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Abstract Hornworts are a deeply diverged lineage of bryophytes and a sister lineage to mosses and liverworts. Hornworts have an array of unique features that can be leveraged to illuminate not only the early evolution of land plants, but also alternative paths for nitrogen and carbon assimilation via cyanobacterial symbiosis and a pyrenoid-based CO2-concentrating mechanism (CCM), respectively. Despite this, hornworts are one of the few plant lineages with limited available genetic tools. Here we report an efficient biolistics method for generating transient expression and stable transgenic lines in the model hornwort, Anthoceros agrestis. An average of 569 (±268) cells showed transient expression per bombardment, with green fluorescent protein expression observed within 48–72 h. A total of 81 stably transformed lines were recovered across three separate experiments, averaging six lines per bombardment. We followed the same method to transiently transform nine additional hornwort species, and obtained stable transformants from one. This method was further used to verify the localization of Rubisco and Rubisco activase in pyrenoids, which are central proteins for CCM function. Together, our biolistics approach offers key advantages over existing methods as it enables rapid transient expression and can be applied to widely diverse hornwort species. 

Summary Despite their key phylogenetic position and their unique biology, hornworts have been widely overlooked. Until recently there was no hornwort model species amenable to systematic experimental investigation.Anthoceros agrestishas been proposed as the model species to study hornwort biology.We have developed anAgrobacterium‐mediated method for the stable transformation ofA. agrestis, a hornwort model species for which a genetic manipulation technique was not yet available.High transformation efficiency was achieved by using thallus tissue grown under low light conditions. We generated a total of 274 transgenicA. agrestislines expressing the β‐glucuronidase (GUS), cyan, green, and yellow fluorescent proteins under control of the CaMV 35S promoter and several endogenous promoters. Nuclear and plasma membrane localization with multiple color fluorescent proteins was also confirmed.The transformation technique described here should pave the way for detailed molecular and genetic studies of hornwort biology, providing much needed insight into the molecular mechanisms underlying symbiosis, carbon‐concentrating mechanism, RNA editing and land plant evolution in general.