title: T-DNA transfer and T-DNA integration efficiencies upon Arabidopsis thaliana root explant cocultivation and floral dip transformation.
abstract: T-DNA transfer and integration frequencies during Agrobacterium-mediated root explant cocultivation and floral dip transformations of Arabidopsis thaliana were analyzed with and without selection for transformation-competent cells. Based on the presence or absence of CRE recombinase activity without or with the CRE T-DNA being integrated, transient expression versus stable transformation was differentiated. During root explant cocultivation, continuous light enhanced the number of plant cells competent for interaction with Agrobacterium and thus the number of transient gene expression events. However, in transformation competent plant cells, continuous light did not further enhance cotransfer or cointegration frequencies. Upon selection for root transformants expressing a first T-DNA, 43-69 % of these transformants showed cotransfer of another non-selected T-DNA in two different light regimes. However, integration of the non-selected cotransferred T-DNA occurred only in 19-46 % of these transformants, indicating that T-DNA integration in regenerating root cells limits the transformation frequencies. After floral dip transformation, transient T-DNA expression without integration could not be detected, while stable T-DNA transformation occurred in 0.5-1.3 % of the T1 seedlings. Upon selection for floral dip transformants with a first T-DNA, 8-34 % of the transformants showed cotransfer of the other non-selected T-DNA and in 93-100 % of them, the T-DNA was also integrated. Therefore, a productive interaction between the agrobacteria and the female gametophyte, rather than the T-DNA integration process, restricts the floral dip transformation frequencies.
authors: Department Plant Systems Biology, VIB, Ghent University, Technologiepark 927, 9052, Ghent, Belgium.|Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052, Ghent, Belgium.
authors: Rim
authors: R
authors: Ghedira
authors: Department Plant Systems Biology, VIB, Ghent University, Technologiepark 927, 9052, Ghent, Belgium.|Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052, Ghent, Belgium.
authors: Sylvie
authors: S
authors: De Buck
authors: Laboratory of Plant Genetics, Institute for Molecular Biology and Biotechnology, Vrije Universiteit Brussel (VUB), 1050, Brussel, Belgium.|Bayer CropScience NV, Technologiepark 38, 9052, Ghent, Belgium.
authors: Frédéric
authors: F
authors: Van Ex
authors: Laboratory of Plant Genetics, Institute for Molecular Biology and Biotechnology, Vrije Universiteit Brussel (VUB), 1050, Brussel, Belgium.
authors: Geert
authors: G
authors: Angenon
authors: Department Plant Systems Biology, VIB, Ghent University, Technologiepark 927, 9052, Ghent, Belgium. ann.depicker@psb.vib-ugent.be.|Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052, Ghent, Belgium. ann.depicker@psb.vib-ugent.be.
authors: Ann
authors: A
authors: Depicker
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics; D017360:Arabidopsis / Q000235:genetics*; D018920:Coculture Techniques; D004269:DNA, Bacterial / Q000235:genetics*; D035264:Flowers / Q000235:genetics; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D018517:Plant Roots / Q000235:genetics; D030821:Plants, Genetically Modified; D014170:Transformation, Genetic
chemical_list: D004269:DNA, Bacterial; C036483:T-DNA
keywords: Agrobacterium; CRE recombinase; Transformation efficiency; Transgene expression
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