An Agrobacterium-mediated stable transformation technique for the hornwort model Anthoceros agrestis

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 agrestis has been proposed as the model species to study hornwort biology. We have developed an Agrobacterium-mediated method for the stable transformation of A. 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 transgenic A. agrestis lines expressing the beta-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.

Automated summary

Significant progress has been made in establishing a transformation method for the hornwort model species A. agrestis, enabling the study of hornwort biology at a molecular and genetic level. This technique has opened up possibilities for understanding molecular mechanisms related to symbiosis, carbon-concentrating mechanism, RNA editing, and overall land plant evolution.