Skip to main content

Thank you for visiting 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.

Generation of marker-free plastid transformants using a transiently cointegrated selection gene


Genetic engineering of higher plant plastids typically involves stable introduction of antibiotic resistance genes as selection markers. Even though chloroplast genes are maternally inherited in most crops1, the possibility of marker transfer to wild relatives2 or microorganisms3 cannot be completely excluded. Furthermore, marker expression can be a substantial metabolic drain4. Therefore, efficient methods for complete marker removal from plastid transformants are necessary. One method to remove the selection gene from higher plant plastids is based on loop-out recombination5, a process difficult to control because selection of homoplastomic transformants is unpredictable. Another method uses the CRE/lox system6,7, but requires additional retransformation and sexual crossing for introduction and subsequent removal of the CRE recombinase. Here we describe the generation of marker-free chloroplast transformants in tobacco using the reconstitution of wild-type pigmentation8 in combination with plastid transformation vectors, which prevent stable integration of the kanamycin selection marker9. One benefit of a procedure using mutants is that marker-free plastid transformants can be produced directly in the first generation (T0) without retransformation or crossing.

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

Access options

Rent or buy this article

Prices vary by article type



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

Figure 1: Cointegrate formation and subsequent recombination events with conventional and alternative plastid transformation vectors.
Figure 2: Analysis of transformants derived from reconstitution of petA and rpoA.
Figure 3: Southern blot analysis of petA-reconstituted lines.
Figure 4: Southern blot analysis of rpoA-reconstituted lines.


  1. Maliga, P., Carrer, H., Kanevski, I., Staub, J. & Svab, Z. Plastid engineering in land plants: a conservative genome is open to change. Phil. Trans. R. Soc. Lond. B 342, 203–208 (1993).

    Article  CAS  Google Scholar 

  2. Daniell, H. Molecular strategies for gene containment in transgenic crops. Nat. Biotechnol. 20, 581–586 (2002).

    Article  CAS  Google Scholar 

  3. Kay, E., Vogel, T.M., Bertolla, F., Nalin, R. & Simonet, P. In situ transfer of antibiotic resistance genes from transgenic (transplastomic) tobacco plants to bacteria. Appl. Environ. Microbiol. 68, 3345–3351 (2002).

    Article  CAS  Google Scholar 

  4. Kuroda, H. & Maliga, P. Sequences downstream of the translation initiation codon are important determinants of translation efficiency in chloroplasts. Plant Physiol. 125, 430–436 (2001).

    Article  CAS  Google Scholar 

  5. Iamtham, S. & Day, A. Removal of antibiotic resistance genes from transgenic tobacco plastids. Nat. Biotechnol. 18, 1172–1176 (2000).

    Article  CAS  Google Scholar 

  6. Corneille, S., Lutz, K., Svab, Z. & Maliga, P. Efficient elimination of selectable marker genes from the plastid genome by the CRE-lox site-specific recombination system. Plant J. 27, 171–178 (2001).

    Article  CAS  Google Scholar 

  7. Hajdukiewicz, P.T., Gilbertson, L. & Staub, J.M. Multiple pathways for Cre/lox-mediated recombination in plastids. Plant J. 27, 161–170 (2001).

    Article  CAS  Google Scholar 

  8. Klaus, S.M., Huang, F.C., Eibl, C., Koop, H.U. & Golds, T.J. Rapid and proven production of transplastomic tobacco plants by restoration of pigmentation and photosynthesis. Plant J. 35, 811–821 (2003).

    Article  CAS  Google Scholar 

  9. Huang, F.C. et al. Efficient plastid transformation in tobacco using the aphA-6 gene and kanamycin selection. Mol. Genet. Genomics 268, 19–27 (2002).

    Article  CAS  Google Scholar 

  10. Staub, J.M. & Maliga, P. Expression of a chimeric uidA gene indicates that polycistronic mRNAs are efficiently translated in tobacco plastids. Plant J. 7, 845–848 (1995).

    Article  CAS  Google Scholar 

  11. Maliga, P. Progress towards commercialization of plastid transformation technology. Trends Biotechnol 21, 20–28 (2003).

    Article  CAS  Google Scholar 

  12. Boynton, J.E. et al. Chloroplast transformation in Chlamydomonas with high velocity microprojectiles. Science 240, 1534–1537 (1988).

    Article  CAS  Google Scholar 

  13. Staub, J.M. & Maliga, P. Extrachromosomal elements in tobacco plastids. Proc. Natl. Acad. Sci. USA 91, 7468–7472 (1994).

    Article  CAS  Google Scholar 

  14. Staub, J.M. & Maliga, P. Marker rescue from the Nicotiana tabacum plastid genome using a plastid/Escherichia coli shuttle vector. Mol. Gen. Genet. 249, 37–42 (1995).

    Article  CAS  Google Scholar 

  15. 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 

  16. Ruf, S., Hermann, M., Berger, I.J., Carrer, H. & Bock, R. Stable genetic transformation of tomato plastids and expression of a foreign protein in fruit. Nat. Biotechnol. 19, 870–875 (2001).

    Article  CAS  Google Scholar 

  17. Eibl, C. et al. In vivo analysis of plastid psbA, rbcL and rpl32 UTR elements by chloroplast transformation: tobacco plastid gene expression is controlled by modulation of transcript levels and translation efficiency. Plant J. 19, 333–345 (1999).

    Article  CAS  Google Scholar 

  18. 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 

  19. Gamborg, O.L., Miller, R.A. & Ojima, K. Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 50, 151–158 (1968).

    Article  CAS  Google Scholar 

  20. Murashige, T. & Skoog, F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473–497 (1962).

    Article  CAS  Google Scholar 

  21. Jefferson, R.A., Bevan, M. & Kavanagh, T. The use of the Escherichia coli beta-glucuronidase as a gene fusion marker for studies of gene expression in higher plants. Biochem. Soc. Trans. 15, 17–18 (1987).

    Article  CAS  Google Scholar 

Download references


The authors thank Christian Eibl and Cornelia Stettner for helpful discussions and careful reading of the manuscript. Expert technical assistance was provided by Carolin Adams, Angela Alkofer and Simin Erschadi. This work was supported in part by Bayerische Forschungsstiftung (grant no. 356/99) and Bayerisches Staatsministerium für Wirtschaft, Verkehr und Technologie (grant no. 3600 - VIII/1e-15520).

Author information

Authors and Affiliations


Corresponding author

Correspondence to Hans-Ulrich Koop.

Ethics declarations

Competing interests

All authors are employees of ICON Genetics, a private biotechnology company.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Klaus, S., Huang, FC., Golds, T. et al. Generation of marker-free plastid transformants using a transiently cointegrated selection gene. Nat Biotechnol 22, 225–229 (2004).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


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