Abstract
Electroporation (electric field-mediated DNA transfer) of tobacco protoplasts in the presence of the linearized plasmid pMON200 has led to the formation of transgenic plants. Defined electric shocks were delivered by capacitive discharges with readily available, low-cost electrical components. This transformation procedure is simple and efficient and may suggest a quick method for determining the appropriate electric fields for new cell systems. An optimal transformation frequency of 2.2 X 10(-4) (based on the number of cells subjected to the shock) was obtained with a single 2000-V/cm, 250-microseconds-duration capacitive discharge. Calli transformed to kanamycin resistance have been regenerated into whole plants. Southern blots of DNA from the transgenic plants demonstrate the integration of the selectable marker gene (neomycin phosphotransferase) at single or multiple genomic sites. In some cases, the plasmid appears to be integrated intact; in others, it is rearranged. The blots also provide evidence of plasmid recircularization and/or the formation of head-to-head and head-to-tail concatemers in most of the plants analyzed. Although some plants apparently have multiple integration sites, analysis of progeny obtained by self-fertilization of the transgenic plants indicates that the kanamycin-resistance marker is inherited as a single dominant gene.
Full text
PDF




Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- A simple and general method for transferring genes into plants. Science. 1985 Mar 8;227(4691):1229–1231. doi: 10.1126/science.227.4691.1229. [DOI] [PubMed] [Google Scholar]
- Chomczynski P., Qasba P. K. Alkaline transfer of DNA to plastic membrane. Biochem Biophys Res Commun. 1984 Jul 18;122(1):340–344. doi: 10.1016/0006-291x(84)90480-7. [DOI] [PubMed] [Google Scholar]
- Deshayes A., Herrera-Estrella L., Caboche M. Liposome-mediated transformation of tobacco mesophyll protoplasts by an Escherichia coli plasmid. EMBO J. 1985 Nov;4(11):2731–2737. doi: 10.1002/j.1460-2075.1985.tb03996.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Folger K. R., Wong E. A., Wahl G., Capecchi M. R. Patterns of integration of DNA microinjected into cultured mammalian cells: evidence for homologous recombination between injected plasmid DNA molecules. Mol Cell Biol. 1982 Nov;2(11):1372–1387. doi: 10.1128/mcb.2.11.1372. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fromm M. E., Taylor L. P., Walbot V. Stable transformation of maize after gene transfer by electroporation. 1986 Feb 27-Mar 5Nature. 319(6056):791–793. doi: 10.1038/319791a0. [DOI] [PubMed] [Google Scholar]
- Fromm M., Taylor L. P., Walbot V. Expression of genes transferred into monocot and dicot plant cells by electroporation. Proc Natl Acad Sci U S A. 1985 Sep;82(17):5824–5828. doi: 10.1073/pnas.82.17.5824. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hinnen A., Hicks J. B., Fink G. R. Transformation of yeast. Proc Natl Acad Sci U S A. 1978 Apr;75(4):1929–1933. doi: 10.1073/pnas.75.4.1929. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Horsch R. B., Fraley R. T., Rogers S. G., Sanders P. R., Lloyd A., Hoffmann N. Inheritance of functional foreign genes in plants. Science. 1984 Feb 3;223(4635):496–498. doi: 10.1126/science.223.4635.496. [DOI] [PubMed] [Google Scholar]
- Kanai R., Edwards G. E. Purification of enzymatically isolated mesophyll protoplasts from c(3), c(4), and crassulacean Acid metabolism plants using an aqueous dextran-polyethylene glycol two-phase system. Plant Physiol. 1973 Nov;52(5):484–490. doi: 10.1104/pp.52.5.484. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McMahon A. P., Flytzanis C. N., Hough-Evans B. R., Katula K. S., Britten R. J., Davidson E. H. Introduction of cloned DNA into sea urchin egg cytoplasm: replication and persistence during embryogenesis. Dev Biol. 1985 Apr;108(2):420–430. doi: 10.1016/0012-1606(85)90045-4. [DOI] [PubMed] [Google Scholar]
- Otten L. A., Schilperoort R. A. A rapid micro scale method for the detection of lysopine and nopaline dehydrogenase activities. Biochim Biophys Acta. 1978 Dec 8;527(2):497–500. doi: 10.1016/0005-2744(78)90363-7. [DOI] [PubMed] [Google Scholar]
- Paszkowski J., Shillito R. D., Saul M., Mandák V., Hohn T., Hohn B., Potrykus I. Direct gene transfer to plants. EMBO J. 1984 Dec 1;3(12):2717–2722. doi: 10.1002/j.1460-2075.1984.tb02201.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Potter H., Weir L., Leder P. Enhancer-dependent expression of human kappa immunoglobulin genes introduced into mouse pre-B lymphocytes by electroporation. Proc Natl Acad Sci U S A. 1984 Nov;81(22):7161–7165. doi: 10.1073/pnas.81.22.7161. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Scangos G., Ruddle F. H. Mechanisms and applications of DNA-mediated gene transfer in mammalian cells - a review. Gene. 1981 Jun-Jul;14(1-2):1–10. doi: 10.1016/0378-1119(81)90143-8. [DOI] [PubMed] [Google Scholar]
- Sengupta-Gopalan C., Reichert N. A., Barker R. F., Hall T. C., Kemp J. D. Developmentally regulated expression of the bean beta-phaseolin gene in tobacco seed. Proc Natl Acad Sci U S A. 1985 May;82(10):3320–3324. doi: 10.1073/pnas.82.10.3320. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
- Stinchcomb D. T., Shaw J. E., Carr S. H., Hirsh D. Extrachromosomal DNA transformation of Caenorhabditis elegans. Mol Cell Biol. 1985 Dec;5(12):3484–3496. doi: 10.1128/mcb.5.12.3484. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Watts J. W., King J. M. A simple method for large-scale electrofusion and culture of plant protoplasts. Biosci Rep. 1984 Apr;4(4):335–342. doi: 10.1007/BF01140497. [DOI] [PubMed] [Google Scholar]