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

• Published in: Theoretical and applied genetics Vol. 117; no. 8; pp. 1325 - 1334
• Main Authors: Moravčíková, Jana, Vaculková, Eva, Bauer, Miroslav, Libantová, Jana
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Berlin/Heidelberg : Springer-Verlag 01-11-2008; Springer-Verlag; Springer; Springer Nature B.V
• Subjects: Agriculture; Arabidopsis Proteins - genetics; Biochemistry; Biological and medical sciences; Biomedical and Life Sciences; Biotechnology; Chimera; Classical genetics, quantitative genetics, hybrids; Cloning; DNA, Bacterial - genetics; DNA, Plant - genetics; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Plant; Genes; Genetic Engineering - methods; Genetic Markers; Genetic Vectors; Genetics of eukaryotes. Biological and molecular evolution; Globulins - genetics; Integrases; Life Sciences; Nicotiana - genetics; Original Paper; Plant Biochemistry; Plant Breeding/Biotechnology; Plant Genetics and Genomics; Plants, Genetically Modified - genetics; Promoter Regions, Genetic; Pteridophyta, spermatophyta; Seed Storage Proteins - genetics; Seeds; Seeds - genetics; Transformation, Genetic; Transgenic plants; Vegetals; Slovakia; Excision Event; Selectable Marker Gene; Germination Assay; nptII Gene; loxP Site; Seeds; Excision; Genetics; Strategy; Agriculture; Transgenic plant; Marker
• ISSN: 0040-5752; 1432-2242
• DOI: 10.1007/s00122-008-0866-4

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.