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

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 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.

Insights from the cold transcriptome of Physcomitrella patens: global specialization pattern of conserved transcriptional regulators and identification of orphan genes involved in cold acclimation

“…Considering also the large amount of orphan genes among the persistent DEGs, we conclude an important role of species- or lineage-specific genes in the acquisition of abiotic stress tolerance in moss. To answer the question of whether these genes are related to poikilohydry and hold the key for stress tolerance requires further investigations, for example via targeted gene replacement (Hohe et al ., 2004). The analysis of novel (not yet analyzed) genes from mosses may also help to improve the resilience of crop plants in a changing climate.…”

A phenol-enriched cuticle is ancestral to lignin evolution in land plants

“…The PpCYP98 disruption construct was excised from the vector backbone by Bam HI digestion, using restriction sites introduced during PCR. Twenty-five microgram of linearized construct were used for PEG-mediated transfection of P. patens protoplast31. Transformants were selected on Knop plates supplemented with 25 mg l −1 geneticin (G418).…”

Origin and function of stomata in the moss Physcomitrella patens

“…Table 1 introducing EcoRI sites to the ends of the PCR product. After cloning to plasmid pJet1.2 (Thermo Fisher) an nptII selection cassette 39 was inserted into this fragment via unique restriction sites for HincII and BcuI, respectively. Before moss transformation the KO construct was released from the vector backbone via EcoRI digest.…”