An optimised transformation protocol forAnthoceros agrestisand three more hornwort species

AbstractLand plants comprise two large monophyletic lineages, the vascular plants and the bryophytes, which diverged from their most recent common ancestor approximately 480 million years ago. Of the three lineages of bryophytes, only the mosses and the liverworts are systematically investigated, while the hornworts are understudied. Despite their importance for understanding fundamental questions of land plant evolution, they only recently became amenable to experimental investigation, withAnthoceros agrestisbeing developed as a hornwort model system. Availability of a high quality genome assembly and a recently developed genetic transformation technique makesA. agrestisan attractive model species for hornworts. Here we describe an updated and optimised transformation protocol forA. agrestiswhich can be successfully used to genetically modify one more strain ofA. agrestisand three more hornwort species,Anthoceros punctatus, Leiosporoceros dussiandPhaeoceros carolinianus.The new transformation method is less laborious, faster and results in the generation of greatly increased numbers of transformants compared to the previous method. We have also developed a new selection marker for transformation. Finally, we report the development of a set of different cellular localisation signal peptides for hornworts providing new tools to better understand hornwort cell biology.