Feasibility of the seed specific cruciferin C promoter in the self excision Cre/loxP strategy focused on generation of marker-free transgenic plants
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.
KeywordsnptII Gene Selectable Marker Gene loxP Site Germination Assay Excision Event
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.
- Coppoolse ER, deVroomen MJ, Roelofs D, Smit J, van Gennip F, Hersmus BJM, Nijkamp JJ, van Haaren MJ (2003) Cre recombinase expression can result in phenotypic aberrations in plants. Plant Mol Biol 51:263–279Google Scholar
- Luo K, Duan H, Zhao D, Zheng X, Deng W, Chen Y, Stewart CN, McAvoy R, Jiang X, Wu Y, He A, Pei Y, Li Y (2007) ‘GM-gene-deletor’: fused loxP-FRT recognition sequences dramatically improve the efficiency of FLP or CRE recombinase on transgene excision from pollen and seed of tobacco plants. Plant Biotechnol J 5:263–274PubMedCrossRefGoogle Scholar