Plant Molecular Biology

, Volume 76, Issue 3–5, pp 443–451

Chloramphenicol acetyltransferase as selectable marker for plastid transformation



Chloroplast transformation remains a demanding technique and is still restricted to relatively few plant species. The limited availability of selectable marker genes and the lack of selection markers that would be universally applicable to all plant species represent some of the most serious technical problems involved in extending the species range of plastid transformation. Here we report the development of the chloramphenicol acetyltransferase gene cat as a new selectable marker for plastid transformation. We show that, by selecting for chloramphenicol resistance, tobacco chloroplast transformants are readily obtained. Transplastomic lines quickly reach the homoplasmic state (typically in one additional regeneration round), accumulate the chloramphenicol acetyltransferase enzyme to high levels and transmit their plastid transgenes maternally into the next generation. No spontaneous antibiotic resistance mutants appear upon chloramphenicol selection. Several lines of evidence support the assumption that plant mitochondria are also sensitive to chloramphenicol suggesting that the chloramphenicol acetyltransferase may be a good candidate selectable marker for plant mitochondrial transformation.


Plastid transformation Nicotiana tabacum Selectable marker Chloramphenicol cat 


  1. Ahmadabadi M, Ruf S, Bock R (2007) A leaf-based regeneration and transformation system for maize (Zea mays L.). Transgenic Res 16:437–448PubMedCrossRefGoogle Scholar
  2. Allison LA, Maliga P (1995) Light-responsive and transcription-enhancing elements regulate the plastid psbD core promoter. EMBO J 14:3721–3730PubMedGoogle Scholar
  3. Ayliffe MA, Timmis JN (1992) Tobacco nuclear DNA contains long tracts of homology to chloroplast DNA. Theor Appl Genet 85:229–238CrossRefGoogle Scholar
  4. Barone P, Zhang X-H, Widholm JM (2009) Tobacco plastid transformation using the feedback-insensitive anthranilate synthase [α]-subunit of tobacco (ASA2) as a new selectable marker. J Exp Bot 60:3195–3202PubMedCrossRefGoogle Scholar
  5. Bock R (2001) Transgenic chloroplasts in basic research and plant biotechnology. J Mol Biol 312:425–438PubMedCrossRefGoogle Scholar
  6. Bock R (2007) Plastid biotechnology: prospects for herbicide and insect resistance, metabolic engineering and molecular farming. Curr Opin Biotechnol 18:100–106PubMedCrossRefGoogle Scholar
  7. Carrer H, Hockenberry TN, Svab Z, Maliga P (1993) Kanamycin resistance as a selectable marker for plastid transformation in tobacco. Mol Gen Genet 241:49–56PubMedCrossRefGoogle Scholar
  8. Denovan-Wright EM, Nedelcu AM, Lee RW (1998) Complete sequence of the mitochondrial DNA of Chlamydomonas eugametos. Plant Mol Biol 36:285–295PubMedCrossRefGoogle Scholar
  9. Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15Google Scholar
  10. Eibl C, Zou Z, Beck A, Kim M, Mullet J, Koop H-U (1999) 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 19:333–345PubMedCrossRefGoogle Scholar
  11. Fromm H, Edelman M, Aviv D, Galun E (1987) The molecular basis for rRNA-dependent spectinomycin resistance in Nicotiana chloroplasts. EMBO J 6:3233–3237PubMedGoogle Scholar
  12. Goldschmidt-Clermont M (1991) Transgenic expression of aminoglycoside adenyl transferase in the chloroplast: a selectable marker for site-directed transformation of Chlamydomonas. Nucleic Acids Res 19:4083–4089PubMedCrossRefGoogle Scholar
  13. Hager M, Biehler K, Illerhaus J, Ruf S, Bock R (1999) Targeted inactivation of the smallest plastid genome-encoded open reading frame reveals a novel and essential subunit of the cytochrome b6f complex. EMBO J 18:5834–5842PubMedCrossRefGoogle Scholar
  14. Harris EH, Boynton JE, Gillham NW (1994) Chloroplast ribosomes and protein synthesis. Microbiol Rev 58:700–754PubMedGoogle Scholar
  15. Huang F-C, Klaus SMJ, Herz S, Zou Z, Koop H-U, Golds TJ (2002) Efficient plastid transformation in tobacco using the aphA-6 gene and kanamycin selection. Mol Genet Genomics 268:19–27PubMedCrossRefGoogle Scholar
  16. Karcher D, Kahlau S, Bock R (2008) Faithful editing of a tomato-specific mRNA editing site in transgenic tobacco chloroplasts. RNA 14:217–224PubMedCrossRefGoogle Scholar
  17. Kuroda H, Maliga P (2001) Complementarity of the 16S rRNA penultimate stem with sequences downstream of the AUG destabilizes the plastid mRNAs. Nucleic Acids Res 29:970–975PubMedCrossRefGoogle Scholar
  18. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedCrossRefGoogle Scholar
  19. Lutz KA, Maliga P (2007) Construction of marker-free transplastomic plants. Curr Opin Biotechnol 18:107–114PubMedCrossRefGoogle Scholar
  20. Maliga P (2004) Plastid transformation in higher plants. Annu Rev Plant Biol 55:289–313PubMedCrossRefGoogle Scholar
  21. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue culture. Physiol Plant 15:473–497CrossRefGoogle Scholar
  22. Nie ZQ, Chang DY, Wu M (1987) Protein-DNA interaction within one cloned chloroplast DNA replication origin of Chlamydomonas. Mol Gen Genet 209:265–269PubMedCrossRefGoogle Scholar
  23. Oey M, Lohse M, Kreikemeyer B, Bock R (2009a) Exhaustion of the chloroplast protein synthesis capacity by massive expression of a highly stable protein antibiotic. Plant J 57:436–445PubMedCrossRefGoogle Scholar
  24. Oey M, Lohse M, Scharff LB, Kreikemeyer B, Bock R (2009b) Plastid production of protein antibiotics against pneumonia via a new strategy for high-level expression of antimicrobial proteins. Proc Natl Acad Sci USA 106:6579–6584PubMedCrossRefGoogle Scholar
  25. Rogalski M, Ruf S, Bock R (2006) Tobacco plastid ribosomal protein S18 is essential for cell survival. Nucleic Acids Res 34:4537–4545PubMedCrossRefGoogle Scholar
  26. Rogalski M, Karcher D, Bock R (2008a) Superwobbling facilitates translation with reduced tRNA sets. Nat Struct Mol Biol 15:192–198PubMedCrossRefGoogle Scholar
  27. Rogalski M, Schöttler MA, Thiele W, Schulze WX, Bock R (2008b) Rpl33, a nonessential plastid-encoded ribosomal protein in tobacco, is required under cold stress conditions. Plant Cell 20:2221–2237PubMedCrossRefGoogle Scholar
  28. Ruf S, Kössel H, Bock R (1997) Targeted inactivation of a tobacco intron-containing open reading frame reveals a novel chloroplast-encoded photosystem I-related gene. J Cell Biol 139:95–102PubMedCrossRefGoogle Scholar
  29. Ruf S, Biehler K, Bock R (2000) A small chloroplast-encoded protein as a novel architectural component of the light-harvesting antenna. J Cell Biol 149:369–377PubMedCrossRefGoogle Scholar
  30. Ruf S, Hermann M, Berger IJ, Carrer H, Bock R (2001) Stable genetic transformation of tomato plastids and expression of a foreign protein in fruit. Nat Biotechnol 19:870–875PubMedCrossRefGoogle Scholar
  31. Ruf S, Karcher D, Bock R (2007) Determining the transgene containment level provided by chloroplast transformation. Proc Natl Acad Sci USA 104:6998–7002PubMedCrossRefGoogle Scholar
  32. Svab Z, Maliga P (1991) Mutation proximal to the tRNA binding region of the Nicotiana plastid 16S rRNA confers resistance to spectinomycin. Mol Gen Genet 228:316–319PubMedCrossRefGoogle Scholar
  33. Svab Z, Maliga P (1993) High-frequency plastid transformation in tobacco by selection for a chimeric aadA gene. Proc Natl Acad Sci USA 90:913–917PubMedCrossRefGoogle Scholar
  34. Svab Z, Maliga P (2007) Exceptional transmission of plastids and mitochondria from the transplastomic pollen parent and its impact on transgene containment. Proc Natl Acad Sci USA 104:7003–7008PubMedCrossRefGoogle Scholar
  35. Svab Z, Hajdukiewicz P, Maliga P (1990) Stable transformation of plastids in higher plants. Proc Natl Acad Sci USA 87:8526–8530PubMedCrossRefGoogle Scholar
  36. Tenson T, Mankin A (2006) Antibiotics and the ribosome. Mol Microbiol 59:1664–1677PubMedCrossRefGoogle Scholar
  37. Vahrenholz C, Riemen G, Pratje E, Dujon B, Michaelis G (1993) Mitochondrial DNA of Chlamydomonas reinhardtii: the structure of the ends of the linear 15.8-kb genome suggests mechanisms for DNA replication. Curr Genet 24:241–247PubMedCrossRefGoogle Scholar
  38. Wurbs D, Ruf S, Bock R (2007) Contained metabolic engineering in tomatoes by expression of carotenoid biosynthesis genes from the plastid genome. Plant J 49:276–288PubMedCrossRefGoogle Scholar
  39. Ye G-N, Hajdukiewicz PTJ, Broyles D, Rodriguez D, Xu CW, Nehra N, Staub JM (2001) Plastid-expressed 5-enolpyruvylshikimate-3-phosphate synthase genes provide high level glyphosate tolerance in tobacco. Plant J 25:261–270PubMedCrossRefGoogle Scholar
  40. Yu F, Liu X, Alsheikh M, Park S, Rodermel S (2008) Mutations in suppressor of variegation1, a factor required for normal chloroplast translation, suppress var2-mediated leaf variegation in Arabidopsis. Plant Cell 20:1786–1804PubMedCrossRefGoogle Scholar
  41. Zhou F, Badillo-Corona JA, Karcher D, Gonzalez-Rabade N, Piepenburg K, Borchers A-MI, Maloney AP, Kavanagh TA, Gray JC, Bock R (2008) High-level expression of HIV antigens from the tobacco and tomato plastid genomes. Plant Biotechnol J 6:897–913PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  1. 1.Max-Planck-Institut für Molekulare PflanzenphysiologiePotsdam-GolmGermany

Personalised recommendations