High Frequency of Single-Copy T-DNA Transformants Produced After Floral Dip in CRE-Expressing Arabidopsis Plants
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.
Key wordsCRE/loxP recombinase Single-copy T-DNA Arabidopsis Floral-dip transformation T-DNA inversion
The authors thank Martine De Cock for the help in preparing the manuscript. This work was supported by grants from the 6th Framework Program of the European Union “GENINTEG” (LSHG-CT2003-503303) and the European Union BIOTECH program (QLRT-2000-00078), with additional cofinancing from the Flemish Community and the Research Foundation Flanders (grant no. G021106).
- 2.Banta, L. M., and Montenegro, M. (2008) Agrobacterium and plant biotechnology in. Agrobacterium, From Biology to Biotechnology (Tzfira, T. and Citovsky, V. eds.), Springer, New York, pp. 73–148.Google Scholar
- 8.De Buck, S., and Depicker, A. (2004) Gene expression and level of expression. in, Handbook of Plant Biotechnology, Vol. 1 (Christou, P. and Klee, H. eds.), John Wiley & Sons, Chichester, pp. 331–345.Google Scholar