An improved and highly standardised transformation procedure allows efficient production of single and multiple targeted gene-knockouts in a moss, Physcomitrella patens.
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 1000 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.
Plant Biotechnology, Freiburg University, Schaenzlestrasse 1, 79104 Freiburg, Germany.
Annette
A
Hohe
Tanja
T
Egener
Jan M
JM
Lucht
Hauke
H
Holtorf
Christina
C
Reinhard
Gabriele
G
Schween
Ralf
R
Reski
D000595:Amino Acid Sequence; D001483:Base Sequence; D019068:Bryopsida / Q000235:genetics*; D018076:DNA, Complementary; D018390:Gene Targeting; D017343:Genes, Plant; D005822:Genetic Vectors; D018745:Genome, Plant; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D008957:Models, Genetic; D030821:Plants, Genetically Modified; D011523:Protoplasts; D011995:Recombination, Genetic; D012689:Sequence Homology, Nucleic Acid; D014170:Transformation, Genetic
D018076:DNA, Complementary; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins