biostudies-literatureNeubauer ASwiss National Science Foundatione11456https://www.ebi.ac.uk/biostudies/studies/S-EPMC9039799biostudies-literatureUnknown10(2)<h4>Premise</h4>A detailed protocol for the protoplast transformation of hornwort tissue is not yet available, limiting molecular biological investigations of these plants and comparative analyses with other bryophytes, which display a gametophyte-dominant life cycle and are critical to understanding the evolution of key land plant traits.<h4>Methods and results</h4>We describe a detailed protocol to isolate and transiently transform protoplasts of the model hornwort <i>Anthoceros agrestis</i>. The digestion of liquid cultures with Driselase yields a high number of viable protoplasts suitable for polyethylene glycol (PEG)-mediated transformation. We also report early signs of protoplast regeneration, such as chloroplast division and cell wall reconstitution.<h4>Conclusions</h4>This protocoApplications in plant sciencesStep-by-step protocol for the isolation and transient transformation of hornwort protoplasts.PMC9039799184826160004Notzold SIRuaud SNeubauer AFrangedakis ESzovenyi PWaller MLi FWBailly AWicke SfalseStep-by-step protocol for the isolation and transient transformation of hornwort protoplasts.<h4>Premise</h4>A detailed protocol for the protoplast transformation of hornwort tissue is not yet available, limiting molecular biological investigations of these plants and comparative analyses with other bryophytes, which display a gametophyte-dominant life cycle and are critical to understanding the evolution of key land plant traits.<h4>Methods and results</h4>We describe a detailed protocol to isolate and transiently transform protoplasts of the model hornwort <i>Anthoceros agrestis</i>. The digestion of liquid cultures with Driselase yields a high number of viable protoplasts suitable for polyethylene glycol (PEG)-mediated transformation. We also report early signs of protoplast regeneration, such as chloroplast division and cell wall reconstitution.<h4>Conclusions</h4>This protoco2022-01-01T00:00:00Z2022 Mar-Apr2025-04-26T15:12:51.312Z2025-04-06T14:51:39.437ZS-EPMC90397993549519210.1002/aps3.11456