Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Removal of antibiotic resistance genes from transgenic tobacco plastids

Nature Biotechnology volume 18, pages 1172–1176 (2000)Cite this article

An Erratum to this article was published on 01 February 2001

Abstract

Removal of antibiotic resistance genes from genetically modified (GM) crops removes the risk of their transfer to the environment or gut microbes. Integration of foreign genes into plastid DNA enhances containment in crops that inherit their plastids maternally. Efficient plastid transformation requires the aadA marker gene, which confers resistance to the antibiotics spectinomycin and streptomycin. We have exploited plastid DNA recombination and cytoplasmic sorting to remove aadA from transplastomic tobacco plants. A 4.9 kbp insert, composed of aadA flanked by bar and uidA genes, was integrated into plastid DNA and selected to remove wild-type plastid genomes. The bar gene confers tolerance to the herbicide glufosinate despite being GC-rich. Excision of aadA and uidA mediated by two 174 bp direct repeats generated aadA-free T0 transplastomic plants containing the bar gene. Removal of aadA and bar by three 418 bp direct repeats allowed the isolation of marker-free T2 plants containing a plastid-located uidA reporter gene.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: pUM71 integration into the tobacco plastid genome (cpDNA) and predicted gene excision events (not to scale).
Figure 2: DNA blot analysis of pUM71 transformants.
Figure 3: Herbicide tolerance of pUM71 transplastomic plants.
Figure 4: Marker gene excision in pUM71 and pUM70 transplastomic plants.
Figure 5: Isolation of marker-free transplastomic plants.
Figure 6: Molecular analyses of aadA-free transplastomic plants.

Similar content being viewed by others

References

  1. Yoder, J.I. & Goldsbrough, A.P. Transformation systems for generating marker-free transgenic plants. Bio-Technology 12, 263–267 ( 1994).

    Article  CAS  Google Scholar 

  2. Maliga, P. Towards plastid transformation in flowering plants. Trends Biotechnol. 11, 101–107 ( 1993).

    Article  CAS  Google Scholar 

  3. Scott, S.E. & Wilkinson, M.J. Low probability of chloroplast movement from oilseed rape (Brassica napus) into wild Brassica rapa. Nat. Biotechnol. 17, 390–392 (1999).

    Article  CAS  Google Scholar 

  4. Daniell, H., Datta, R., Varma, S., Gray, S. & Lee, S.B. Containment of herbicide resistance through genetic engineering of the chloroplast genome. Nat. Biotechnol. 16, 345–348 (1998).

    Article  CAS  Google Scholar 

  5. Shinozaki, K. et al. The complete nucleotide sequence of the tobacco chloroplast genome: its gene organization and expression. EMBO J. 5, 2043–2049 (1986).

    Article  CAS  Google Scholar 

  6. Boynton, J.E. et al. Chloroplast transformation in Chlamydomonas with high velocity microprojectiles. Science 240, 1534– 1538 (1988).

    Article  CAS  Google Scholar 

  7. Svab, Z., Hajdukiewicz, P. & Maliga, P. Stable transformation of plastids in higher plants. Proc. Natl. Acad Sci. USA 87, 8526– 8530 (1990).

    Article  CAS  Google Scholar 

  8. McBride, K.E. et al. Amplification of a chimeric Bacillus gene in chloroplast leads to an extraordinary level of insecticidal protein in tobacco. Bio-Technology 13, 362–365 (1995).

    CAS  PubMed  Google Scholar 

  9. Kota, M. et al. Overexpression of the Bacillus thuringiensis (Bt) Cry2Aa2 protein in chloroplasts confers resistance to plants against susceptible and Bt -resistant insects. Proc. Natl. Acad. Sci. USA 96, 1840–1845 (1999).

    Article  CAS  Google Scholar 

  10. Staub, J.M. et al. High-yield production of a human therapeutic protein in tobacco chloroplasts . Nat. Biotechnol. 18, 333– 338 (2000).

    Article  CAS  Google Scholar 

  11. Svab, Z. & Maliga, P. High frequency plastid transformation in tobacco by selection for a chimeric aadA gene. Proc. Natl. Acad.Sci. USA 90, 913– 917 (1993).

    Article  CAS  Google Scholar 

  12. Khan, M.S. & Maliga, P. Fluorescent antibiotic resistance marker for tracking plastid transformation in higher plants. Nat. Biotechnol. 17, 910–915 (1999).

    Article  CAS  Google Scholar 

  13. Sikdar, S.R., Serino, G., Chaudhuri, S. & Maliga, P. Plastid transformation in Arabidopsis thaliana. Plant Cell Rep. 18, 20–24 ( 1998).

    Article  CAS  Google Scholar 

  14. Sidorov, V.A. et al. Stable chloroplast transformation in potato: use of green fluorescent protein as a plastid marker. Plant J. 19, 209–216 (1999).

    Article  CAS  Google Scholar 

  15. Jefferson, R. Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol. Biol. Rep. 5, 387–405 (1987).

    Article  CAS  Google Scholar 

  16. Goldschmidt-Clermont, M. Transgenic expression of aminoglycoside adenine transferase in the chloroplast: a selectable marker for site-directed transformation in Chlamydomonas. Nucleic Acids Res. 19, 4083–4089 (1991).

    Article  CAS  Google Scholar 

  17. White, J., Chang, S.Y.P. & Bibb, M.J. A cassette containing the BAR gene of Streptomyces hygroscopicus: a selectable marker for plant transformation. Nucleic Acids Res. 18, 1062 ( 1990).

    Article  CAS  Google Scholar 

  18. Sugiura, M., Shinozaki, K., Zaita, N., Kusuda, M. & Kumano, M. Clone bank of the tobacco (Nicotiana tabacum) chloroplast genome as a set of overlapping restriction endonuclease fragments: mapping of 11 ribosomal protein genes. Plant Sci. 44, 211–217 (1986).

    Article  CAS  Google Scholar 

  19. Deblock, M. et al. Engineering herbicide resistance by expression of a detoxifying enzyme . EMBO J. 6, 2513–2518 (1987).

    Article  CAS  Google Scholar 

  20. Staub, J.M. & Maliga, P. Accumulation of D1 polypeptide in tobacco plastids is regulated via the untranslated region of the psbA messenger RNA. EMBO J. 12, 601– 606 (1993).

    Article  CAS  Google Scholar 

  21. Zoubenko, O.V., Allison, L.A., Svab, Z. & Maliga, P. Efficient targeting of foreign genes into the tobacco plastid genome. Nucleic Acids Res. 22, 3819–3824 (1994).

    Article  CAS  Google Scholar 

  22. Fischer, N., Stampacchia, O., Redding, K. & Rochaix, J.D. Selectable marker recycling in the chloroplast. Mol. Gen. Genet. 251, 373–380 ( 1996).

    Article  CAS  Google Scholar 

  23. Frey, J. Genetic flexibility of plant chloroplasts. Nature 398 , 115–116 (1999).

    Article  CAS  Google Scholar 

  24. Birky, C.W. The partitioning of cytoplasmic organelles at cell divsion. Int. Rev. Cytol. S15, 49–89 ( 1983).

    Google Scholar 

  25. Zubko, M.K. & Day, A. Stable albinism induced without mutagenesis: a model for ribosome-free plastid inheritance. Plant J. 15, 265–271 ( 1998).

    Article  CAS  Google Scholar 

  26. Guda, C., Lee, S.B. & Daniell, H. Stable expression of a biodegradable protein-based polymer in tobacco chloroplasts. Plant Cell Rep. 19, 257–262 (2000).

    Article  CAS  Google Scholar 

  27. Sambrook, J., Fritsch, E.F. & Maniatis, T. Molecular cloning: a laboratory manual, Edn. 2. (Cold Spring Harbor Press, Cold Spring Harbor, NY; 1989).

    Google Scholar 

Download references

Acknowledgements

We are grateful to Drs. M.J. Bibb (Norwich), M. Goldschmidt-Clermont (Geneva), M. Sugiura (Nagoya), and Mrs. J. White (Norwich) for generous gifts of plasmids. We thank Dr. M.K. Zubko for tissue culture advice and the EM graphics unit for assistance. A.D. was supported by grants from the BBSRC and Royal Society. S.I. is the recipient of a Royal Thai Government PhD Scholarship and an Overseas Research Student Award.

Author information

Authors and Affiliations

  1. 3.614 Stopford Building, School of Biological Sciences, Manchester University, Oxford Road, Manchester , M13 9PT, UK

    Siriluck Iamtham & Anil Day

Authors
  1. Siriluck Iamtham
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Anil Day
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Anil Day.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Iamtham, S., Day, A. Removal of antibiotic resistance genes from transgenic tobacco plastids . Nat Biotechnol 18, 1172–1176 (2000). https://doi.org/10.1038/81161

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/81161

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing