Excision of Plastid Marker Genes Using Directly Repeated DNA Sequences

  • Elisabeth A. Mudd
  • Panagiotis Madesis
  • Elena Martin Avila
  • Anil Day
Part of the Methods in Molecular Biology book series (MIMB, volume 1132)


Excision of marker genes using DNA direct repeats makes use of the predominant homologous recombination pathways present in the plastids of algae and plants. The method is simple, efficient, and widely applicable to plants and microalgae. Marker excision frequency is dependent on the length and number of directly repeated sequences. When two repeats are used a repeat size of greater than 600 bp promotes efficient excision of the marker gene. A wide variety of sequences can be used to make the direct repeats. Only a single round of transformation is required, and there is no requirement to introduce site-specific recombinases by retransformation or sexual crosses. Selection is used to maintain the marker and ensure homoplasmy of transgenic plastid genomes. Release of selection allows the accumulation of marker-free plastid genomes generated by marker excision, which is spontaneous, random, and a unidirectional process. Positive selection is provided by linking marker excision to restoration of the coding region of an herbicide resistance gene from two overlapping but incomplete coding regions. Cytoplasmic sorting allows the segregation of cells with marker-free transgenic plastids. The marker-free shoots resulting from direct repeat-mediated excision of marker genes have been isolated by vegetative propagation of shoots in the T0 generation. Alternatively, accumulation of marker-free plastid genomes during growth, development and flowering of T0 plants allows the collection of seeds that give rise to a high proportion of marker-free T1 seedlings. The simplicity and convenience of direct repeat excision facilitates its widespread use to isolate marker-free crops.

Key words

Chloroplast transformation DNA direct repeats Herbicide tolerant plants Homologous recombination Homoplasmic Heteroplasmic Marker gene excision 



Work in the authors’ laboratory was supported by research grants BB/E020445 and BB/I011552 from the Biotechnology and Biological Sciences Research Council (UK).


  1. 1.
    Svab Z, Hajdukiewicz P, Maliga P (1990) Stable transformation of plastids in higher plants. Proc Natl Acad Sci U S A 87:8526–8530PubMedCentralPubMedCrossRefGoogle Scholar
  2. 2.
    O’Neill C, Horvath GV, Horvath E, Dix PJ, Medgyesy P (1993) Chloroplast transformation in plants: polyethylene glycol (PEG) treatment of protoplasts is an alternative to biolistic delivery systems. Plant J 3:729–738PubMedCrossRefGoogle Scholar
  3. 3.
    Staub JM, Maliga P (1992) Long regions of homologous DNA are incorporated into the tobacco plastid genome by transformation. Plant Cell 4:39–45PubMedCentralPubMedGoogle Scholar
  4. 4.
    Day A, Goldschmidt-Clermont M (2011) The chloroplast transformation toolbox: selectable markers and marker removal. Plant Biotechnol J 9:540–553PubMedCrossRefGoogle Scholar
  5. 5.
    Goldschmidt-Clermont M (1991) Transgenic expression of aminoglycoside adenine transferase in the chloroplast: a selectable marker of site-directed transformation of chlamydomonas. Nucleic Acids Res 19:4083–4089PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Svab Z, Maliga P (1993) High-frequency plastid transformation in tobacco by selection for a chimeric aadA gene. Proc Natl Acad Sci U S A 90:913–917PubMedCentralPubMedCrossRefGoogle Scholar
  7. 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. 8.
    Huang FC, Klaus SM, Herz S, Zou Z, Koop HU, Golds TJ (2002) Efficient plastid transformation in tobacco using the aphA-6 gene and kanamycin selection. Mol Genet Genomics 268:19–27PubMedCrossRefGoogle Scholar
  9. 9.
    Li W, Ruf S, Bock R (2011) Chloramphenicol acetyltransferase as selectable marker for plastid transformation. Plant Mol Biol 76:443–451Google Scholar
  10. 10.
    Kay E, Vogel TM, Bertolla F, Nalin R, Simonet P (2002) In situ transfer of antibiotic resistance genes from transgenic (transplastomic) tobacco plants to bacteria. Appl Environ Microbiol 68:3345–3351PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Ceccherini MT, Pote J, Kay E, Van VT, Marechal J, Pietramellara G, Nannipieri P, Vogel TM, Simonet P (2003) Degradation and transformability of DNA from transgenic leaves. Appl Environ Microbiol 69:673–678PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Pontiroli A, Rizzi A, Simonet P, Daffonchio D, Vogel TM, Monier JM (2009) Visual evidence of horizontal gene transfer between plants and bacteria in the phytosphere of transplastomic tobacco. Appl Environ Microbiol 75:3314–3322PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Goldstein DA, Tinland B, Gilbertson LA, Staub JM, Bannon GA, Goodman RE, McCoy RL, Silvanovich A (2005) Human safety and genetically modified plants: a review of antibiotic resistance markers and future transformation selection technologies. J Appl Microbiol 99:7–23PubMedCrossRefGoogle Scholar
  14. 14.
    Sacchettini JC, Rubin EJ, Freundlich JS (2008) Drugs versus bugs: in pursuit of the persistent predator Mycobacterium tuberculosis. Nat Rev Microbiol 6:41–52PubMedCrossRefGoogle Scholar
  15. 15.
    Kubanova A, Frigo N, Kubanov A, Sidorenko S, Lesnaya I, Polevshikova S, Solomka V, Bukanov N, Domeika M, Unemo M (2010) The Russian gonococcal antimicrobial susceptibility programme (RU-GASP) - national resistance prevalence in 2007 and 2008, and trends during 2005–2008. Euro Surveill 15:10–14Google Scholar
  16. 16.
    Fuchs RL, Ream JE, Hammond BG, Naylor MW, Leimgruber RM, Berberich SA (1993) Safety assessment of the neomycin phosphotransferase II (NPTII) protein. Bio/Technology 11:1543–1547PubMedCrossRefGoogle Scholar
  17. 17.
    Boynton JE, Gillham NW, Harris EH, Hosler JP, Johnson AM, Jones AR, Randolph-Anderson BL, Robertson D, Klein TM, Shark KB et al (1988) Chloroplast transformation in Chlamydomonas with high velocity microprojectiles. Science 240:1534–1538PubMedCrossRefGoogle Scholar
  18. 18.
    Klaus SM, Huang FC, Eibl C, Koop HU, Golds TJ (2003) Rapid and proven production of transplastomic tobacco plants by restoration of pigmentation and photosynthesis. Plant J 35:811–821PubMedCrossRefGoogle Scholar
  19. 19.
    Kode V, Mudd EA, Iamtham S, Day A (2006) Isolation of precise plastid deletion mutants by homology-based excision: a resource for site-directed mutagenesis, multi-gene changes and high-throughput plastid transformation. Plant J 46:901–909PubMedCrossRefGoogle Scholar
  20. 20.
    Barone P, Zhang XH, Widholm JM (2009) Tobacco plastid transformation using the feedback-insensitive anthranilate synthase [alpha]-subunit of tobacco (ASA2) as a new selectable marker. J Exp Bot 60:3195–3202PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    Klaus SM, Huang FC, Golds TJ, Koop HU (2004) Generation of marker-free plastid transformants using a transiently cointegrated selection gene. Nat Biotechnol 22:225–229PubMedCrossRefGoogle Scholar
  22. 22.
    Ye GN, Colburn SM, Xu CW, Hajdukiewicz PT, Staub JM (2003) Persistence of unselected transgenic DNA during a plastid transformation and segregation approach to herbicide resistance. Plant Physiol 133:402–410PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Day A, Madesis P (2007) DNA replication, recombination, and repair in plastids. In: Bock R (ed) Cell and molecular biology of plastids. Springer, Berlin, pp 65–119CrossRefGoogle Scholar
  24. 24.
    Kowalczykowski SC (2000) Initiation of genetic recombination and recombination-dependent replication. Trends Biochem Sci 25:156–165PubMedCrossRefGoogle Scholar
  25. 25.
    Cerutti H, Osman M, Grandoni P, Jagendorf AT (1992) A homolog of Escherichia coli RecA protein in plastids of higher plants. Proc Natl Acad Sci U S A 89:8068–8072PubMedCentralPubMedCrossRefGoogle Scholar
  26. 26.
    Nakazato E, Fukuzawa H, Tabata S, Takahashi H, Tanaka K (2003) Identification and expression analysis of cDNA encoding a chloroplast recombination protein REC1, the chloroplast RecA homologue in Chlamydomonas reinhardtii. Biosci Biotechnol Biochem 67:2608–2613PubMedCrossRefGoogle Scholar
  27. 27.
    Shedge V, Arrieta-Montiel M, Christensen AC, Mackenzie SA (2007) Plant mitochondrial recombination surveillance requires unusual RecA and MutS homologs. Plant Cell 19:1251–1264PubMedCentralPubMedCrossRefGoogle Scholar
  28. 28.
    Rowan BA, Oldenburg DJ, Bendich AJ (2010) RecA maintains the integrity of chloroplast DNA molecules in Arabidopsis. J Exp Bot 61:2575–2588PubMedCentralPubMedCrossRefGoogle Scholar
  29. 29.
    Marechal A, Parent JS, Veronneau-Lafortune F, Joyeux A, Lang BF, Brisson N (2009) Whirly proteins maintain plastid genome stability in Arabidopsis. Proc Natl Acad Sci U S A 106:14693–14698PubMedCentralPubMedCrossRefGoogle Scholar
  30. 30.
    Newman SM, Harris EH, Johnson AM, Boynton JE, Gillham NW (1992) Nonrandom distribution of chloroplast recombination events in Chlamydomonas reinhardtii—evidence for a hotspot and an adjacent cold region. Genetics 132:413–429PubMedCentralPubMedGoogle Scholar
  31. 31.
    Staub JM, Maliga P (1995) Marker rescue from the Nicotiana tabacum genome using a plastid Escherichia coli shuttle vector. Mol Gen Genet 249:37–42PubMedCrossRefGoogle Scholar
  32. 32.
    Khakhlova O, Bock R (2006) Elimination of deleterious mutations in plastid genomes by gene conversion. Plant J 46:85–94PubMedCrossRefGoogle Scholar
  33. 33.
    Birky CW (2001) The inheritance of genes in mitochondria and chloroplasts: laws, mechanisms, and models. Annu Rev Genet 35:125–148PubMedCrossRefGoogle Scholar
  34. 34.
    Lutz KA, Maliga P (2008) Plastid genomes in a regenerating tobacco shoot derive from a small number of copies selected through a stochastic process. Plant J 56:975–983PubMedCrossRefGoogle Scholar
  35. 35.
    Dufourmantel N, Dubald M, Matringe M, Canard H, Garcon F, Job C, Kay E, Wisniewski JP, Ferullo JM, Pelissier B, Sailland A, Tissot G (2007) Generation and characterization of soybean and marker-free tobacco plastid transformants over-expressing a bacterial 4-hydroxyphenylpyruvate dioxygenase which provides strong herbicide tolerance. Plant Biotechnol J 5:118–133PubMedCrossRefGoogle Scholar
  36. 36.
    Lelivelt CLC, McCabe MS, Newell CA, deSnoo CB, van Dun KMP, Birch-Machin I, Gray JC, Mills KHG, Nugent JM (2005) Stable plastid transformation in lettuce (Lactuca sativa L.). Plant Mol Biol 58:763–774PubMedCrossRefGoogle Scholar
  37. 37.
    Zoubenko OV, Allison LA, Svab Z, Maliga P (1994) Efficient targeting of foreign genes into the tobacco plastid genome. Nucleic Acids Res 22:3819–3824PubMedCentralPubMedCrossRefGoogle Scholar
  38. 38.
    Iamtham S, Day A (2000) Removal of antibiotic resistance genes from transgenic tobacco plastids. Nat Biotechnol 18:1172–1176PubMedCrossRefGoogle Scholar
  39. 39.
    Cerutti H, Johnson AM, Boynton JE, Gillham NW (1995) Inhibition of chloroplast DNA recombination and repair by dominant negative mutants of Escherichia coli RecA. Mol Cell Biol 15:3003–3011PubMedCentralPubMedGoogle Scholar
  40. 40.
    Fischer N, Stampacchia O, Redding K, Rochaix JD (1996) Selectable marker recycling in the chloroplast. Mol Gen Genet 251:373–380PubMedCrossRefGoogle Scholar
  41. 41.
    Kindle KL, Richards KL, Stern DB (1991) Engineering the chloroplast genome: techniques and capabilities for chloroplast transformation in Chlamydomonas reinhardtii. Proc Natl Acad Sci U S A 88:1721–1725PubMedCentralPubMedCrossRefGoogle Scholar
  42. 42.
    Ahlert D, Ruf S, Bock R (2003) Plastid protein synthesis is required for plant development in tobacco. Proc Natl Acad Sci U S A 100:15730–15735PubMedCentralPubMedCrossRefGoogle Scholar
  43. 43.
    Serino G, Maliga P (1997) A negative selection scheme based on the expression of cytosine deaminase in plastids. Plant J 12:697–701PubMedCrossRefGoogle Scholar
  44. 44.
    Corneille S, Lutz K, Svab Z, Maliga P (2001) Efficient elimination of selectable marker genes from the plastid genome by the CRE-lox site-specific recombination system. Plant J 27:171–178PubMedCrossRefGoogle Scholar
  45. 45.
    Hajdukiewicz PTJ, Gilbertson L, Staub JM (2001) Multiple pathways for Cre/lox-mediated recombination in plastids. Plant J 27:161–170PubMedCrossRefGoogle Scholar
  46. 46.
    Lestrade C, Pelissier B, Roland A, Dubald M (2009) Construct for obtaining transplastomic plant or plant cell comprises at least chimeric gene encoding selectable marker and chimeric color gene, or chimeric gene encoding luminescent protein, or chimeric gene encoding a negative marker Bayer Cropscience Ag. Patent Application WO2010079117Google Scholar
  47. 47.
    Kuroda H, Maliga P (2003) The plastid clpP1 protease gene is essential for plant development. Nature 425:86–89PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Elisabeth A. Mudd
    • 1
  • Panagiotis Madesis
    • 1
  • Elena Martin Avila
    • 1
  • Anil Day
    • 1
  1. 1.Faculty of Life SciencesThe University of ManchesterManchesterUK

Personalised recommendations