Summary
In P. hybrida and B. nigra an enhancement of transformation rates (direct gene transfer) of about six to seven-fold was obtained after irradiation of protoplasts with 12.5 Gy (X-ray). The effect of protoplast irradiation was similar in experiments where protoplasts were irradiated 1h before transformation (X-ray/DNA) or 1h after completion of the transformation procedure (DNA/X-ray). Increased X-ray doses up to 62.5 Gy resulted in further enhancement of percentages of transformed colonies, indicating a correlation between relative transformation frequencies (RTF) and the doses applied. Estimation of degradation rates of plasmid sequences in plant protoplasts yielded a reduction of plasmid concentration to 50% 8–12 h after transformation. In 1-day-old protoplasts, the level of plasmid fragments dropped to 0%–10% compared to 1h after transformation. The results demonstrate that the integration rates of plasmid sequences into the plant genome may in part be governed by DNA repair mechanisms. This could be an explanation for the observed genotypic dependence of transformation rates in different plant species and plant genotypes. Gene copy number reconstructions revealed enhanced integration rates of plasmid sequences in transformed colonies derived from irradiated protoplasts.
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Balks N, Zakai N, Friedberg D, Loyter A (1988) Linear forms of plasmid DNA are superior to supercoiled structures as active templates for gene expression in plant protoplasts. Plant Mol Biol 11:517–527
Bergounioux C, Perennes C, Miege C, Gadal P (1986) Male sterility effect upon protoplast division in Petunia hybrida. Protoplasma 130:138–144
Bergounioux C, Perennes C, Brown SC, Sarda C, Gadal P (1988) Relation between protoplast division, cell cycle stage and, nuclear chromatin structure. Protoplasma 142:127–136
Binding H (1974) Regeneration von haploiden und diploiden Pflanzen aus Protoplasten von Petunia hybrida. Z Pflanzenphysiol 74:327–356
Chuong PV, Pauls KP, Beversdorf WD (1987) Plant regeneration from Brassica nigra (L.) Koch stem protoplasts. In Vitro Cell Develop Biol 23:449–452
Cook R, Meyer Y (1981) Hormonal control of tobacco protoplast nucleic acid metabolism during in vitro culture. Planta 152:1–7
Damm B, Schmidt R, Willmitzer L (1989) Efficient transformation of Arabidopsis thaliana using direct gene transfer to protoplasts. Mol Gen Genet 217:6–12
Glimelius K (1984) High growth rate and regeneration capacity of hypocotyl protoplasts in some Brassicaceae. Physiol Plant 61:38–44
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 (1987a) Stable transformation of moth bean Vigna aconitifolia via direct gene transfer. Plant Cell Rep 6:313–317
Köhler F, Golz C, Eapen S, Schieder O (1987b) Influence of plant cultivar and plasmid-DNA on transformation rates in tobacco and moth bean. Plant Sci 53:87–91
Köhler F, Cardon G, Pöhlmann M, Gill R, Schieder O (1989) Enhancement of transformation rates in higher plants by low-dose irradiation: are DNA repair systems involved in the incorporation of exogenous DNA into the plant genome? Plant Mol Biol 12:189–199
Korr H, Koeser K, Oldenkott S, Schmidt H, Schulze B (1989) X-ray dose effect relationship on unscheduled DNA synthesis in mouse brain cells studied in vivo. Radiat Environ Biophys 28:13–26
Krüger-Lebus S, Potrykus I (1987) A simple and efficient method for direct gene transfer to Petunia hybrida without electroporation. Plant Mol Biol Rep 5:289–294
Li L, Kohlenbach HW (1982) Somatic embryogenesis in quite a direct way in cultures of mesophyll protoplasts of Brassica napus L. Plant Cell Rep 1:209–211
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press. Cold Spring Harbor/NY
Murashige T, Skoog F (1962) Revised media for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15:473–497
Murray MG, Thompson WF (1980) Rapid isolation of highmolecular-weight plant DNA. Nucleic Acids Res 8:4321–4325
Nagata T, Takebe I (1971) Plating of isolated tobacco mesophyll protoplasts on agar medium. Planta 99:12–22
Negrutiu I, Shillito R, Potrykus I, Biasini G, Sala F (1987) Hybrid genes in the analysis of transformation conditions. Plant Mol Bioll 8:363–373
Paszkowski J, Shillito R, Saul M, Mandak V, Hohn T, Hohn B, Potrykus I (1984) Direct gene transfer to plants. EMBO J 3:2717–2722
Paszkowski J, Baur M, Bogucki A, Potrykus I (1988) Gene targeting in plants. EMBO J 7:4021–4026
Pröls M, Töpfer R, Schell J, Steinbiß HH (1988) Transient gene expression in tobacco protoplasts: I. Time course of CAT appearance. Plant Cell Rep 7:221–224
Sacristan MD, Gerdemann-Knörck M, Schieder O (1989) Incorporation of hygromycin resistance in Brassica nigra and its transfer to B. napus through asymmetric protoplast fusion. Theor Appl Genet 78:194–200
Schieder O (1984) Isolation and culture of protoplasts: Datura. In: Vasil (ed) Cell culture and somatic cell genetics of plants, vol 1. Academic Press, Orlando, pp 350–355
Schocher RJ, Shillito RD, Saul MW, Paszkowski J, Potrykus I (1986) Co-transformation of unlinked foreign genes into plants by direct gene transfer. Bio/Technol 4:1093–1096
Shillito RD, Saul M, Paszkowski J, Potrykus I (1985) High efficiency direct gene transfer to plants. Bio/Technol 3:1099–1103
Tyagi S, Spörlein B, Tyagi AK, Herrmann RG, Koop HU (1989) PEGand electroporation-induced transformation in Nicotiana tabacum: influence of genotype on transformation frequencies. Theor Appl Genet 78:287–292
Wilson SM, Thorpe TA, Moloney MM (1989) PEG-mediated expression of GUS and CAT genes in protoplasts from embryogenic suspension cultures of Picea glauca. Plant Cell Rep 7:704–707
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Communicated by G. Wenzel
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Köhler, F., Benediktsson, I., Cardon, G. et al. Effect of various irradiation treatments of plant protoplasts on the transformation rates after direct gene transfer. Theoret. Appl. Genetics 79, 679–685 (1990). https://doi.org/10.1007/BF00226883
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DOI: https://doi.org/10.1007/BF00226883