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.     

Somatic hybridization between male sterile Nicotiana tabacum and N. glutinosa through protoplast fusion

Summary Protoplasts derived from suspension cultured cells of cytoplasmic male sterile Nicotiana tabacum (N. debneyi cytoplasm) and of fertile N. glutinosa were fused with the aid of polyethylene glycol (PEG). Out of 1,089 colonies developed from PEG-treated protoplasts, 29 restored whole plants.A somatic hybrid plant was selected on the basis of isoelectrofocusing analysis of Fraction I protein in leaves of regenerated plants. A newly created hybrid contained small subunits of both parents but only a N. glutinosa type large subunit.Male sterile character was conserved in a hybrid plant while leaf morphology was intermediate between the parents. By tobacco mosaic virus infection tests, the hybrid's leaves showed resistant symptoms, hypersensitive local lesions, which were due to N. glutinosa nuclear genome expression.Abbreviations PEG Polyethylene glycol - TMV Tobacco mosaic virus     

A somatic hybrid plant obtained by protoplast fusion between navel orange (Citrus sinensis) and satsuma mandarin (C. unshiu)

A somatic hybrid plant was obtained by protoplast fusion between navel orange and satsuma mandarin. Protoplasts isolated from nucellar calli of navel orange and from leaves of satsuma mandarin were fused by the PEG method. The fusion products were cultured in a Murashige & Tucker medium containing 0.6 M sucrose. In this medium, some colonies developed into whole plants through embryogenesis. One of the regenerated plants was shown to be a hybrid, which was proven by restriction endonuclease analysis of nuclear ribosomal DNA. The chromosome number of the hybrid was 36. Both parents have a chromosome number 2n=18.     

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.     

Fertile somatic hybrids between Petunia hybrida and a wild species,Petunia variabilis

Somatic hybrid plants were regenerated following electrofusion between leaf mesophyll protoplasts of P. hybrida (2n = 14) and a wild sexually incompatible species, P. variabilis (2n = 18). The selection of hybrids was based on the hybrid vigour, expressed both in the growth of the callus and at the shoot formation stage, resulting from the combination of parental genomes. Calli exhibiting vigorous growth were selected, and upon transfer to regeneration medium gave rise to shoots. Four regenerated plants from three calli had morphological characteristics intermediate between those of the parents. The hybrid nature of these plants was confirmed by chromosome counts as well as isozyme and DNA analyses. They had amphidiploid chromosome numbers (2n = 32) and were fertile. Following self-pollination and backcrossing with P. variabilis, large numbers of F₂ and BC₁ seedlings were obtained.     

The role of irradiation dose and DNA content of somatic hybrid calli in producing asymmetric plants between an interspecific tomato hybrid and eggplant

Highly asymmetric somatic hybrid plants were obtained by PEG/DMSO fusion of gamma-irradiated mesophyll protoplasts of the kanamycin-resistant (KmR⁺) interspecific hybrid Lycopersicon esculentum x L. pennellii (EP) with mesophyll protoplasts of Solanum melongena (eggplant, E). Elimination of the EP chromosomes was obtained by irradiating the donor genome with different doses of gamma rays (100, 250, 500, 750 and 1000 Gy). The selection of somatic hybrid calli was based on kanamycin resistance; EP and E protoplasts did not divide due to the irradiation treatment and sensitivity to kanamycin, respectively. KmR⁺ calli were recovered following all irradiation doses of donor EP protoplasts. The hybrid nature of the recovered calli was confirmed by PCR amplification of the NptII gene, RAPD patterns and Southern hybridizations using potato ribosomal DNA and pTHG2 probes. Ploidy levels of calli confirmed as hybrid were further analyzed by flow cytometry. Such analyses revealed that the vast majority of hybrid calli that did not regenerate shoots were 5–9n polyploids. The three asymmetric somatic hybrid plants obtained were regenerated only from callus with a ploidy level close to 4n, and such calli occurred only when the donor EP had been exposed to 100 Gy. The amount of DNA in somatic hybrid calli, from 100-Gy exposure, was found by dot blot hybridization with the species-specific probe, pTHG2, to be equivalent with 3.1–25.8% of the tomato genome. Thus, DNA contained in 3.8–13.2 average-size tomato chromosomes was present in these hybrid calli. The asymmetric somatic hybrid plants had the eggplant morphology and were regenerated from one hybrid callus that contained an amount of tomato DNA equivalent to 6.29 average-size tomato chromosomes.     

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.     

Valine-resistant plants from in vitro selected tobacco cells

Summary Growth inhibition of protoplasts and cells of tobacco (Nicotiana tabacum L.) occurs when 0.1 to 10 mM L-valine is present in the culture medium but not if L-isoleucine is simultaneously added. Mesophyll protoplasts from haploid tobacco were submitted to ultraviolet irradiation and cell colonies derived from these protoplasts were plated in a medium containing valine. Plants were regenerated from five presumptive resistant calli and retested for valine-resistance. Among the recovered plants two were definitely resistant to valine. This trait was transmitted in sexual crosses as a mendelian character.     

Fertile somatic hybrids between transgenicNicotiana tabacum and transgenicN. debneyi selected by dual-antibiotic resistance

Summary A simple, yet effective selection system was used to produce fertile somatic hybrids betweenNicotiana tabacum andN. debneyi. This approach utilized transgenic antibiotic-resistantN. tabacum andN. Debneyi as donor plants for mesophyll protoplast fusions. Thirteen somatic hybrid plants were regenerated from calli capable of growth on medium containing both antibiotics. The majority of the hybrids displayed a range of leaf and floral morphologies and growth habits that were intermediate to those of the parental species, and had chromosome numbers varying from amphidiploid (2n = 96) to hypoaneuploid (2n = 60). Isoenzyme and RFLP analysis demonstrated the presence and expression of nuclear genes from both parents in all of the hybrids. Most plants are fully fertile. Thus, these plants differ from the malesterile tobacco cybrids and alloplasmic lines produced by transferring theN. debneyi cytoplasm to tobacco. A nonrandom pattern of cytoplasmic segregation in the fusion products occurred with a bias towards the presence ofN. debneyi cp and mtDNA. Evidence for the presence of rearranged or recombinant cp and mtDNA in some of the hybrids was obtained. The somatic hybrids were successfully backcrossed to theN. tabacum parent and are now being tested for immunity to black root rot, a trait specific toN. debneyi, but not existent in theN. tabacum parental line.     

Detection of the Nicotiana rustica chloroplast genome coding for the large subunit of Fraction I protein in a somatic hybrid in which only the N. tabacum chloroplast genome appeared to have been expressed

Nine plants were produced from anthers of a somatic hybrid which had been obtained by fusion of Nicotiana tabacum L. and N. rustica L. protoplants. As determined by electrofocusing, the Fraction I protein of the original somatic hybrid had largesubunit polypeptides exclusively of the N. tabacum type. Two of the plants regenerated from anthers contained Fraction-I-protein large subunits exclusively of the N. rustica type. Since each plant was regenerated from a single cell, the somatic hybrid must have had cells containing both the N. tabacum and N. rustica chloroplast genome although the latter was not expressed. Possibilities to account for this non-expression of a chloroplast genome in the somatic hybrid are discussed.     

Hybrids between tobacco crown gall cells and normal somatic cells of Atropa belladonna

Protoplast fusion of Nicotiana tabacum (B6S3) crown gall cells and Atropa belladonna leaf mesophyll cells was carried out. Hybrids were selected for their capacity to grow on hormone-free media and to green in light. Shoots incapable of rhizogenesis were regenerated on the same media and grafted onto normal plants of different species. 57 hybrid cell lines differing in their genetic constitution were produced. Analysis of hybrid lines involved the determination of the lysopine dehydrogenase (LpDH) activity and the molecular forms of esterase and amylase, a restriction analysis of chloroplast DNA and a cytogenetic study.Abbreviations LS-H Linsmaier and Skoog (1965) hormone-free medium - LpDH lysopine dehydrogenase     

Asymmetric somatic hybridization between Triticum aestivum L. and Haynaldia villosa Schur

Suspension cell-derived protoplasts of wheat, inactivated with different concentrations (0-2.5mol/L) of IOA, were fused by PEG method with the Haynaldia villosa protoplasts which originated from the calli 4-5d after subculture and were irradiated with 60Co-γ ray. Cell colonies, calli or regenerated plants were obtained from different combinations of fusion. The calli and plants were verified to be hybrids by chromosome counting, isozyme analysis and morphological inspection.     

Asymmetric hybridization between Nicotiana tabacum and N. repanda by donor recipient protoplast fusion: transfer of TMV resistance

Summary Genetically asymmetric hybrids were recovered by fusion of Nicotiana tabacum protoplasts with irradiated protoplasts of kanamycin-resistant, nopalineproducing plants of N. repanda. Hybrid calli were selected by culture on media containing kanamycin and were regenerated. These plants were morphologically similar to N. tabacum but produced nopaline, indicating they retained genes from N. repanda. Esterase isozyme profiles also indicated that the plants are somatic hybrids, but are more similar to N. tabacum than N. repanda. Chromosome counts showed most of the hybrids had 55–62 chromosomes, which is consistent with extensive, although incomplete elimination of N. repanda chromosomes. The hybrids were largely male sterile, but about half of them set seed when crossed with N. tabacum. Chromosome numbers of the progeny and the pattern of inheritance of kanamycin resistance indicated the continued elimination of N. repanda genetic material in these backcrosses. The N. repanda parent used in these fusions gave a hypersensitive response to TMV, whereas the N. tabacum parent was TMV sensitive. When inoculated with TMV, plants from two hybrid clones gave a hypersensitive response. Plants from the other clones became systemically infected with the virus.     

Production of a triple mutant,chlorophyll-deficient,streptomycin-, and kanamycin-resistant Nicotiana tabacum,and its use in intergeneric somatic hybrid formation with Solanum melongena

Summary In order to produce a triple mutant, sexual crosses between a chlorophyll-deficient, streptomycin-resistant mutant of Nicotiana tabacum (SA) and a kanamycin-resistant transformant of N. tabacum (KR.) were carried out. From the offspring of this cross, a triple mutant (KR-SA) was selected. In N. tabacum KR-SA, chlorophyll deficiency is due to recessive mutation in the nuclear genome, streptomycin resistance is due to a dominant mutation in the chloroplast genome, and kanamycin resistance is shown to be a dominant nuclear marker. Cell suspension protoplasts of N. tabacum KRSA were fused with callus protoplasts of Solanum melongena by dextran treatment. Somatic hybrid plants were selected for streptomycin resistance and the ability to produce clorophyll in regenerated plants. By using this selection system, green plants were recovered from two colonies. When these green plants were then tested for kanamycin resistance, all analyzed plants carried this trait. In addition, the hybrid nature of these plants was confirmed by investigation of the peroxidase isozyme. The present results show that the use of N. tabacum KR-SA in studies of somatic hybridization makes it possible to select somatic hybrid plants easily and provides information of the N. tabacum genome.Chemical Regulation of Biomechanism, The Institute of Physical and Chemical Research, Wako 351-01, Japan     

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

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

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

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

Cybrids of Nicotiana tabacum and Petunia hybrida have an intergeneric mixture of chloroplasts from P. hybrida and mitochondria identical or similar to N. tabacum

Summary The mitochondrial genomes of cybrids of Nicotiana tabacum containing chloroplasts of Petunia hybrida were characterized by restriction endonuclease digestion and agarose gel electrophoresis. Cybrids that displayed normal growth and development contained mitochondrial DNA indistinguishable from N. tabacum mitochondrial DNA. Cybrids that displayed abnormal growth and development contained mitochondrial DNA that differed from N. tabacum either by possessing a few additional fragments, by lacking a few fragments, or both. In spite of these differences, the mitochondrial DNA of cybrids showing abnormal growth and development was much more similar to N. tabacum than to P. hybrida mitochondrial DNA. In those cybrids that contained P. hybrida chloroplasts and N. tabacum mitochondria, cotransfer of cytoplasmic organelles did not occur. Although P. hybrida chloroplasts can interact compatibly with the N. tabacum nucleus, no cybrids were found in which P. hybrida mitochondria coexisted with the N. tabacum nucleus.     

Direct Gene Transfer in Psophocarpus tetragonolobus Resistance to Kanamycin

Epicotyl-derived protoplasts of Psophocarpus tetragonolobuswere isolated and regenerated to plants. These protoplasts weretransformed to kanamycin resistance following uptake of plasmid(pABDl or pHP23) DNA in combination with PEG treatment. Protoplast-derivedtransformed colonies were selected on kanamycin (75 mg l^–1).The transformed calli expressed NPT II activity and also exhibitedthe presence of the plasmid gene integrated into the plant genome.However, none of the transformed clones showed regenerationof shoot buds. Psophocarpus tetragonolobus, winged bean, naked DNA transformation, protoplast culture, regenerated plants     

Production of asymmetric somatic hybrid plants between Cichorium intybus L. and Helianthus annuus L.

In order to obtain male-sterile asymmetric somatic hybrids between chicory (Cichorium intybus L.) and a sunflower (Helianthus annuus L.) male-sterile cytoplasmic line, mesophyll chicory protoplasts inactivated with iodoacetic acid and hypocotyl sunflower protoplasts irradiated with γ-rays have been fused, using PEG and applying two different procedures. Thirty three plants were regenerated from putative hybrid calli. A cytological analysis of their root-tip cells indicated that most of them had 18 chromosomes, the same number as chicory. Through Southern hybridisation on total DNA using the maize mitochondrial specific gene probes Cox I, Cox II and Cob, three plants were identified as cytoplasmic asymmetric hybrids, as shown by hybridisation bands specific for both chicory and sunflower. One of the regenerated plants produced a novel pattern of hybridisation that was not detected in either parent. When hybridisation of total DNA was carried out with an atpA mitochondrial gene probe the same three cybrids presented both the fertile chicory fragment and the male-sterile sunflower fragment. Finally, Southern hybridisation with an ORF 522 probe, which in sunflower is co-transcribed with the atpA gene, confirmed the hybrid nature of the three plants. The morphology of the cybrids resembled the parental chicory phenotype, and at anthesis their anthers produced fewer pollen grains which could not germinate either ”in vitro” or ”in situ.” Cybrid plants grown in the field produced seeds when free-pollination occurred. Received: 26 April 2000 / Accepted: 28 August 2000     

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.