Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Asymmetric hybridization in Nicotiana by fusion of irradiated protoplasts

  • 48 Accesses

  • 74 Citations


Mesophyll protoplasts of a kanamycin-resistant, nopaline-positive Nicotiana plumbaginifolia seed line were inactivated by γ-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.

This is a preview of subscription content, log in to check access.


Km r :

kanamycin resistant

Km s :

kamacysin sensitive

Nop + :

nopaline positive

Nop :

nopaline negative


  1. Bates GW (1985) Electrical fusion for optimal formation of protoplast heterokaryons in Nicotiana. Planta 165:217–224

  2. Bates GW, Hasenkampf CA (1985) Culture of plant somatic hybrids following electrical fusion. Theor Appl Genet 70:227–233

  3. Dellaporta SL, Wood J, Hicks JB (1985) Maize DNA miniprep. In: Malmberg R, Messing J, Sussex I (eds) Molecular biology of plants. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp 36–37

  4. Dudits D, Hadlaczky G, Bajszar GY, Koncz C, Lazar G, Horvath G (1979) Plant regeneration from intergeneric cell hybrids. Plant Sci Lett 15:101–112

  5. Dudits D, Fejer O, Hadlaczky G, Koncz C, Lazar GB, Horvath G (1980) Intergeneric gene transfer mediated by plant protoplast fusion. Mol Gen Genet 179:283–288

  6. Evans DA, Flick CE, Jensen RA (1981) Disease resistance: incorporation into sexually incompatible somatic hybrids of the genus Nicotiana. Science 213:907–909

  7. Evans DA, Bravo JE, Gleba YY (1983) Somatic hybridization, fusion methods, recovery of hybrids, and genetic analysis. Int Rev Cytol Suppl 16:143–159

  8. Galun E, Aviv D (1983) Cytoplasmic hybridization: genetic and breeding applications. In: Evans DA, Sharp WR, Ammirato PV, Yamada Y (eds) Handbook of plant cell culture, vol 1. Techniques for propagation and breeding. Macmillan Publishing Co, New York, pp 358–392

  9. Gleba YY, Sytnik KM (1984) Protoplast fusion, genetic engineering in higher plants. Springer-Verlag, Berlin

  10. Goodspeed TH (1954) The genus Nicotiana. Chronica Botanica Co, Waltham, Massachusetts

  11. Gupta PP, Gupta M, Schieder O (1982) Correction of nitrate reductase defect in auxotrophic plant cells through protoplast-mediated intergeneric gene transfers. Mol Gen Genet 188:378–383

  12. Gupta PP, Schieder O, Gupta M (1984) Intergeneric nuclear gene transfer between somatically and sexually incompatible plants through asymmetric protoplast fusion. Mol Gen Genet 197:30–35

  13. Horsch RB, Fraley RT, Rogers SG, Sanders PR, Lloyd A, Hoffmann N (1984) Inheritance of functional foreign genes in plants. Science 223:496–498

  14. Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG, Fraley RT (1985) A simple and general method for transferring genes into plants. Science 227:1229–1231

  15. Klobutcher LA, Ruddle FH (1981) Chromosome mediated gene transfer. Ann Rev Biochem 50:533–554

  16. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning. Cold Spring Harbor Laboratories, Cold Spring Harbor, New York

  17. Menczel L, Nagy F, Kiss ZR, Maliga P (1981) Streptomycin resistant and sensitive somatic hybrids of Nicotiana tabacum + Nicotiana knightiana: correlation of resistance to N. tabacum plastids. Theor Appl Genet 59:191–195

  18. Menczel L, Galiba G, Nagy F, Maliga P (1982) Effect of radiation dosage on efficiency of chloroplast transfer by protoplast fusion in Nicotiana. Genetics 100:487–495

  19. Menczel L, Polsby LS, Steinback KE, Maliga P (1986) Fusion-mediated transfer of triazine-resistant chloroplasts: characterization of Nicotiana tabacum cybrid plants. Mol Gen Genet 205:201–205

  20. Moav R (1961) Genetic instability in Nicotiana hybrids: II. Studies of the Ws(pbg) locus of N. plumbaginifolia in N. tabacum nuclei. Am J Bot 47:87–93

  21. Nagy JI, Maliga P (1976) Callus induction and plant regeneration from mesophyll protoplasts of Nicotiana sylvestris. Z Pflanzenphysiol 78:453–455

  22. Nagy F, Lazar G, Maliga P (1983) A heteroplasmic state induced by protoplast fusion is a necessary condition for detecting rearrangements in Nicotiana mitochondrial DNA. Theor Appl Genet 66:203–207

  23. Otten L, Schilperoort RA (1978) A rapid micro scale method for the detection of lysopine and nopaline dehydrogenase activities. Biochim Biophys Acta 527:497–500

  24. Pandey KK (1975) Sexual transfer of specific genes without gametic fusion. Nature 256:310–313

  25. Schieder O, Vasil IK (1980) Protoplast fusion and somatic hybridization. Int Rev Cytol Suppl 11:21–46

  26. Shillito RD, Paszkowski J, Potrykus I (1983) Agarose plating and a bead type culture technique enable and stimulate development of protoplast-derived colonies in a number of plant species. Plant Cell Rep 2:244–247

  27. Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517

  28. Szabados L, Hadlaczky GY, Dudits D (1981) Uptake of isolated plant chromosomes by plant protoplasts. Planta 151:141–145

Download references

Author information

Additional information

Communicated by P. Maliga

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Bates, G.W., Hasenkampf, C.A., Contolini, C.L. et al. Asymmetric hybridization in Nicotiana by fusion of irradiated protoplasts. Theoret. Appl. Genetics 74, 718–726 (1987). https://doi.org/10.1007/BF00247548

Download citation

Key words

  • Gene transfer
  • Asymmetric hybrids
  • Kanamycin resistance
  • Tobacco
  • Fertility