Summary
Cloning of genes by transformation with genomic banks and rescue of a phenotype has been extensively used in bacterial systems. This approach has not been possible in plant systems because of the large genome sizes and poor transformation frequencies of most plant species. Recent advances in plant transformation permit the generation of large numbers of transformants in petunia. We have used this system to rescue a model gene encoding resistance to kanamycin by “shotgun” cloning. The gene encoding neomycin phosphotransferase (NPTII) was introduced into the genome of Arabidopsis thaliana by Agrobacterium tumefaciens-mediated transformation. A genomic bank of DNA from this tissue was constructed in a cosmid vector containing features which would allow its use in plant transformation. The unselected genomic bank was mobilized from Escherichia coli to A. tumefaciens and used to retransform petunia leaf discs. The rescued gene was identified by its ability to confer a kanamycin-resistant phenotype in petunia tissue. The presence of the NPTII gene was confirmed by nopaline assay and Southern blot analysis. This experiment demonstrates the feasibility of gene rescue, in certain circumstances, in plants.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
An G, Watson B, Stachel S, Gordon M, Nester E (1985) New cloning vehicles for transformation of higher plants. EMBO J 4:277–284
Bennett M, Smith J, Heslop-Harrison J (1982) Nuclear DNA amounts in angiosperms. Proc R Soc Lond [Biol] 216:179–199
Chang C, Meyerowitz E (1986) Molecular cloning and DNA sequence of the Arabidopsis thaliana alcohol dehydrogenase gene. Proc Natl Acad Sci USA 83:1408–1412
Crawford N, Campell W, Davis R (1986) Nitrate reductase from squash: cDNA cloning and nitrate regulation. Proc Natl Acad Sci USA 83:8073–8076
Ditta G, Stanfield S, Corbin D, Helinski D (1980) Broad host range DNA cloning systems for gram negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci USA 77:7347–7351
Ditta G, Schmidhauser T, Yakobson E, Lu P, Liang X-W, Finlay DR, Guiney D, Helinski DR (1985) Plasmids related to the broad host range vector pRK290 useful for gene cloning and for monitoring gene expression. Plasmid 13:149–153
Estelle M, Somerville C (1986) The mutants of Arabidopsis. Trends Genet 2:89–93
Fraley R, Rogers S, Horsch R, Eichholz D, Flick J, Fink C, Hoffmann N, Sanders P (1985) The SEV system: a new disarmed Ti plasmid vector for plant transformation. Bio/Technology 3:629–635
Garfinkel D, Simpson R, Ream L, White F, Gordon M, Nester E (1981). Genetic analysis of crown gall: fine structure map of the T-DNA by site-directed mutagenesis. Cell 27:143–153
Horsch R, Klee H (1986) Rapid assay of foreign gene expression in leaf discs transformed by Agrobacterium tumefaciens: sole of T-DNA borders in the transfer process. Proc Natl Acad Sci USA 83:4428–4432
Horsch R, Fry J, Hoffmann N, Wallroth M, Eichholz D, Rogers S, Fraley R (1985) A simple and general method for transferring genes into plants. Science 227:1229–1231
Kislev N, Rubenstein I (1980) Utility of ethidium bromide in the extraction from whole plants of high molecular weight maize DNA. Plant Physiol 66:1140–1143
Klee H, Yanofsky N, Nester E (1985) Vectors for transformation of higher plants. Bio Technology 3:637–642
Leutwiler L, Hough-Evans B, Meyerowitz E (1984) The DNA of Arabidopsis thaliana. Mol Gene Genet 194:15–23
Lloyd A, Barnason A, Rogers S, Byrne M, Fraley R, Horsch R (1986) Transformation of Arabidopsis thaliana with Agrobacterium tumefaciens. Science 234:464–466
McCormick S, Niedermeyer J, Fry J, Barnason A, Horsch R, Fraley R (1986) Leaf disc transformation of cultivated tomato (L. esculentum) using Agrobacterium tumefaciens. Plant Cell Rep 5:81–84
Meyerowitz E, Pruitt R (1986) Arabidopsis thaliana and plant molecular genetics. Science 229:1214–1218
Miller J (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, NY
Otten L, Schilperoort R (1978) A rapid microscale method for the detection of lysopine and nopaline dehydrogenase activities. Biochim Biophys Acta 527:497–500
Pruitt R, Meyerowitz E (1986) Characterization of the genome of Arabidopsis thaliana. J Mol Biol 187:169–183
Schmidhauser T, Helinski D (1985) Regions of broad host-range plasmid RK2 involved in replication and stable maintenance in nine species of gram-negative bacteria. J Bacteriol 164:446–455
Sciaky D, Montoya A, Chilton M-D (1978) Fingerprints of Agrobacterium Ti plasmids. Plasmid 1:238–253
Shah D, Horsch R, Klee H, Kishore G, Winter J, Tumer N, Hironaka C Sanders P, Gasser C, Aykent S, Siegal N, Rogers S, Fraley R (1986) Engineering herbicide tolerance in transgenic plants. Science 233:478–481
Simoens C, Alliotte T, Mendel R, Muller A, Schiemann J, Van Lijsebettens M, Schell J, Van Montagu M, Inze D (1986) A binary vector for transferring genomic libraries to plants. Nucleic Acids Res 14:8073–8090
Simpson R, Spielmann A, Margossian L, McKnight T (1986) A disarmed binary vector from Agrobacterium tumefaciens functions in Agrobacterium rhizogenes. Plant Mol Biol 6:403–415
Southern E (1975) Detection of specific DNA sequences among fragments separated by gel electrophoresis. J Mol Biol 98:503–517
van den Elzen P, Lee K, Townsend J, Bedbrook J (1985) Simple binary vectors for DNA transfer to plant cells. Plant Mol Biol 5:149–154
Velten J, Schell J (1985) Selection-expression plasmid vectors for use in genetic transformation of higher plants. Nucleic Acids Res 13:6981–6998
Wang K, Herrera-Estrella L, Van Montagu M, Zambryski P (1984) Right 25 bp terminus of the nopaline T-DNA is essential for and determines direction of DNA transfer from Agrobacterium to the plant genome. Cell 38:455–462
Wullems G, Molendijk L, Ooms G, Schilperoort R (1981) Differential expression of crown gall tumor markers in transformants obtained after in vitro Agrobacterium tumefaciens-induced transformation of cell wall regenerating protoplasts derived from Nicotiana tabacum. Proc Natl Acad Sci USA 78:4344–4348
Yadav N, Vanderleyden J, Bennett D, Barnes W, Chilton M-D (1982) Short direct repeats flank the T-DNA on a nopaline Ti plasmid. Proc Natl Acad Sci USA 79:6322–6326
Author information
Authors and Affiliations
Additional information
Communicated by R.B. Goldberg
Rights and permissions
About this article
Cite this article
Klee, H.J., Hayford, M.B. & Rogers, S.G. Gene rescue in plants: A model system for “shotgun” cloning by retransformation. Molec Gen Genet 210, 282–287 (1987). https://doi.org/10.1007/BF00325695
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00325695