Research Article

Current Genetics

, Volume 44, Issue 6, pp 339-347

First online:

An improved and highly standardised transformation procedure allows efficient production of single and multiple targeted gene-knockouts in a moss, Physcomitrella patens

  • Annette HoheAffiliated withPlant Biotechnology, Freiburg UniversityDepartment of Plant Propagation, Institute of Vegetable and Ornamental Crops
  • , Tanja EgenerAffiliated withPlant Biotechnology, Freiburg UniversityLaboratory of Molecular Biotechnology, Centre for Biotechnology UNIL-EPFL and Institute of Animal Biology, Université de Lausanne
  • , Jan M. LuchtAffiliated withPlant Biotechnology, Freiburg UniversityGinkgo communication
  • , Hauke HoltorfAffiliated withPlant Biotechnology, Freiburg UniversityPlant Genetics, Institute for Plant Sciences
  • , Christina ReinhardAffiliated withPlant Biotechnology, Freiburg University
  • , Gabriele SchweenAffiliated withPlant Biotechnology, Freiburg University
  • , Ralf ReskiAffiliated withPlant Biotechnology, Freiburg University Email author 

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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.


Ammonium tartrate Bioreactor culture Functional genomics Gene-targeting Homologous recombination