| Affiliation: | (1) Plant Biotechnology, Freiburg University, Schaenzlestrasse 1, 79104 Freiburg, Germany;(2) Present address: Department of Plant Propagation, Institute of Vegetable and Ornamental Crops, Kuehnhaeuser Strasse 101, 99189 Kuehnhausen, Germany;(3) Present address: Laboratory of Molecular Biotechnology, Centre for Biotechnology UNIL-EPFL and Institute of Animal Biology, Université de Lausanne, 1015 Lausanne, Switzerland;(4) Present address: Ginkgo communication, Unterer Batterieweg 113, 4059 Basel, Switzerland;(5) Present address: Plant Genetics, Institute for Plant Sciences, ETH Centre, LFW, Universitaetsstrasse 2, 8092 Zurich, Switzerland |
| Abstract: | The moss Physcomitrella patens is the only land plant known to date with highly efficient homologous recombination in its nuclear DNA, making it a unique model for plant functional genomics approaches. For high-throughput production of knockout plants, a robust transformation system based on polyethylene glycol-mediated transfection of protoplasts was developed and optimised. Both the DNA conformation and pre-culture of plants used for protoplast isolation significantly affected transformation efficiencies. Employing a newly developed PCR high-throughput method, the gene-targeting efficiency in more than 1,000 plants transformed with different cDNA-based knockout constructs was determined and analysed with regard to the length and intron/exon structure of the homologous gene locus. Different targeting constructs, each containing an identical selectable marker gene, were applied as batch DNA in a single transformation experiment and resulted in double-knockout plants. Thus, the fast and efficient generation of multiple targeted gene-knockouts is now feasible in Physcomitrella.Communicated by U. Kück |
