LAUSR

Abstract Premise Hornworts belong to a unique lineage of bryophytes with critical traits for elucidating the evolution of land plants; however, the development of functional genetic tools for hornworts has been hampered by their relatively slow gametophytic growth. Methods To identify the external factors that influence the development of hornwort gametophytes and potentially augment their growth, we evaluated the contributions of several culture medium components on the axenic gametophytic growth of Anthoceros agrestis, a model hornwort. A streamlined growth assay utilizing semiautomated image analysis was developed to rapidly quantify and compare tissue development spanning four weeks of culture on solidified medium. Results The addition of sucrose, ammonium nitrate, activated charcoal, pH buffering, and growth regulators (2,4‐dichlorophenoxyacetic acid, 6‐benzylaminopurine, and thidiazuron) affected gametophyte tissue survival, growth patterns, and the rate of thalli growth. Subsequently, an optimized medium composition and growth regimen for accelerating A. agrestis gametophytic growth were formulated, which at four weeks of culture increased the tissue wet weight by 2.1‐ to 8.5‐fold compared with other previously utilized hornwort growth media. Discussion Our protocol for generating vigorous starting material and accelerated tissue regeneration is pertinent for advancing gene function characterization and genome editing in hornworts.