An improved and highly standardised transformation procedure allows efficient production of single and multiple targeted gene-knockouts in a moss, Physcomitrella patens
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- Hohe, A., Egener, T., Lucht, J.M. et al. Curr Genet (2004) 44: 339. doi:10.1007/s00294-003-0458-4
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.