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. 1984 Aug;3(8):1681–1689. doi: 10.1002/j.1460-2075.1984.tb02032.x

Expression of foreign genes in regenerated plants and in their progeny

Marc De Block 1, Luis Herrera-Estrella 1, Marc Van Montagu 1, Jeff Schell 1,2, Patricia Zambryski 1
PMCID: PMC557582  PMID: 16453538

Abstract

Chimeric genes comprised of the nopaline synthase promoter and bacterial coding sequences specifying resistance to kanamycin, chloramphenicol or methotrexate, were inserted into the non-oncogenic Ti plasmid vector pGV3850 by recombination (through homologous pBR322 sequences present in the chimeric gene constructs and pGV3850). These co-integrates in Agrobacterium were used to infect single plant protoplasts of Nicotiana by co-cultivation. The resistance traits allowed the selection of transformed calli in tissue culture in the presence of the appropriate antibiotic. Furthermore, as a non-oncogenic Ti plasmid was used for the protoplast transformation, phenotypically normal and fertile plants could be regenerated from the resistant calli. We have shown that these fully differentiated plant tissues exhibit functional expression of resistance traits (KmR and CmR). All plants carrying the chimeric genes developed normally, flowered, and set seeds. The inheritance of several of these resistance traits was analyzed and shown to be Mendelian. These results are model experiments to demonstrate that genes of interest can be systematically transferred to the genome of plants using non-oncogenic Ti plasmid derivatives; and that transformed plants are capable of normal growth and differentiation, thus providing a natural environment for the study of gene expression and development of plant cells.

Keywords: Agrobacterium, plant cell transformation, selectable markers, Ti plasmid vectors, chimeric genes

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bevan M. W., Chilton M. D. T-DNA of the Agrobacterium Ti and Ri plasmids. Annu Rev Genet. 1982;16:357–384. doi: 10.1146/annurev.ge.16.120182.002041. [DOI] [PubMed] [Google Scholar]
  2. Bolivar F. Construction and characterization of new cloning vehicles. III. Derivatives of plasmid pBR322 carrying unique Eco RI sites for selection of Eco RI generated recombinant DNA molecules. Gene. 1978 Oct;4(2):121–136. doi: 10.1016/0378-1119(78)90025-2. [DOI] [PubMed] [Google Scholar]
  3. Caplan A., Herrera-Estrella L., Inzé D., Van Haute E., Van Montagu M., Schell J., Zambryski P. Introduction of genetic material into plant cells. Science. 1983 Nov 18;222(4625):815–821. doi: 10.1126/science.222.4625.815. [DOI] [PubMed] [Google Scholar]
  4. Colbère-Garapin F., Horodniceanu F., Kourilsky P., Garapin A. C. A new dominant hybrid selective marker for higher eukaryotic cells. J Mol Biol. 1981 Jul 25;150(1):1–14. doi: 10.1016/0022-2836(81)90321-1. [DOI] [PubMed] [Google Scholar]
  5. Fraley R. T., Rogers S. G., Horsch R. B., Sanders P. R., Flick J. S., Adams S. P., Bittner M. L., Brand L. A., Fink C. L., Fry J. S. Expression of bacterial genes in plant cells. Proc Natl Acad Sci U S A. 1983 Aug;80(15):4803–4807. doi: 10.1073/pnas.80.15.4803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gamborg O. L., Miller R. A., Ojima K. Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res. 1968 Apr;50(1):151–158. doi: 10.1016/0014-4827(68)90403-5. [DOI] [PubMed] [Google Scholar]
  7. Herrera-Estrella L., Block M. D., Messens E., Hernalsteens J. P., Montagu M. V., Schell J. Chimeric genes as dominant selectable markers in plant cells. EMBO J. 1983;2(6):987–995. doi: 10.1002/j.1460-2075.1983.tb01532.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Maliga P., Sz-Breznovits A., Márton L. Streptomycin-resistant plants from callus culture of haploid tobacco. Nat New Biol. 1973 Jul 4;244(131):29–30. doi: 10.1038/newbio244029a0. [DOI] [PubMed] [Google Scholar]
  10. Marcoli R., Iida S., Bickle T. A. The DNA sequence of an IS/-flanked transposon coding for resistance to chloramphenicol and fusidic acid. FEBS Lett. 1980 Jan 28;110(1):11–14. doi: 10.1016/0014-5793(80)80011-1. [DOI] [PubMed] [Google Scholar]
  11. Otten L., De Greve H., Hernalsteens J. P., Van Montagu M., Schieder O., Straub J., Schell J. Mendelian transmission of genes introduced into plants by the Ti plasmids of Agrobacterium tumefaciens. Mol Gen Genet. 1981;183(2):209–213. doi: 10.1007/BF00270619. [DOI] [PubMed] [Google Scholar]
  12. Rao R. N., Rogers S. G. Plasmid pKC7: a vector containing ten restriction endonuclease sites suitable for cloning DNA segments. Gene. 1979 Sep;7(1):79–82. doi: 10.1016/0378-1119(79)90044-1. [DOI] [PubMed] [Google Scholar]
  13. Simpson R. B., O'Hara P. J., Kwok W., Montoya A. L., Lichtenstein C., Gordon M. P., Nester E. W. DNA from the A6S/2 crown gall tumor contains scrambled Ti-plasmid sequences near its junctions with plant DNA. Cell. 1982 Jul;29(3):1005–1014. doi: 10.1016/0092-8674(82)90464-0. [DOI] [PubMed] [Google Scholar]
  14. Southern P. J., Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet. 1982;1(4):327–341. [PubMed] [Google Scholar]
  15. Van Haute E., Joos H., Maes M., Warren G., Van Montagu M., Schell J. Intergeneric transfer and exchange recombination of restriction fragments cloned in pBR322: a novel strategy for the reversed genetics of the Ti plasmids of Agrobacterium tumefaciens. EMBO J. 1983;2(3):411–417. doi: 10.1002/j.1460-2075.1983.tb01438.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Yadav N. S., Vanderleyden J., Bennett D. R., Barnes W. M., Chilton M. D. Short direct repeats flank the T-DNA on a nopaline Ti plasmid. Proc Natl Acad Sci U S A. 1982 Oct;79(20):6322–6326. doi: 10.1073/pnas.79.20.6322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Zambryski P., Depicker A., Kruger K., Goodman H. M. Tumor induction by Agrobacterium tumefaciens: analysis of the boundaries of T-DNA. J Mol Appl Genet. 1982;1(4):361–370. [PubMed] [Google Scholar]
  18. Zambryski P., Joos H., Genetello C., Leemans J., Montagu M. V., Schell J. Ti plasmid vector for the introduction of DNA into plant cells without alteration of their normal regeneration capacity. EMBO J. 1983;2(12):2143–2150. doi: 10.1002/j.1460-2075.1983.tb01715.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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