Biolistics-mediated transformation of hornworts and its application to study pyrenoid protein localization.

The article focuses on the development of a biolistic (particle bombardment) transformation method for hornworts, a lineage of bryophytes important for studying land plant evolution and carbon/nitrogen assimilation mechanisms. Using the model hornwort *Anthoceros agrestis*, the method enables efficient transient expression of green fluorescent protein (GFP) within 48–72 hours and recovery of stable transgenic lines after 8–10 weeks, with an average of six stable lines per bombardment. The approach was successfully applied to transiently transform nine additional hornwort species and to stably transform *Anthoceros fusiformis*, demonstrating broad applicability across the hornwort phylogeny. Furthermore, the method facilitated fluorescent protein tagging and localization of key pyrenoid proteins—Rubisco small subunit (RbcS) and Rubisco activase (RCA)—which are central to the CO₂-concentrating mechanism (CCM) unique to hornworts. This biolistic protocol provides a valuable genetic tool for functional genomics and comparative studies in hornworts, supporting future research into their unique biology and potential biotechnological applications.