Theoretical and Applied Genetics

, 117:1325 | Cite as

Feasibility of the seed specific cruciferin C promoter in the self excision Cre/loxP strategy focused on generation of marker-free transgenic plants

  • Jana Moravčíková
  • Eva Vaculková
  • Miroslav Bauer
  • Jana LibantováEmail author
Original paper


This work is focused on the generation of selectable marker-free transgenic tobacco plants using the self excision Cre/loxP system that is controlled by a strong seed specific Arabidopsis cruciferin C (CRUC) promoter. It involves Agrobacterium-mediated transformation using a binary vector containing the gus reporter gene and one pair of the loxP sites flanking the cre recombinase and selectable nptII marker genes (floxed DNA). Surprisingly, an ectopic activation of CRUC resulting in partial excision of floxed DNA was observed during regeneration of transformed cells already in calli. The regenerated T0 plants were chimeric, but no ongoing ectopic expression was observed in these one-year-long invitro maintained plants. The process of the nptII removal was expected in the seeds; however, none of the analysed T0 transgenic lines generated whole progeny sensitive to kanamycin. Detailed analyses of progeny of selected T0-30 line showed that 10.2% GUS positive plants had completely removed nptII gene while the remaining 86.4% were still chimeras. Repeated activation of the cre gene in T2 seeds resulted in increased rate of marker-free plants, whereas four out of ten analysed chimeric T1 plants generated completely marker-free progenies. This work points out the feasibility as well as limits of the CRUC promoter in the Cre/loxP strategy.


nptII Gene Selectable Marker Gene loxP Site Germination Assay Excision Event 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



We thank Drs. Mlynárová and Nap for providing plasmids pFLUAR101, pLM91-containing CRUC-cre expression unit and for helpful discussion. We thank Dr. Salaj for help with microscopic techniques, and Dr. Matušíková for her kind proof reading of the manuscript. The authors thank Anna Fábelová for invitro plant care. The study was funded by Scientific Grant Agency of the Ministry of Education of Slovak Republic and Slovak Academy of Sciences 2/0011/08.

Supplementary material

122_2008_866_MOESM1_ESM.tif (9.7 mb)
Electronic Supplementary Material S1 Examples of Southern blot analysis of parental T0 plants. a VspI-digested DNA probed with the GUS specific probe. The bands of a 2.9 kb correspond to the internal gus fragment. All additional bands (>1.5 kb) correspond to the border fragments and indicate the number of independent transgene copies. The absence of the internal 2.9 kb gus fragment in the plant T0-8 indicates an incomplete gus gene integration. b EcoRI-digested DNA probed with the NPTII specific probe. The bands (>2.1 kb) correspond to the number of independent transgene copies. NT – non-transgenic control plant, asterisks indicate single copy transformants used in further analyses. (TIFF 9924 kb)
122_2008_866_MOESM2_ESM.tif (6.2 mb)
Electronic Supplementary Material S2 PCR detection of premature excision in T0 plants. Photographs of ethidium bromide-stained 1% agarose gels carrying PCR products obtained from the single-copy T0 tobacco plants. a PCR with the primers P7/P6 that amplified a 0.59 kb fragment indicating excision of the nptII gene (NPT-). b PCR with the primers P5/P6 that amplified a 1.8 kb fragment corresponding to the nptII gene (NPT+). c PCR products obtained on the same plant set with the actin primers. The lane M contains 1 kb DNA ladder (Fermentas) as a size marker, next follow the PCR products of individual T0 plants, the last lane represents PCR product of plasmid pEV2 (EV2). (TIFF 6356 kb)
122_2008_866_MOESM3_ESM.tif (3.5 mb)
Electronic Supplementary Material S3 PCR analyses of premature excision in developing transgenic calli. Genomic DNA from transgenic calli was subjected to PCR analysis with the primers P7/P6 that amplified a 0.59 kb PCR fragment indicating the nptII gene excision (NPT-) and the primers P5/P6 that amplified a 1.8 kb fragment corresponding to the nptII gene (NPT+). The actin primers were used as a PCR control. The lane M contains 1 kb DNA ladder (Fermentas) as a size marker, C – transgenic calli, NT – non-transformed control, EV2 – plasmid pEV2. (TIFF 3590 kb)


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Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Jana Moravčíková
    • 1
  • Eva Vaculková
    • 1
  • Miroslav Bauer
    • 2
  • Jana Libantová
    • 1
    Email author
  1. 1.Institute of Plant Genetics and BiotechnologySlovak Academy of SciencesNitra 1Slovak Republic
  2. 2.Research Institute of Animal ProductionNitraSlovak Republic

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