Summary
Experimental parameters for direct gene transfer with recombinant DNA encoding neomycin phosphotransferase II (NPTII) under control of eukaryotic expression signals were established. The introduced gene was shown by the growth of transformants on media containing kanamycin, by genomic blotting and by assaying NPTII activity. Leaf protoplasts from three green genotypes of varieties xanthii and petit havanna, and from four plastome-encoded albino genotypes of Nicotiana tabacum were analyzed with respect to cell division kinetics and yield of kanamycin-tolerant colonies after direct gene transfer. No clear correlation was found between the time of onset of cell division and transformation frequency.
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References
Aviv D, Galun E (1985) An in vitro procedure to assign pigment mutations in Nicotiana to either the chloroplast or the nucleus. J Hered 76:135–137
Beck E, Ludwig G, Auerswald EA, Reiss B, Schaller H (1982) Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5. Gene 19:327–336
Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523
Davey MR, Cocking EC, Freeman J, Pearce N, Tudor I (1980) Transformation of Petunia protoplasts by isolated Agrobacterium plasmids. Plant Sci Lett 18:307–313
Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:151–158
Hain R, Stabel P, Czernilofsky AP, Steinbiss HH, 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
Köhler F, Golz C, Eapen S, Kohn H, Schieder O (1987) Stable transformation of moth bean Vigna aconitifolia via direct gene transfer. Plant Cell Rep 6:313–317
Koop HU, Schweiger HG (1985) Regeneration of plants from individually cultivated protoplasts using an improved microculture system. J Plant Physiol 121:245–257
Krens FA, Molendijk L, Wullems GJ, Schilperoort RA (1982) In vitro transformation of plant protoplasts with Ti-plasmid DNA. Nature 296:72–74
Lörz H, Baker B, Schell J (1985) Gene transfer to cereal cells mediated by protoplast transformation. Mol Gen Genet 199:178–182
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor/NY
Meyer P, Walgenbach E, Bussmann K, Hombrecher G, Saedler H (1985) Synchronized tobacco protoplasts are efficiently transformed by DNA. Mol Gen Genet 201:513–518
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–479
Negrutiu I, Shillito R, Potrykus I, Biasini G, Sala F (1987) Hybrid genes in the analysis of transformation conditions. I. Setting up a simple method for direct gene transfer in plant protoplasts. Plant Mol Biol 8:363–373
Okada K, Takebe I, Nagata T (1986) Introduction of functional RNA into plant protoplasts by electroporation. Mol Gen Genet 205:398–403
Paszkowski J, Pisan B, Shillito RD, Hohn T, Hohn B, Potrykus I (1984) Direct gene transfer to plants. EMBO J 3:2717–2722
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
Reiss B, Sprengel HW, Schaller H (1984) A new sensitive method for qualitative and quantitative assay of neomycin phosphotransferase in crude cell extracts. Gene 30:211–218
Riggs CD, Bates GW (1986) Stable transformation of tobacco by electroporation: Evidence for plasmid concatenation. Proc Natl Acad Sci USA 83:5602–5606
Rogers DO, Bendich AJ (1985) Extraction of DNA from milligram amounts of fresh, herbarium and mummified plant tissues. Plant Mol Biol 5:69–76
Schreier PH, Seftor EA, Schell J, Bohnert HJ (1985) The use of nuclear-encoded sequences to direct the light-regulated synthesis and transport of a foreign protein into plant protoplasts. EMBO J 4:25–32
Shillito RD, Saul M, Paszkowski J, Müller M, Potrykus I (1985) High efficiency gene transfer to plants. Bio/Technol 3:1099–1103
Töpfer R, Matzeit V, Gronenborn B, Schell J, Steinbiß HH (1987) A set of plant expression vectors for transcriptional and translational fusions. Nucleic Acid Res 15:5890
Uchimiya H, Fushimi T, Hashimoto H, Harada H, Syono K, Sugawara Y (1986) Expression of a foreign gene in callus derived from DNA treated protoplasts of rice (Oryza sativa L.). Mol Gen Genet 204:204–207
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Communicated by G. Wenzel
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Tyagi, S., Spörlein, B., Tyagi, A.K. et al. PEG- and electroporation-induced transformation in Nicotiana tabacum: influence of genotype on transformation frequencies. Theoret. Appl. Genetics 78, 287–292 (1989). https://doi.org/10.1007/BF00288813
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DOI: https://doi.org/10.1007/BF00288813