High frequency of single-copy T-DNA transformants produced after floral dip in CRE-expressing Arabidopsis plants

Annelies De Paepe, Sylvie De Buck, Jonah Nolf, Ann Depicker
Methods in Molecular Biology 2012, 847: 317-33
Transgenic plants that harbor a single copy of the introduced transgene are preferable to those with multiple transgene copies because multiple T-DNA copies correlate with expression variability and susceptibility to silencing. Especially after the commonly used floral-dip Agrobacterium-mediated transformation method, the frequency of single-copy transformants is low. The CRE/loxP recombinase-based strategy to resolve complex T-DNA loci has proven to be successful to efficiently obtain single-copy T-DNA transformants by directly transforming loxP-containing T-DNA vectors in CRE-expressing Arabidopsis thaliana plants. This chapter describes in detail how to transform three available loxP-containing T-DNA vectors into CRE-producing Arabidopsis C24 plants and subsequently how to analyze the transgenic plants for the T-DNA locus structure.

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