Summary
The combination in the nuclear genome of a dominant resistance marker (to select against unfused wild-type cells) and a recessive deficiency marker (to select against unfused mutant cells) in a cell line should provide a system for selecting fusion hybrids between the mutant line and any wild-type line. To test this idea, we fused protoplasts from a non-morphogenic cell line of Nicotiana tabacum which was kanamycin resistant (by transformation) and deficient in nitrate reductase (NR-K+) with protoplasts from N. tabacum cv. Petit Havana clone SR1, which provided resistance against streptomycin as an additional selectable marker (NR+K-SR+). Putative hybrids were selected using a culture medium containing no available reduced nitrogen source and 50 mg/l kanamycin sulphate. After regeneration into plants, the hybrid character was demonstrated from: (i) the morphological variation of the regenerants; (ii) the chromosome number; (iii) the ability to grow on medium without a reduced nitrogen source and containing kanamycin sulphate at 50 mg/l; (iv) the presence of nitrate reductase activity; (v) the presence of the gene coding for neomycin phosphotransferase, which provides resistance to kanamycin sulphate; (vi) callus formation from leaves on medium containing 1 g/l streptomycin or 50 mg/l kanamycin sulphate; (vii) F1 plants containing nitrate reductase and the gene for neomycin phosphotransferase. Fusions between the mutant cell line (NR-K+) and three wild-type tobacco species and subsequent cultivation on medium containing no available nitrogen source but 50 mg/l kanamycin sulphate resulted in callus formation with all combinations, while hybrid plants were only regenerated when N. sylvestris was the fusion partner.
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References
Egger A (1986) Einfluß von NO2 auf Enzyme der assimilatorischen Sulfat- und Nitratreduktion in Nadeln von Fichte (Picea abies L.). Lizentiatsarbeit University of Bern, Switzerland
Fromm ME, Taylor LP, Walbot U (1986) Stable transformation of maize after gene transfer by electroporation. Nature 319:791–793
Gamborg OL (1970) The effect of amino acids and ammonium on the growth of plant cells in suspension culture. Plant Physiol 45:372–375
Hain R, Stabel P, Czernilofsky AP, Steinbiss L, Herrera-Estrella L, Schell J (1985) Uptake, integration, expression and genetic transmission of a selectable chimaeric gene by plant protoplasts. Mol Gen Genet 199:161–168
Hamill JD, Pental D, Cocking EC, Müller AJ (1983) Production of a nitrate reductase deficient streptomycin resistant mutant of Nicotiana tabacum for somatic hybridization studies. Heredity 50:197–200
Harms CT (1983) Somatic hybridization by plant protoplast fusion. In: Potrykus I, Harms CT, Hinnen A, Hütter R, King PJ, Shillito RD (eds) Protoplasts 1983, Lecture Proceedings. Birkhäuser, Basel p 69
Lazar GB (1983) Recent developments in plant protoplast fusion and selection technology. In: Potrykus I, Harms CT, Hinnen A, Hütter R, King PJ, Shillito RD (eds) Protoplasts 1983, Lecture Proceedings. Birkhäuser, Basel, p 61
Linsmaier EM, Skoog F (1965) Organic growth factor requirements of tobacco tissue cultures. Physiol Plant 18:100–127
Lörz H, Baker B, Schell J (1985) Gene transfer to cereal cells mediated by protoplast transformation. Mol Gen Genet 199:178–182
Maliga P, Sz-Breznovits A, Marton L (1973) Streptomycin-resistant plants from callus culture of haploid tobacco. Nature New Biol 244:29–30
Mendel RR, Müller AJ (1979) Nitrate reductase deficient mutant cell lines of Nicotiana tabacum. Further biochemical characterization. Mol Gen Genet 77:145–152
Mendel RR, Alikulov ZA, Lvov NP, Müller AJ (1981) Presence of the molybdenum cofactor in nitrate reductase — deficient mutant cell lines of Nicotiona tabacum. Mol Gen Genet 181:395–399
Müller AJ, Grafe R (1978) Isolation and characterization of cell lines of Nicotiana tabacum lacking nitrate reductase. Mol Gen Genet 161:67–76
Neyra CA, Hageman RH (1975) Nitrate uptake and induction of nitrate reductase in excised roots. Plant Physiol 56:692–695
Paszkowski J, Saul MW (1986) Direct gene transfer to plants. Methods Enzymol 118:668–684
Paszkowski J, Shillito RD, Saul M, Mandak V, Hohn T, Hohn B, Potrykus I (1984) Direct gene transfer to plants. EMBO J 3:2717–2722
Pental D, Hamill JD, Pirrie A, Cocking EC (1986) Somatic hybridization of Nicotiana tabacum and Petunia hybrida. Mol Gen Genet 202:342–347
Potrykus I, Shillito RD (1986) Protoplasts: Isolation, culture, plant regeneration. Methods Enzymol 118:549–578
Potrykus I, Jia J, Lazar GB, Saul M (1984) Hyoscyamus muticus + Nicotiana tabacum fusion hybrids selected via auxotroph complementation. Plant Cell Rep 3:68–71
Potrykus I, Shillito RD, Saul MW, Paszkowski J (1985) Direct gene transfer. State of the art and future potential. Plant Mol Biol Rep 3:117–128
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
Shillito RD, Saul MW, Paszkowski J, Müller M, Potrykus I (1985) High efficiency direct gene transfer to plants. Biotechnology 3:1099–1103
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Brunold, C., Krüger-Lebus, S., Saul, M.W. et al. Combination of kanamycin resistance and nitrate reductase deficiency as selectable markers in one nuclear genome provides a universal somatic hybridizer in plants. Mol Gen Genet 208, 469–473 (1987). https://doi.org/10.1007/BF00328141
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DOI: https://doi.org/10.1007/BF00328141