High frequency of single-copy T-DNA transformants produced by floral dip in CRE-expressing Arabidopsis plants.
For genetic transformation of plants, floral dip with Agrobacterium often results in integration of multiple T-DNA copies at a single locus and frequently in low and unstable transgene expression. To obtain efficient single-copy T-DNA transformants, two CRE/loxP recombinase-based simplifying strategies for complex T-DNA loci were compared. A T-DNA vector with oppositely oriented loxP sites was transformed into CRE-expressing and wild-type control Arabidopsis thaliana plants. Of the primary CRE-expressing transformants, 55% harboured a single copy of the introduced T-DNA, but only 15% in the wild-type plants. However, 73% of the single-copy transformants in the CRE background showed continuous somatic inversion of the DNA segment between the two loxP sites. To avoid inversion of the loxP-flanked T-DNA segment, two T-DNA vectors harbouring only one loxP site were investigated for their suitability for CRE/loxP recombinase-mediated resolution upon floral-dip transformation into CRE-expressing plants. On average, 70% of the transformants in the CRE background were single-copy transformants, whereas the single-copy T-DNA frequency was only 11% for both vectors in the wild-type background. Both resolution strategies yielded mostly Cre transformants in which the 35S-driven transgene expression was stable and uniform in the progeny and remarkably, also in Cre transformants with multiple T-DNA copies. Therefore, a role is proposed for the CRE recombinase in preventing inverted T-DNA repeat formation or modifying the locus chromatin structure, resulting in a reduced sensitivity for silencing.
De Paepe
Annelies
A
Department of Plant Systems Biology, Flanders Institute for Biotechnology (VIB), 9052 Gent, Belgium.
De Buck
Sylvie
S
Hoorelbeke
Katleen
K
Nolf
Jonah
J
Peck
Ingrid
I
Depicker
Anna
A
D017360:Arabidopsis / Q000235:genetics*; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics*; D018744:DNA, Plant / Q000235:genetics; D035264:Flowers / Q000235:genetics; D018628:Gene Dosage; D018506:Gene Expression Regulation, Plant; D005822:Genetic Vectors; D019426:Integrases / Q000235:genetics*; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified / Q000235:genetics*; D010957:Plasmids; D012231:Rhizobium / Q000235:genetics; D014170:Transformation, Genetic; D019076:Transgenes
D004269:DNA, Bacterial; D018744:DNA, Plant; C036483:T-DNA; C045073:Cre recombinase; D019426:Integrases