Determination of the T-DNA transfer and the T-DNA integration frequencies upon cocultivation of Arabidopsis thaliana root explants.
Using the Cre/lox recombination system, we analyzed the extent to which T-DNA transfer to the plant cell and T-DNA integration into the plant genome determine the transformation and cotransformation frequencies of Arabidopsis root cells. Without selection for transformation competence, the stable transformation frequency of shoots obtained after cocultivation and regeneration on nonselective medium is below 0.5%. T-DNA transfer and expression occur in 5% of the shoots, indicating that the T-DNA integrates in less than 10% of the transiently expressing plant cells. A limited fraction of root cells, predominantly located at the wounded sites and in the pericycle, are competent for interaction with agrobacteria and the uptake of a T-DNA, as demonstrated by histochemical GUS staining. When selection for transformation competence is applied, the picture is completely different. Then, approximately 50% of the transformants show transient expression of a second, nonselected T-DNA and almost 50% of these cotransferred T-DNAs are integrated into the plant genome. Our results indicate that both T-DNA transfer and T-DNA integration limit the transformation and cotransformation frequencies and that plant cell competence for transformation is based on these two factors.
De Buck
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Vakgroep Moleculaire Genetica, Departement Plantengenetica, Vlaams Interuniversitair Instituut voor Biotechnologie, Universiteit Gent, Belgium.
De Wilde
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C
Van Montagu
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M
Depicker
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A
D016960:Agrobacterium tumefaciens / Q000235:genetics*; D017360:Arabidopsis / Q000166:cytology / Q000235:genetics* / Q000382:microbiology; D004269:DNA, Bacterial / Q000235:genetics* / Q000502:physiology; D017353:Gene Deletion; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D019426:Integrases / Q000235:genetics / Q000378:metabolism; D018517:Plant Roots / Q000166:cytology / Q000235:genetics* / Q000382:microbiology; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D014170:Transformation, Genetic; D014764:Viral Proteins
D004269:DNA, Bacterial; C036483:T-DNA; D014764:Viral Proteins; C045073:Cre recombinase; D019426:Integrases; D005966:Glucuronidase