Abstract
We developed a highly efficient transformation protocol for the PEG-mediated direct transfer of plasmid DNA into protoplasts of haploid Datura innoxia. Vectors harbouring a neomycin phosphotransferase II gene or a hygromycin B phosphotransferase gene under the control of different promoters were used in the transformation experiments. Various amounts of plasmid DNA were applied without any carrier DNA to show the direct influence of the plasmid DNA concentration on the transformation efficiency. Approximately 95% of the selected calli were regenerated to plants; 20% of them remained haploid. Total DNA of different transgenic plants was analysed with regard to the integration pattern of the plasmid DNA. Plants carrying only one or two copies of the vector DNA were observed as well as individuals with multi-copy integration (up to ten or more copies).
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Abbreviations
- ATF/RTF:
-
absolute/relative transformation frequency
- BAP:
-
6-benzylaminopurine
- CaMV:
-
cauliflower mosaic virus
- CTAB:
-
N-cetyl-N,N,N-trimethyl-ammonium bromide
- HPT:
-
hygromycin B phosphotransferase gene
- PEG:
-
polyethyleneglycol
- MES:
-
2-(N-morpholino) ethanesulfonic acid
- NPT II:
-
neomycin phosphotransferase II gene
References
Baker B, Coupland G, Fedoroff NV, Starlinger P, Schell J (1987) Phenotypic assay for excision of the maize controlling element Ac in tobacco. EMBO J 6(6): 1547–1554
Benediktsson I, Köhler F, Schieder O (1991) Transient and stable expression of marker genes in cotransformed Petunia protoplasts in relation to X-ray and UV-irradiation. Transgenic Res 1: 38–44
Bilang R, Shigeru I, Peterhans A, Potrykus I, Paszkowski J (1991) The 3′ terminal region of the hygromycin-B-resistance gene is important for its activity in Escherichia coli and Nicotiana tabacum. Gene 100: 247–250
Binding H (1974) Regeneration von haploiden und diploiden Pflanzen aus Protoplasten von Petunia hybrida L. Z Pflanzenphysiol 74: 327–356
Damm B, Schmidt R, Willmitzer, L (1989) Efficient transformation of Arabidopsis thaliana using direct gene transfer to protoplasts. Mol Gen Genet 217: 6–12
Feinberg AP, Vogelstein B (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132: 6–13
Gamborg O L, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Experim Cell Res 50: 151–158
Guha S, Maheswari SC (1964) In vitro production of embryos from anthers of Datura. Nature 204: 497
Karesch H, Bilang R, Scheid OM, Potrykus I (1991) Direct gene transfer to protoplasts of Arabidopsis thaliana. Plant Cell Rep 9: 571–574
Köhler F, Golz C, Schieder O (1987) Influence of plant cultivar and plasmid-DNA on transformation rates in tobacco and moth bean. Plant Sci 53: 87–91
Köhler F, Benediktsson I, Cardon G, Andreo CS, Schieder O (1990) Effect of various irradiation treatments of plant protoplasts on the transformation rates after direct gene transfer. Theor Appl Gen 79: 679–685
Negrutiu I, Mouras A, Horth M, Jacobs M (1987) Direct gene transfer to plants: Present developments and some future prospectives. Plant Physiol Biochem 25 (4): 493–503
Paszkowski J, Baur M, Bogucki, A, Potrykus I (1988) Gene targeting in plants. EMBO J 7 (13): 4021–4026
Rogers SO, Bendich, AJ (1985) Extraction of DNA from milligram amounts of fresh, herbarium and mummified plant tissues. Plant Mol Biol 5: 69–76
Shillito RD, Saul MW, Paszkowski J, Müller M, Potrykus I (1985) High efficiency direct gene transfer to plants. Bio/Technology 3: 1099–1103
Schieder O (1975) Regeneration von haploiden und diploiden Datura innoxia Mill. Mesophyll-Protoplasten zu Pflanzen. Z Pflanzenphysiol 76: 462–466
Schieder O (1978) Somatic hybrids of Datura innoxia Mill. Datura discolor Bernh. and Datura innoxia Mill. + Datura stramonium L. var. tatula L. I. Selection and characterization. Mol Gen Genet 162: 113–119
Schieder O (1984) Isolation and culture of protoplasts: Datura. In: Cell Culture and Somatic Cell Genetics of Plants, Vol 1, Vasil I (ed), Orlando: Academic Press, pp 350–355
Vasil IK (1984) Cell Culture and Somatic Cell Genetics of Plants, Vol 1. Academic Press, Orlando
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Schmidt-Rogge, T., Meixner, M., Srivastava, V. et al. Transformation of haploid Datura innoxia protoplasts and analysis of the plasmid integration pattern in regenerated transgenic plants. Plant Cell Reports 12, 390–394 (1993). https://doi.org/10.1007/BF00234698
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DOI: https://doi.org/10.1007/BF00234698