenerated were morphologically more similar to N. tabacum than to N. plumbaginifolia, and many were indistinguishable from N. tabacum. It was found that 37 plants displayed one or two esterases characteristic of N. plumbaginifolia in addition to a full set of esterases from N. tabacum. Based on their esterases, we have classified these plants as somatic hybrids. However, irradiation has clearly reduced the amount of N. plumbaginifolia genetic material that they retain; 24 plants were found that had only N. tabacum esterases but that produced nopaline and were kanamycin resistant. Genomic DNA from several of these plants was probed by Southern blotting for the presence of the authentic neomycin phosphotransferase gene (kanamycin-resistance gene) — all were found to contain the gene. These plants were classified as asymmetric hybrids. Finally, 25 plants were regenerated that were kanamycin sensitive, negative for nopaline, and contained only N. tabacum esterases. All of the regenerated plants, including this final category, were male sterile. As transferring the N. plumbaginifolia cytoplasm to an N. tabacum nuclear background results in an alloplasmic form of male sterility, all of the plants regenerated in this study appear to be cybrids irrespective of their nuclear constitution. Chromosome analysis of the asymmetric hybrids showed that most of them contained one more chromosome than is normal for N. tabacum. The somatic hybrids examined all had several additional chromosomes. Although male sterile, the asymmetric hybrids were female fertile to varying degrees and were successfully backcrossed with N. tabacum. Analysis of the resultant F₁ progeny indicated that the kanamycin-resistance gene from N. plumbaginifolia is partially unstable during meiosis, as would be expected for factors inherited on an unpaired chromosome.Abbreviations Km ^r kanamycin resistant - Km ^s kamacysin sensitive - Nop ⁺ nopaline positive - Nop ^– nopaline negative     

Functional cybrid plants possessing a Nicotiana genome and an Atropa plastome

Summary Mesophyll protoplasts of plastome chlorophyll-deficient, streptomycin-resistant Nicotiana tabacum were fused with those of wild type Atropa belladonna using the polyethylene-glycol/high pH/high Ca⁺⁺/dimethylsulfoxide method. Protoplasts were cultured in nutrient media suitable for regeneration of tobacco but not Atropa cells. In two experiments, a total of 41 cell lines have been selected as green colonies. Cytogenetic (chromosomal number and morphology) and biochemical (isozyme analyses of esterase, amylase and peroxidase) studies were used to evaluate the nuclear genetic constitution of regenerated plants. To study plastid genetic constitution, restriction endonuclease analysis of chloroplast DNA was performed. Three groups of regenerants have been identified: (a) nuclear hybrids (4 cell lines); (b) Atropa plants, most probably arising from rare surviving parental protoplasts (4 lines) and (c) Nicotiana/Atropa cybrids possessing a tobacco genome and an Atropa plastome (33 lines). Most of cybrids obtained were diploid, morphologically normal plants phenotypically similar to tobacco. Some plants flowered and yielded viable seeds. Part of cybrid regenerants were variegated, variegation being transmitted to sexual progeny. Electron microscopic analysis of the mesophyll cells of variegated leaves revealed the presence of heteroplastidic cells. Analysis of thylakoid membrane polypeptides shows that in the cybrids the content of at least one of the major polypeptides, presumably a chlorophyll a/b binding protein is drastically reduced.     

A light sensitive recipient for the effective transfer of chloroplast and mitochondrial traits by protoplast fusion in Nicotiana

Summary A light sensitive mutant was used as a recipient in the transfer of chloroplasts from a wildtype donor. Gamma irradiated (lethal dose) mesophyll protoplasts of Nicotiana gossei were fused with mesophyll protoplasts of a N. plumbaginifolia line carrying light sensitive plastids from a N. tabacum mutant. After fusion, colonies containing wild-type plastids from the cytoplasm donor were selected by their green colour. Most of the regenerated plants had N. plumbaginifolia morphology, but were a normal green in colour. The presence of donor-type plastids was confirmed by the restriction pattern of chloroplast DNA in each plant analysed. These cybrids were fully male sterile with an altered flower morphology typical of certain types of alloplasmic male sterility in Nicotiana. The use of the cytoplasmic light sensitive recipient proved to be suitable for effective interspecific transfer of wild-type chloroplasts. The recombinant-type mitochondrial DNA restriction patterns and the male sterility of the cybrids indicated the co-transfer of chloroplast and mitochondrial traits. On leave from: Department of Genetics, Section of Biosciences, Martin Luther University, Domplatz 1, DDR-4020 Halle/ S., German Democratic Republic     

Transmission genetics of the somatic hybridization process in Nicotiana

Summary Callus protoplasts of a Nicotiana tabacum chlorophyll-deficient mutant were fused with mesophyll protoplasts from one of following five sources: 4 cmsanalogs of tobacco bearing the cytoplasms of N. plumbaginifolia, N. suaveolens, N. repanda, and N. undulata, respectively, as well as wild species N. glauca. In another series of experiments, callus protoplasts from the chlorophyll-deficient genome Su/Su mutant of tobacco were fused with mesophyll protoplasts of the wild species N. glauca and those of a plastome chlorophyll-deficient tobacco mutant. The screening of hybrids consisted of visual identification followed by mechanical isolation and cloning of heteroplasmic fusion products in microdroplets of nutrient medium. Studies of regenerated plants included the analyses of gross morphology of plants, leaf and flower morphology, analysis of chromosome size and morphology and chromosome numbers, studies of multiple molecular forms of esterase and amylase, analysis of chloroplast DNA restriction patterns and analyses of chlorophyll-deficiency controlled by Su and P ^– genes. The study of progeny of 41 clones representing all species' combinations demonstrated that regenarants of most (63%) clones from intraspecific (for nuclear genes) combinations were cybrid forms, whereas in the case of the fusion N. tabacum + N. glauca, the true nuclear hybrids prevailed and the proportion of cybrids did not exceed 26%. Clones regenerating both hybrid and cybrid plants from the same fusion product were also found.     

Interspecific protoplast fusion to rescue a cytoplasmic lincomycin resistance mutation into fertile Nicotiana plumbaginifolia plants

Summary A lincomycin-resistant cell line, LR105, was isolated in a mutagenized (0.1 mM N-ethyl-N-nitrosourea) callus culture initiated from a haploid Nicotiana sylvestris plant. The regenerated plants had an abnormal morphology and did not set viable seeds.Transfer of lincomycin resistance was attempted from the original N. sylvestris nuclear background into Nicotiana plumbaginifolia by protoplast fusion, since it was expected that resistance would be cytoplasmically coded. LR105 protoplasts were irradiated with a lethal dose (120 J kg^-1; ⁶⁰ Co source), fused with sensitive N. plumbaginifolia protoplasts and the colonies grown from the fused population were screened for lincomycin resistance. Expression of resistance was expected only if the cytoplasm of the irradiated cells had mixed with nonirradiated cytoplasm, and was reactivated as a result of cell fusion (Menczel et al. 1982).Plants were regenerated in 44 resistant clones. Plants in 41 clones had a N. plumbaginifolia nuclear genome. In three clones somatic hybrids were obtained. The resistant N. plumbaginifolia cybrid plants were fertile, unlike the original LR105 plants. Lincomycin resistance was inherited maternally in the eight clones in which crosses were made. In these clones the introduction of N. sylvestris chloroplasts into a N. plumbaginifolia nuclear background was confirmed by the SmaI restriction endonuclease pattern of the chloroplast DNA. The involvement of chloroplast DNA in determining lincomycin resistance is therefore implied.     

Spontaneous extensive chromosome elimination in somatic hybrids between somatically congruent species Nicotiana tabacum L. and Atropa belladonna L.

Summary Mesophyll protoplasts of the kanamycin-resistant nightshade, Atropa belladonna, were fused with mesophyll protoplasts of the phosphinothricin resistant-tobacco, Nicotiana tabacum. A total of 447 colonies resistant to both inhibitors was selected. Most of them regenerated shoots with morphology similar to one of the earlier obtained and described symmetric somatic hybrids Nicotiana + Atropa. However, three colonies (0.2%) regenerated vigorously growing tobacco-like shoots; they readily rooted, and after transfer to soil, developed into normal, fertile plants. Unlike their tobacco parental line, BarD, the obtained plants are resistant to kanamycin [they root normally in the presence of kanamycin (200 mg/1)] and possess activity of neomycin phosphotransferase (NPT II) with the same electrophoretic mobility as the one of the nightshade line. According to Southern blot hybridization analysis carried out with the use of radioactively labeled cloned fragments of the Citrus lemon ribosomal DNA repeat, as well as with Nicotiana plumbaginifolia genus-specific, interspersed repeat Inp, the kanamycin-resistant plants under investigation have only species-specific hybridizing bands from tobacco. Cytological analysis of the chromosome sets shows that plants of all three lines possess 48 large chromosomes similar to Nicotiana tabacum ones (2n = 48), and one small extra chromosome (chromosome fragment) similar to Atropa belladonna ones (2n = 72). Available data allow the conclusion that highly asymmetric, normal fertile somatic hybrids with a whole diploid Nicotiana tabacum genome and only part (not more than 2.8%) of an Atropa belladonna genome have been obtained without any pretreatment of a donor genome, although both these species are somatically congruent.     

Metabolic complementation for a single gene function associated with partial and total loss of donor DNA in interspecific somatic hybrids

Summary We report here on the obtainment of interspecific somatic, asymmetric, and highly asymmetric nuclear hybrids via protoplast fusion. Asymmetric nuclear hybrids were obtained after fusion of mesophyll protoplasts from a nitrate reductase-deficient cofactor mutant of N. plumbaginifolia with irradiated (100 krad) kanamycin resistant leaf protoplasts of a haploid N. tabacum. Selection for nitrate reductase (NR) and/or kanamycin (Km) resistance resulted in the production of three groups of plants (NR⁺, NR⁺, Km^R, and NR^-Km^R). Cytological analysis of some hybrid regenerants showed the presence of numerous tobacco chromosomes and chromosome fragments, besides a polyploid N. plumbaginifolia genome (tetra or hexaploid). All the regenerants tested were male sterile but some of them could be backcrossed to the recipient partner. In a second experiment, somatic and highly asymmetric nuclear hybrids were obtained after fusion of mesophyll protoplasts from the universal hybridizer of N. plumbaginifolia with suspension protoplasts of a tumor line of N. tabacum. Selection resulted in two types of colonies: nonregenerating hybrid calli turned out to be true somatic hybrids, while cytological analysis of regenerants obtained on morphogenic calli did not show any presence of donor-specific chromosomes. Forty percent of the hybrid regenerants were completely fertile, while the others could only be backcrossed to the recipient N. plumbaginifolia. Since the gene we selected for is not yet cloned, we were not able to demonstrate the transfer of genetic material at the molecular level. However, since no reversion frequency for the nitrate reductase mutant is known, and due to a detailed cytological knowledge of both fusion partners, we feel confident in speculating that intergenomic recombination between N. plumbaginifolia and N. tabacum has occurred.     

Cytoplasmic hybridization in Nicotiana: mitochondrial DNA analysis in progenies resulting from fusion between protoplasts having different organelle constitutions

Summary Our previous studies indicated that fusion products with one functional nucleus but organelles of the two fusion partners (i.e. heteroplastomic cybrids) could be obtained by fusing X-irradiated (cytoplasmic donor) with non-irradiated (recipient) Nicotiana protoplasts. The present report deals with the analysis of mitochondria in cybrid populations resulting from the fusion of donor Nicotiana tabacum protoplasts with recipient protoplasts having a N. Sylvestris nucleus but chloroplasts of an alien Nicotiana species, and exhibiting cytoplasmic male sterility. The two fusion parents showed significant differences in restriction patterns of their chloroplast and mitochondrial DNA. Four groups of cybrid plants were obtained by this fusion. All had N. sylvestris nuclei but contained either donor or recipient chloroplasts and had either sterile or fertile anthers. There was no correlation between anther fertility and chloroplasts type. The mitochondrial DNA restriction patterns of sterile cybrids were similar to the respective patterns of the sterile fusion partner while the mitochondrial DNA restriction patterns of the fertile cybrids were similar to the respective patterns of the fertile fusion partner. The results indicate an independent assortment of chloroplasts and mitochondria from the heteroplastomic fusion products.     

GFP expression as an indicator of somatic hybrids between transgenic Satsuma mandarin and calamondin at embryoid stage

Somatic hybridization was performed via electrofusion between embryogenic suspension-derived protoplasts of transgenic green fluorescent protein (GFP) Satsuma mandarin (Citrus unshiu Marc. cv. Guoqing No. 1) (G1) callus and mesophyll protoplasts of calamondin (Citrus microcarpa Bunge), and three embryoids expressing GFP under UV light were obtained after 60 days of culture. The three embryoids were considered not as diploid cybrids but true allotetraploid somatic hybrids, as it was based on: (1) citrus heterokaryons are generally more vigorous and have higher capacity for embryogenesis as compared with unfused and homo-fused embryogenic callus protoplasts; (2) the callus line of G1 Satsuma mandarin has lost the embryogenesis capacity; and (3) citrus diploid cybrids produced by symmetric fusion always possess nuclear genome of mesophyll parent, and calamondin without GFP gene was used as leaf parent in this study. Subsequent flow cytometry, simple sequence repeat and cleaved amplified polymorphic sequence analysis of one regenerated callus mass and three resulting plants validated this supposition, i.e., the callus was derived from transgenic G1 callus protoplasts, and the three plants were true allotetraploid somatic hybrids possessing nuclear genomic DNA of both parents and cytoplasmic DNA from callus parent. The potential of transgenic GFP citrus callus as suspension parent in citrus somatic fusion to study the mechanism of cybrid formation, create new citrus cybrids, and transfer organelle-encoded agronomic traits was also discussed.     

Hybrid genes in the analysis of transformation conditions. 3. Temporal/spatial fate of NPTII gene integration,its inheritance and factors affecting these processes in Nicotiana plumbaginifolia

Freshly isolated haploid mesophyll protoplasts of Nicotiana plumbaginifolia were transformed for kanamycin resistance. In 38% of the 224 transformants analysed, transmission of the NPTII gene occurred as a homozygous trait, while 62% of the transformants were heterozygous for the trait. In the first case, the foreign DNA integration predominantly (95%) resulted in monogenic inheritance. The second group was characterized by a significant (46%) proportion of multiple insertions. However, there was no clear-cut difference in the integration pattern between the two groups. Furthermore, transformation rates were increased by 4- to 10-fold when transformed diploid protoplasts were treated with UV light or with 3-aminobenzamide. The number of insertion sites was also increased by these treatments. These results shed further light on the fate of the foreign DNA in transformed plants and on means to control or manipulate the integration event(s).     

Intra- and interspecific gametosomatic hybridisation within the genus Petunia

Following PEG and high pH induced fusion, intraspecific gametosomatic hybrid plants (pollen tetrad protoplasts of a normal purple flowered variety of P. hybrida fused with cell suspension protoplasts of a nuclear albino mutant of the variety Blue Lace) and interspecific gametosomatic hybrid plants (tetrad protoplasts (as above) fused with cell suspension protoplasts of a nuclear albino mutant of P. parviflora) were recovered. Hybrid plants of both combinations possessed an intermediate vegetative and floral morphology with chromosome numbers of 2n=3x=21 and 2n=3x=25 respectively. Hybrid cells were in both systems identified as green colonies against an albino background as a result of complementation to chlorophyll proficiency. Pollen tetrad protoplasts did not divide. The production of such plants at the intra- and interspecific level in Petunia has shown that the concept of gametosomatic hybridisation can be extended to genera other than Nicotiana. An alternative selection strategy is available to that as used earlier for Nicotiana.     

Electrical fusion for optimal formation of protoplast heterokaryons in Nicotiana

The electrical fusion of protoplasts has been studied in order to maximize the formation of heterokaryons for culture. Heterokaryons of Nicotiana tabacum L. mesophyll protoplasts and N. plumbaginifolia Viviani supension-cell protoplasts were identified in fixed and stained as well as living material; a quantitative fusion index was thereby developed. With this index the efficiencies of various electric fields and fusion-chamber designs have been determined. Optimal fusion was obtained with an alternating-current (AC) field of 150 V/cm and direct-current (DC) square-wave pulses of 1000 V/cm. A new, simple-to-use, largescale fusion chamber is described in which batches of up to 5·10⁵ protoplasts (0.5 ml of cells at 10⁶/ml) can be fused in 5–7 min with efficiencies approaching 40%. Half of the fusion products are heterokaryons, thus fusion is random. Of the fusion products, 60% are bi- or trinucleate. Using fusion procedures similar to those described here Bates and C. Hasenkampf (1985, Theor. Appl. Genet., in press) have recovered viable somatic hybrids which have been regenerated.Abbreviations AC alternating current - DC direct current - PEG polyethylene glycol     

Protoplast fusion mediated transfer of oligomycin resistance from Nicotiana sylvestris to Solanum tuberosum by intergeneric cybridization

Summary We have successfully bridged the intergeneric barriers between Nicotiana and Solanum with respect to chondriome transfer. To enable this transfer we utilized the donor-recipient protoplast-fusion procedure. Consequently protoplasts of a Nicotiana sylvestris line with putativly oligomycin-resistant mitochondria (line Oli ^R 38) were used as irradiated chondriome donors and iodoacetate-treated protoplasts of Solanum tuberosum cv. Desiree served as recipients. The plated fusion products as well as their derived colonies and calli were exposed to gradually increasing levels of oligomycin. The resulting plantlets had potato morphology and were analyzed with respect to their mitochondrial DNA and chloroplast DNA. Fifteen out of 50 regenerated plants were verified as true cybrids. Detailed analyses of one cybrid revealed chondriome components from the oligomycin-resistant donor line, Oli ^R 38, but retention of the plastome of potato. This cybrid was oligomycin-resistant as revealed by root-culture analysis. It was thus verified that due to selection, chondriome components could be transferred from a N. sylvestris donor into a cybrid having all the phenotypic features controlled by the nucleus of the recipient fusion partner (S. tuberosum).     

Somatic hybridization between potato and Nicotiana plumbaginifolia

Summary Electrofusion was carried out between mesophyll protoplasts from the transformed diploid S. tuberosum clone 413 (2n=2x=24) which contains various genetic markers (hormone autotrophy, opine synthesis, kanamycin resistance, -glucuronidase activity) and mesophyll protoplasts of a diploid wild-type clone of N. plumbaginifolia (2n=2x=20). Hybrid calli were obtained after continuous culture on selection medium containing kanamycin. Parental chromosome numbers, determined at 2 months after fusion, revealed hybrid-specific differences between the individual calli. On the basis of these differences three categories of hybrids were distinguished. Category I hybrids contained between 8 and 24 potato chromosomes and more than 20 N. plumbaginifolia chromosomes; category II hybrids had between 1 and 20 N. plumbaginifolia chromosomes and more than 24 potato chromosomes; category III hybrids contained diploid or subdiploid numbers of chromosomes from both parents. The hybrids were evenly distributed over the three categories. After a 1-year culture of 24 representative hybrid callus lines on selection medium the karyotype of 10 hybrids remained stable, whereas 8 hybrids showed polyploidization of the genome of one parent, together with no or minor changes of the chromosome numbers of the other parent. Six hybrids showed slight changes in the hybrid karyotype. The elimination of chromosomes of a particular parent was not correlated to their metaphase location. The processes of spontaneous biparental chromosome elimination leading to the production of asymmetric hybrids of different categories are discussed.     

Effect of radiation dosage on efficiency of chloroplast transfer by protoplast fusion in Nicotiana

Chloroplasts of Nicotiana tabacum SR1 were transferred into Nicotiana plumbaginifolia by protoplast fusion. The protoplasts of the organelle donor were irradiated with different lethal doses using a ⁶⁰Co source, to facilitate the elimination of their nuclei from the fusion products. After fusion induction, clones derived from fusion products and containing streptomycin-resistant N. tabacum SR1 chloroplasts were selected by their ability to green on a selective medium. When N. tabacum protoplasts were inactivated by iodoacetate instead of irradiation, the proportion of N. plumbaginifolia nuclear segregant clones was low (1–2%). Irradiation markedly increased this value: Using 50, 120, 210 and 300 J kg^-1 doses, the frequency of segregant clones was 44, 57, 84 and 70 percent, respectively. Regeneration of resistant N. plumbaginifolia plants with SR1 chloroplasts indicated that plastids can be rescued from the irradiated cells by fusion with untreated protoplasts. Resistant N. plumbaginifolia plants that were regenerated (43 clones studied) had diploid (2n = 2X = 20) or tetraploid chromosome numbers and were identical morphologically to parental plants. The absence of aneuploids suggests that in these clones irradiation resulted in complete elimination of the irradiated N. tabacum nuclei. Resistance is inherited maternally (five clones tested). The demonstration of chloroplast transfer and the presence of N. tabacum plastids in the N. plumbaginifolia plants was confirmed by chloroplast DNA fragmentation patterns after EcoRI digestion.     

Lipofectin: direct gene transfer to higher plants using cationic liposomes

Summary It has recently been shown that lipofectin, a commercially available preparation of cationic liposomes is capable of animal and plant cell line transfection. Here, it is analyzed with respect to its toxicity for higher plant protoplasts and used for transient expression and stable transformation experiments with mesophyll protoplasts of Nicotiana tabacum and Nicotiana plumbaginifolia. Transient expression of the -glucuronidase gene (GUS) under control of the CaMV-35S-promoter was lower than after introduction of the same gene by polyethylene glycol. By transferring the neomycin phosphotransferase gene (NPTII) and subsequent culture and regeneration under selection with kanamycin, stably transformed plants were recovered after using Lipofectin in various protocols with or without additional application of electroporation. Efficiencies of stable transformation were comparable to those achieved with PEG and/or electroporation. Confirmation of transformants included assaying the enzyme activity of the gene product, genomic blotting, and transfer of the resistant phenotype to the progeny produced from selfed primary transformants.     

Cybridization in Nicotiana tabacum L. using double inactivation of parental protoplasts and post-fusion selection based on nuclear-encoded and chloroplast-encoded marker genes

An effective selection system preceded by double inactivation of parental protoplasts was used to transfer Nicotiana suaveolens Leh. cytoplasmic male sterility into a commercial tobacco (N. tabacum L.) breeding line. Mesophyll protoplasts from transformed plants of N. tabacum cultivar WZ2-3-1-1 possessing a neomycin phosphotransferase II gene were used as the nuclear donors, while those isolated from N. suaveolens plants carrying a chloroplast mutation for resistance to spectinomycin, induced using nitrosomethyl urea, were the cytoplasm donors in somatic cybridizations. Prior to fusion, nuclear donor protoplasts were inactivated with iodoacetamide or rhodamine 6G, while those of the cytoplasm donor were inactivated by X-irradiation. The resultant microcalli were cultured on a shoot regeneration medium containing both kanamycin and spectinomycin to select cybrids. Only regenerants that had typical characteristics of the N. tabacum cultivar were selected for transfer to the glasshouse. Four putative cytoplasmic male-sterile (CMS) plants, out of a total of 44 regenerated plants transferred to the glasshouse, were obtained. Intraspecific somatic transfers of the CMS trait between N. tabacum cultivars with distinctlydifferent morphologies using single inactivation and nonselective shoot regeneration medium were demonstrated. The implications of the results for practical tobacco breeding as a means of circumventing lengthy backcrossing procedures are discussed.