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.
Similar content being viewed by others
References
Akda S, Hirai A (1986) Studies on the mode of separation of chloroplast genomes in parasexual hybrid calli. III. Random separation of two types of chloroplast genomes in hybrid callus. Jpn J Genet 61:437–445
Asahi T, Kumashiro I, Kubo T (1988) Constitution of mitochondrial and chloroplast genomes in male-sterile tobacco obtained by protoplast fusion ofNicotiana tabacum andN. debneyi. Plant Cell Physiol 29:43–49
Aviv D, Galun E (1987) Chondriome analysis in sexual progenies ofNicotiana cybrids. Theor Appl Gent 73:821–826
Aviv D, Bleichman S, Arzee-Gonen P, Galun E (1984) Intersectional cytoplasmic hybrids inNicotiana. Theor Appl Genet 67:499–504
Belliard G, Vedel F, Pelletier G (1979) Mitochondrial recombination in cytoplasmic hybrids ofNicotiana tabacum by protoplast fusion. Nature 281:401–403
Boer PJH, McIntosh JE, Gray MW, Bonen L (1985) The wheat mitochondrial gene for apocytochrome b: absence of a prokaryotic ribosome binding site. Nucleic Acids Res 13:2281–2292
Bonen L, Bird S (1988) Sequence analysis of the wheat mitochondrialatp6 gene reveals a fused upstream reading frame and markedly divergent N termini among plant ATP6 proteins. Gene 73:47–56
Bonen L, Boer PH, Gray MW (1984) The wheat cytochrome oxidase subunit II gene has an intron insert and three radical amino acid changes relative to maize. EMBO J 3:2531–2536
Bonnett HT, Glimelius K (1983) Somatic hybridization inNicotiana: behavior of organelles after fusion of protoplasts from male-fertile and male-sterile cultivars. Theor Appl Genet 65:213–217
Chen K, Wildman SG, Smith HH (1977) Chloroplast DNA distribution in parasexual hybrids as shown by polypeptide composition of Fraction 1 protein. Proc Natl Acad Sci USA 74:5109–5112
Dijak M, Sproule A, Keller WA, Pandeya RS, Gleddie S (1991) Transformation ofNicotiana tabacum, N.debneyi andN. rustica: Inheritance and protoplast expression of antibiotic resistance. Plant Cell Tiss Org Cult 25:189–197
Douglas GC, Keller WA, Setterfield G (1981a) Somatic hybridization betweenN. rustica andN. tabacum. II. Protoplast fusion and selection and regeneration of hybrid plants. Can J Bot 59:220–227
Douglas GC, Wetter LR, Nakamura C, Keller WA, Setterfield G (1981b) Somatic hybridization betweenNicotiana rustica andN. tabacum. III. Biochemical, morphological, and cytological analysis of somatic hybrids. Can J Bot 59:228–237
Evans DA (1983) Agricultural applications of plant protoplast fusion. Biotechnology 1:253–261
Feinberg AP, Vogelstein B (1983) A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6–13
Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:151–158
Gerlach WL, Bedbrook JR (1979) Cloning and characterization of ribosomal genes from wheat and barley. Nuclic Acids Res 7:1869–1885
Glimelius K, Chen K, Bonnett HT (1981) Somatic hybridization inNicotiana: segregation of organellar traits among hybrid and cybrid plants. Planta 153:504–510
Glimelius K, Djupsjobacka M, Fellner-Feldegg H (1986a) Selection and enrichment of plant protoplast heterokaryons of Brassicaceae by flow sorting. Plant Sci 45:133–141
Glimelius D, Fahleson J, Sjodin G, Sundberg E, Djupsjobacka M, Fellner-Feldegg H, Bonnett HT (1986b) Somatic hybridization and cybridization as potential methods for widening of the gene pool of crops within Brassicaceae and Solanaceae. In: Horn W, Jensen CJ, Odenbach W, Schnieder O (eds) Genetic manipulation in plant breeding. de Gruyter, Berlin New York, pp 663–682
Hakansson G, Mark F van der, Bonnett HT, Glimelius K (1988) Variant mitochondrial protein and DNA patterns associated with cytoplasmic male-sterile lines ofNicotiana. Theor Appl Genet 76:431–437
Komari T, Saito Y, Nakakido F, Kumashiro T (1989) Efficient selection of somatic hybrids inNicotiana tabacum L. using a combination of drug resistance markers introduced by transformation. Theor Appl Genet 77:547–552
Kumashiro T, Kubo T (1986) Cytoplasm transfer ofNicotiana debneyi toN. tabacum by protoplast fusion. Jpn J Breed 36:39–48
Masson J, Lancelin D, Bellini C, Lecerf M, Guerche P, Pelletier G (1989) Selection of somatic hybrids between diploid clones of potato (Solanum tuberosum L.) transformed by direct gene transfer. Theor Appl Genet 78:153–159
Medgyesy P, Fejes E, Maliga P (1985) Interspecific chloroplast recombination in aNicotiana somatic hybrid. Proc Natl Acad Sci USA 82:6990–6964
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497
Nagata T, Takebe I (1971) Plating of isolated tobacco mesophyll protoplasts on agar medium. Planta 99:12–20
Sand SA, Christoff GT (1972) Cytoplasmic-chromosomal interactions and altered differentiation in tobacco. J Hered 64:24–30
Thanh ND, Medgyesy P (1989) Limited chloroplast gene transfer via recombination overcomes plastome-genome incompatibility betweenNicotiana tabacum andSolanum tuberosum. Plant Mol Biol 12:87–93
Thomas MR, Johnson LB, White FF (1990) Selection of interspecific somatic hybrids ofMedicago by usingAgrobacterium-transformed tissues. Plant Sci 69:189–198
Author information
Authors and Affiliations
Additional information
Communicated by P. Maliga
Rights and permissions
About this article
Cite this article
Sproule, A., Donaldson, P., Dijak, M. et al. Fertile somatic hybrids between transgenicNicotiana tabacum and transgenicN. debneyi selected by dual-antibiotic resistance. Theoret. Appl. Genetics 82, 450–456 (1991). https://doi.org/10.1007/BF00588598
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00588598