Research | Published:

Transposition Mediated Re–positioning and Subsequent Elimination of Marker Genes from Transgenic Tomato

Subjects

Abstract

We describe a new plant transformation vector system which utilizes the transposition functions of the maize Ac/Ds transposable element family to re-position transgenes in transgenic crop plants. The practical applications of the system are two-fold. It allows the production of plants which exhibit a range of different stabilizable transgene expression levels following a single primary transformation event, and it allows for the elimination of specific transgene sequences—such as a selectable marker gene—subsequent to the transformation event. We have demonstrated the system using the NptII selectable marker gene and a Ds element containing the GUS reporter gene. Progeny plants were recovered from primary transformants from which either the NptII gene or the Ds/GUS element have been eliminated. We also show that the expression level of the GUS gene within both individual and amplified Ds elements can vary as a function of their position in the genome following transposition.

References

  1. 1

    Dean, C., Jones, J., Favreau, M., Dunsmuir, P. and Bedbrook, J. 1988. Influence of flanking sequences on variability in expression levels of an introduced gene in transgenic tobacco plants. Nucl. Acids Res. 16: 9267–9283.

  2. 2

    Bryant, J. and Leather, S. 1992. removal of selectable marker genes from transgenic plants: needless sophistication or social necessity? TIBTECH 10: 274–275.

  3. 3

    Nap, J-P., Bijvoet, J. and Stiekema, W.J. 1992. Biosafety of kanamycin-resistant transgenic plants. Transgenic Research 1: 239–249.

  4. 4

    Flavell, R.B., Dart, E., Fuchs, R.L. and Fraley, R.T. 1992. Selectable marker genes: Safe for plants? Bio/Technology 10: 141–144.

  5. 5

    Gressel, J. 1992. Indiscriminate use of selectable markers—sowing wild oats? TIBTECH 10: 382.

  6. 6

    Dale, P.J. 1992. Spread of engineered genes to wild relatives. Plant Physiol. 100: 13–15.

  7. 7

    Russell, S.H., Hoopes, J.L. and Odell, J.T. 1992. Directed excision of a transgene from the plant genome. Mol. Gen. Genet. 234: 49–59.

  8. 8

    Dale, E.C. and Ow, D.W. 1991. Gene transfer with subsequent removal of the selection gene from the host genome. Proc. Nat. Acad. Sci. USA 88: 10558–10562.

  9. 9

    McKnight, T.D., Lillis, M.T. and Simpson, R.B. 1987. Segregation of genes transferred to one plant cell from two separate Agrobacterium strains. Plant Mol. Biol. 8: 439–445.

  10. 10

    McClintock, B. 1948. Mutable loci in maize. Carnegie Inst. of Wash. Year Book 47: 155–169.

  11. 11

    Fedoroff, N., Wessler, S. and Shure, M. 1983. Isolation of the transposable maize controlling elements Ac and Ds. Cell 35: 235–242.

  12. 12

    Balcells, L., Swinburne, J. and Coupland, G. 1991. Transposons as tools for the isolation of plant genes. TIBTECH 9: 31–37.

  13. 13

    Lassner, M.W., Palys, J. and Yoder, J.I. 1989. Genetic transactivation of Dissociation elements in transgenic tomato plants. Mol. Gen. Genet. 218: 25–32.

  14. 14

    Scofield, S.R., Harrison, K., Nurrish, S.J. and Jones, J.D.G. 1992. Promoter fusions to the Activator transposase gene cause distinct patterns of Dissociation excision in tobacco cotyledons. Plant Cell 4: 573–582.

  15. 15

    Swinburne, J., Balcells, L., Scofield, S.R., Jones, J.D.G. and Coupland, G. 1992. Elevated levels of Activator transposase mRNA are associated with high frequencies of Dissociation Excision in Arabidopsis. Plant Cell 4: 583–595.

  16. 16

    Masterson, R.V., Furtek, D.B., Grevelding, C. and Schell, J. 1989. A maize Ds transposable element containing a didydrofolate reductase gene transposes in Nicotiana tabacum and Arabidopsis thaliana. Mol. Gen. Genet. 219: 461–466.

  17. 17

    Dooner, H.K., Keller, J., Harper, E. and Ralston, E. 1991. Variable patterns of transposition of the maize element Activator in tobacco. Plant Cell 3: 473–482.

  18. 18

    Greenblatt, I. 1984. A chromosome replication pattern deduced from pericarp phenotypes resulting from movements of the transposable element Modulator in maize. Genetics 108: 471–485.

  19. 19

    Jones, J.D.G., Carland, F., Lim, E., Ralston, E. and Dooner, H.K. 1990. Preferential transposition of the maize element Activator to linked chromosomal locations in tobacco. Plant Cell 2: 701–707.

  20. 20

    Belzile, F., Lassner, M.W., Tong, Y., Khush, R. and Yoder, J.I. 1989. Sexual transmission of transposed Activator elements in transgenic tomatoes. Genetics 123: 181–189.

  21. 21

    Goldsbrough, A. and Bevan, M. 1991. New patterns of gene activity in plants detected using an Agrobacterium vector. Plant Mol. Biol. 16: 263–269.

  22. 22

    Yoder, J.I. 1990. Rapid proliferation of the maize transposable element Activator in transgenic tomato. Plant Cell 2: 723–730.

  23. 23

    Marion-Poll, A., Marin, E., Bonnefoy, N. and Pautot, V., 1993. Tranposition of the maize autonomous element Activator in transgenic Nicotiana plumbaginifolia plants. Mol. Gen. Genet. 238: 209–217.

  24. 24

    Hardeman, K.J. and Chandler, V.L. 1989. Characterization of bzl mutants isolated from mutator stocks with high and low numbers of Mul elements. Developmental Genet. 10: 460–472.

  25. 25

    Topping, J.F., Wei, W. and Lindsay, K. 1991. Functional tagging of regulatory elements in the plant genome. Development 112: 1009–1019.

  26. 26

    Bevan, M.W. 1984. Agrobacterium vectors for plant transformation. Nucl. Acid Res. 12: 8711–8721.

  27. 27

    Thomas, C.M., Meyer, R. and Helinski, D.R. 1980. Regions of broad-host-range plasmid RK2 which are essential for replication and maintenance. J. Bacteriol. 141: 213–222.

  28. 28

    Hirsch, P.R., Wang, C.L. and Woodland, M.J. 1986. Construction of a Tn5 derivative determining resistance to gentamycin and spectinomycin using a fragment cloned from R1033. Gene 48: 203–209.

  29. 29

    Peralta, E.G., Hellmiss, R. and Dean, W. 1986. Overdrive, a T-DNA transmission enhancer on the A. tumefaciens tumour-inducing plasmid. EMBO. J. 5: 1137–1142.

  30. 30

    Jones, J.D.G., Shlumukov, L., Carand, F., English, J., Scofield, S.R., Bishop, G.J. and Harrison, K. 1992. Effective vectors for transformation, expression of heterologous genes, and assaying exision in transgenic plants. Transgenic Research 1: 285–297.

  31. 31

    Yoder, J.I., Palys, J., Alpert, K. and Lassner, M. 1988. Ac transposition in transgenic tomato plants. Mol. Gen. Genet. 213: 291–296.

  32. 32

    Jefferson, R.A., Goldsbrough, A. and Bevan, M.W. 1990. Transcriptional regulation of a patatin-1 gene in potato. Plant Mol. Biol. 14: 995–1006.

  33. 33

    Cooley, M.B., D'Souza, M. and Kado, C.I. 1991. The VirC and VirD operons of the Agrobacterium Ti Plasmid are regulated by the ros chromosomal gene: Analysis of the cloned ros gene. J. Bacteriol. 173: 2608–2616.

  34. 34

    Hoekema, A., Hirsch, P.R., Hooykaas, P.J.J. and Schilperoort, R.A. 1983. A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature 303: 179–180.

  35. 35

    Bernatzky, R. and Tanksley, S.D. 1986. Genetics of actin-related sequences in tomato. Theor. Appl. Genet. 72: 314–321.

  36. 36

    Sambrook, J., Fritsch, E.F. and Maniatis, T. 1989. Molecular Cloning. Cold Spring Harbor Laboratory Press.

  37. 37

    Jefferson, R.A. 1987. Assaying chimeric genes in plants: The GUS gene fusion system. Plant Mol. Biol. Rep. 5: 387–405.

Download references

Author information

Rights and permissions

To obtain permission to re-use content from this article visit RightsLink.

About this article

Publication history

  • Received

  • Accepted

  • Issue Date

DOI

https://doi.org/10.1038/nbt1193-1286

Further reading