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.     

Intertribal hybrid cell lines of Atropa belladonna (X) Nicotiana chinensis obtained by cloning individual protoplast fusion products

Summary After fusion of isolated mesophyll protoplasts of belladonna (Atropa belladonna) with callus protoplasts of Chinese tobacco (Nicotiana chinensis) followed by mechanical isolation and cloning of individual heteroplasmic fusion products, 13 cell clones were obtained. The hybrid nature of most of the clones has been confirmed by biochemical (studies of amylase isozymes), cytogenetic (size and morphology of chromosomes) and physiological (peculiarities of cell-growth in vitro) analyses. Study of chromosomes and isozyme patterns in the hybrid cell lines revealed the presence of both parental genomes, without an indication of chromosome elimination, six months after hybridization. In 4 cell lines shootlike structures and plantlets have been produced by means of transfer to organogenesis-inducing media. The data obtained are interpreted as new evidence for the possibility of using non-sexual hybridization for the production of intergeneric, intertribal plant hybrids which cannot be obtained by sexual crossing. From these results the potential of Atropa (X) Nicotiana hybrids as a model system for genetic studies of distantly related plant species is discussed.This work is part of a joint project between Institute of Botany of the Ukrainian Academy of Sciences, Kiev, USSR, and Institute of Pharmaceutical Biology, University of Munich, FRG     

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.     

Somatic hybridization of Nicotiana tabacum and Petunia hybrida

Summary Leaf mesophyll protoplasts of a nitrate reductase deficient streptomycin resistant mutant of Nicotiana tabacum were fused with cell suspension protoplasts of wild type Petunia hybrida. Somatic hybrid cell colonies were selected for streptomycin resistance and nitrate reductase proficiency. Six independent cell lines, capable of growth in selection medium, were analysed by electrophoresis of callus peroxidases and leucine aminopeptidases and also by hybridization with rDNA and a chloroplast encoded gene as molecular probes. The results show that all six lines represented nuclear somatic hybrids, possessing the chloroplast of N. tabacum, at an early stage of development. However, after 6–12 months in culture, genomic incompatibility was observed resulting in the loss of most of the tobacco nuclear genome in the majority of the cell lines. One of the latter cell lines regenerated plants which possessed the chloroplast of N. tabacum in a predominantly P. hybrida nuclear background.     

Transient GFP expression in Nicotiana plumbaginifolia suspension cells: the role of gene silencing,cell death and T-DNA loss

The transient nature of T-DNA expression was studied with a gfp reporter gene transferred to Nicotiana plumbaginifolia suspension cells fromAgrobacterium tumefaciens. Individual GFP-expressing protoplasts were isolated after 4 days' co-cultivation. The protoplasts were cultured without selection and 4 weeks later the surviving proto-calluses were again screened for GFP expression. Of the proto-calluses initially expressing GFP, 50% had lost detectable GFP activity during the first 4 weeks of culture. Multiple T-DNA copies of the gfp gene were detected in 10 of 17 proto-calluses lacking visible GFP activity. The remaining 7 cell lines contained no gfp sequences. Our results confirm that transiently expressed T-DNAs can be lost during growth of somatic cells and demonstrate that transiently expressing cells frequently integrate multiple T-DNAs that become silenced. In cells competent for DNA uptake, cell death and gene silencing were more important barriers to the recovery of stably expressing transformants than lack of T-DNA integration.     

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.     

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.     

Incorporation of hygromycin resistance in Brassica nigra and its transfer to B. napus through asymmetric protoplast fusion

Summary With the idea to develop a selection system for asymmetric somatic hybrids between oilseed rape (Brassica napus) and black mustard (B. nigra), the marker gene hygromycin resistance was introduced in this last species by protoplast transformation with the disarmed Agrobacterium tumefaciens strain C58 pGV 3850 HPT. The B. nigra lines used for transformation had been previously selected for resistance to two important rape pathogens (Phoma lingam, Plasmodiophora brassicae). Asymmetric somatic hybrids were obtained through fusion of X-ray irradiated (mitotically inactivated) B. nigra protoplasts from transformed lines as donor with intact protoplasts of B. napus, using the hygromycin resistance as selection marker for fusion products. The somatic hybrids hitherto obtained expressed both hygromycin phosphotransferase and nopaline synthase genes. Previous experience with other plant species had demonstrated that besides the T-DNA, other genes of the donor genome can be co-transferred. In this way, the produced hybrids constitute a valuable material for studying the possibility to transfer agronomically relevant characters — in our case, diseases resistances — through asymmetric protoplast fusion.     

Asymmetric somatic hybrid plants between an interspecific Lycopersicon hybrid and Solanum melongena

Summary Asymmetric somatic hybrid plants were obtained by a modified PEG/DMSO fusion procedure between protoplasts derived from suspension cells of an interspecific tomato hybrid, Lycopersicon esculentum x L. pennellii, and mesophyll protoplasts of Solanum melongena, eggplant. The tomato hybrid was previously transformed with Agrobacterium tumefaciens and contained the kanamycin-resistance marker gene. Prior to fusion, the donor protoplasts of the tomato hybrid were gamma irradiated at 9.0 krad. Thus, non-division of irradiated tomato hybrid protoplasts coupled with kanamycin sensitivity of eggplant enabled selection of somatic cell hybrids. Forty-nine calli selected post-fusion regenerated leaf-like structures in the presence of 50 mg/l kanamycin. However, only four of the 49 calli regenerated intact shoots which rooted in the presence of 50 mg/l kanamycin and were later transferred to the greenhouse. Analysis of phosphoglucoisomerase and peroxidase isozymes, and Southern hybridization with a nuclear-specific pea 45 S ribosomal RNA gene confirmed somatic hybrid status. Cytology revealed that the four hybrid plants had chromosome numbers of 45, 60, 42 and 57, respectively; they were all sterile.     

The use of genetic markers selected in vitro for the isolation and genetic verification of intraspecific somatic hybrids of Nicotiana tabacum L.

Summary A scheme employing genetic markers obtained by in vitro selection was developed for the stringent isolation of hybrid somatic cells of Nicotiana tabacum. Mesophyll protoplasts that carried two dominant alleles of nuclear genes conferring resistance to the herbicide picloram (pmR1) and the ability to utilize glycerol as the sole source of carbon (Gut) were fused with suspension-culture protoplasts that were marked with the dominant nuclear allele (HuR9) conferring resistance to hydroxyurea. Putative somatic hybrid cell lines were identified by selecting for the Gut and HuR9 markers, followed by an assay for the unselected marker PmR1. Plants regenerated from six of these cell lines were proved to be true somatic hybrids by demonstrating the segregation of each of the three parental markers in the progeny of crosses of those plants with normal seed-derived plants.     

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.     

Culture of plant somatic hybrids following electrical fusion

Summary Electrically-induced protoplast fusion has been used to produce somatic hybrids between Nicotiana plumbaginifolia and Nicotiana tabacum. Following fusion of suspension culture protoplasts (N. plumbaginifolia) with mesophyll protoplasts (N. tabacum) heterokaryons were identified visually and their development was followed in culture. Because electrical fusion is a microtechnique, procedures were developed for culturing the heterokaryons in small numbers and at low density. The fusion and culture procedures described are rapid, uncomplicated and repeatable. Good cell viabilities indicate that the fusion procedure is not cytotoxic. Fused material was cultured 1–2 days at high density in modified K3 medium (Nagy and Maliga 1976). The heterokaryons were isolated manually and grown, at low density in conditioned media. Calli have been regenerated. Esterase isozyme patterns confirm the hybrid character of calli and clonally-derived plantlets recovered from these fusions.     

Subcellular location of lincomycin resistance in Nicotiana mutants

Summary Lincomycin-resistant Nicotiana plumbaginifolia plastid mutants were considered also to carry mitochondrial mutations on the basis of their ability to grow in the dark under selective conditions. To clarify the role of mitochondria, individual protoplasts of the green, lincomycin-resistant N. plumbaginifolia mutant LR400 were microfused with protoplasts of the N. tabacum plastid albino line 92V37, which possesses N. undulata cytoplasm. The production of lincomycin-resistant albino cybrid lines, with N. undulata plastids and recombinant mitochondria, strongly indicated a determining role for mitochondria in the lincomycin resistance. Sequence analysis of the region encompassing putative mutation sites in the 26S rRNA genes from the LR400 and several other lincomycin-resistant N. plumbaginifolia mutants revelaed, however, no differences from the wild-type sequence. As an alternative source of the resistance of the fusion products, the N. tabacum fusion partner was also taken into account. Surprisingly, a natural lincomycin resistance of tobacco was detected, which was inherited as a dominant nuclear trait. This result compromises the interpretation of the fusion data suggested above. Thus, to answer the original question definitively, the mutant LR400 was crossed as a female parent with a N. plumbaginifolia line carrying streptomycin-resistant N. tabacum plastids. Calli were then induced from the seedlings. Occasional paternal plastid transmissions were selected as streptomycin-resistant calli on selective medium. These cell lines were shown by restriction enzyme analysis to contain paternal plastids and maternal mitochondria. They were tested for greening and growing ability in the presence of lincomycin. These resistance traits proved to be genetically linked and exclusively located in the plastids.EMBL accession number X68710     

Translocation events demonstrated by molecular,in situ hybridization and chromosome pairing analyses in highly asymmetric somatic hybrid plants

Cytological analyses show rearranged chromosomes in some highly asymmetric nuclear hybrids obtained after fusion of mesophyll protoplasts ofNicotiana plumbaginifolia (wild type) with γ-irradiated (100 krad), kanamycin-resistant mesophyll protoplasts ofPetunia hybrida. Molecular, cytogenetic andin situ hybridization analyses performed on the asymmetric somatic hybrid P₁, previously identified as having a clearly metacentric chromosome besides a nearly completeNicotiana chromosome complement, are reported. Meiotic analysis andin situ hybridization experiments using ribosomal DNA as a probe showed that this metacentric chromosome represents a translocation of a chromosome fragment onto chromosome 9 ofN. plumbaginifolia. Southern hybridization with an rDNA probe showed that onlyNicotiana-specific rDNA was present.In situ hybridization experiments, using total genomic DNA ofP. hybrida as a probe, demonstrated that the translocated fragment representedPetunia DNA.     

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.     

Regeneration and characterization of plants produced from mature tobacco pollen protoplasts via gametosomatic hybridization

Summary Mature pollen protoplasts (n) isolated from kanamycin resistant plants of Nicotiana tabacum (2n = 4x = 48) were fused with somatic mesophyll protoplasts (2n) of Nicotiana plumbaginifolia (2n = 20) to produce plants. A total of 3.6·10⁶ mature pollen protoplasts were fused with 7·10⁶ mesophyll protoplasts using a PEG/Ca²⁺ method. Mature pollen protoplasts did not divide in our culture conditions, and N. plumbaginifolia protoplasts stopped dividing when the protoplast-derived colonies were transferred to a selection medium containing paromomycine (20 mg·l^-1). A total of 133 actively growing colonies were recovered on the selection medium containing kanamycin (100 mg·l^-1). Plants from twenty resulting cell lines were confirmed as hybrids (17) or cybrids (3) based on leaf and floral morphology and fertility analysis. Isozyme pattern analysis confirmed the nuclear hybrid and cybrid nature, respectively, for 2 and 3 typical gametosomatic selected plants. Root tip squashes of 6 of the gametosomatic hybrid plants revealed chromosome numbers ranging from 44 to 68; the 3 selected cybrid plants had 48 chromosomes. Evidence for organelle transmission from the mesophyll partner in the gametosomatic plants is shown. From the analysis it can be concluded that the gametosomatic fusion involving mature pollen protoplasts (n) carrying a dominant selection marker can be convenient for synthesis of either hybrids or cybrids. Such gametosomatic fusion is therefore considered as a new approach towards the production of androgenetic plants with a choosen cytoplasm.Abbreviations AAT aspartate aminotransferase - BCP bromocresol purple - EST esterase - MES 2-(N-morpholino) ethanesulfonic acid - AP acid phosphatase - PEG polyethyleneglycol - PER peroxydase     

Chloroplast segregation in somatic hybrids of Nicotiana plumbaginifolia and N. sylvestris having different ratios of parental nuclear genomes

Summary Fusion of mesophyll protoplasts of haploid Nicotiana plumbaginifolia (P) and N. sylvestris (S) resulted in the production of somatic hybrid plants of various ploidy levels. Analysis of the restriction fragment patterns of chloroplast DNA from 118 plants belonging to genome constitutions PS, PPS, PSS, and PPSS revealed that two had a pattern corresponding to a mixture of parental DNA while all the others had the pattern of either N. plumbaginifolia or N. sylvestris. In the latter case, the ratio of the two parental types fits 1∶1 in all the four genome constitutions studied. Since the protoplasts used in the fusion experiment were physiologically similar and the hybrid cells were not deliberately selected, these results suggest that chloroplast segregation in the somatic hybrids is independent of the chloroplast input of the fusion partners and the nuclear background of the fusion products.     

Somatic hybridization between birdsfoot trefoil (Lotus corniculatus L.) and L. conimbricensis Willd

Summary Somatic hybrid plants were produced by fusion of birdsfoot trefoil (Lotus corniculatus) cv Leo and L. conimbricensis Willd. protoplasts. Birdsfoot trefoil etiolated hypocotyl protoplasts were inactivated with iodoacetate to inhibit cell division prior to fusion with L. conimbricensis suspension culture protoplasts. L. conimbricensis protoplasts divided to form callus which did not regenerate plants. Thus, plant regeneration from protoplast-derived callus was used to tentatively identify somatic hybrid cell lines. Plants regenerated from three cell lines exhibited additive combinations of parental isozymes of phosphoglucomutase, and L. conimbricensis-specific esterases indicating that they were somatic hybrids. The somatic chromosome number of one somatic hybrid was 36. The other somatic hybrid exhibited variable chromosome numbers ranging from 33 to 40. These observations approximate the expected combination of the birdsfoot trefoil (2n=4x=24) and L. conimbricensis (2n=2x=12) genomes. Somatic hybrid flowers were less yellow than birdsfoot trefoil flowers and had purple keel tips, a trait inherited from the white flowered L. conimbricensis. Somatic hybrids also had inflorescence structure that was intermediate to the parents. Fifteen somatic hybrid plants regenerated from the three callus lines were male sterile. Successul fertilization in backcrosses with birdsfoot trefoil pollen has not yet been obtained suggesting that the hybrids are also female sterile. This is the first example of somatic hybridization between these two sexually incompatible Lotus species.Formerly USDA-ARS, St. Paul, Minn, USA     

Chromosomes in somatic hybrids between Nicotiana plumbaginifolia and a monoploid potato

Summary Leaf mesophyll protoplasts of the monohaploid potato (Solanum tuberosum L.) clone H7322 were fused with callus protoplasts of nitrate reductase deficient (NR^–) mutants Cnx 20 and NA 36 of Nicotiana plumbaginifolia. Somatic hybrid lines were selected for nitrate reductase proficiency. All callus lines tested appeared to be stable for the retention of the potato chromosome carrying the compensating NR gene when grown for over 1.5 years in the absence of nitrate. Shoots were regenerated from six different fusion lines of Cnx 20 + H7322 24 months after fusion. Chromosomal analysis in callus cultures revealed that in both fusion combinations 40–120 N. plumbaginifolia chromosomes were present, as were 9–20 potato chromosomes. Cells with 17 potato chromosomes in combination with a relatively small number (31) of N. plumbaginifolia chromosomes were found in one line. Preferential loss of species-specific chromosomes was not observed. Analysis of regenerating tissue from three lines of Cnx 20 + H7322 revealed that after 24 months of culture intra- and intergeneric translocations, fragments and deletions were present. Elimination of the potato and N. plumbaginifolia chromosomes had taken place before and after genome doubling.     

Analysis of nuclear and organellar DNA of somatic hybrid calli and plants between Lycopersicon spp. and Nicotiana spp.

Protoplast fusion experiments between Lycopersicon esculentum or L. peruvianum and Nicotiana tabacum or N. plumbaginifolia were performed to investigate the possibility of producing symmetric and asymmetric somatic hybrids between these genera. These fusions, which involved 1.7 × 10⁸ protoplasts, yielded 35 viable hybrid calli. Plant regeneration was successful with two calli. One of these regenerants flowered, but developed no fruits. Analysis of the nuclear DNA by means of dot blot hybridization with species-specific repetitive DNA probes combined with flow cytometry, revealed that the nuclei of most hybrid calli contained the same absolute amount of Nicotiana DNA as the Nicotiana parent or (much) less, whereas the amount of Lycopersicon DNA per nucleus was 2–5 times that of the parental genotype. Eighteen of the 34 hybrids analyzed possessed Lycopersicon chloroplast DNA (cpDNA), whereas the other 16 had DNA from Nicotiana chloroplasts. The cpDNA type was correlated with the nuclear DNA composition; hybrids with more than 2C Nicotiana nuclear DNA possessed Nicotiana chloroplasts, whereas hybrids with 2C or less Nicotiana nuclear DNA contained Lycopersicon chloroplasts. Mitochondrial DNA (mtDNA) composition was correlated with both nuclear DNA constitution and chloroplast type. Hybrids possessed only or mainly species-specific mtDNA fragments from the parent predominating in the nucleus and often providing the chloroplasts. The data are discussed in relation to somatic incompatibility which could explain the low frequency at which hybrids between Lycopersicon and Nicotiana species are obtained and the limited morphogenetic potential of such hybrids.