A simple, highly efficient Agrobacterium tumefaciens‐mediated moss transformation system with broad applications

Mosses, particularly desiccation-tolerant (DT) species, are important model organisms for studying genes involved in plant development and stress resistance. The lack of a simple and efficient stable moss transformation system has hindered progress in deciphering the genetic mechanisms underlying traits of interest in these organisms. Here, we present an Agrobacterium tumefaciens-mediated transformation system for DT mosses that uses Agrobacterium strain EHA105 harboring the binary vector pCAMBIA1301-GUS. This system achieved transformation efficiencies of 74% and 81% in Physcomitrium patens and Bryum argenteum protonemata, respectively, without the need for culture and callus formation prior to regeneration. We detected GUS enzyme activity in the regenerated transgenic moss via histochemical staining. Southern blot, PCR, and RT-qPCR analyses confirmed the presence of the GUS gene. In addition, we successfully used this system to transform wild DT Syntrichia caninervis. Furthermore, P. patens and B. argenteum transformed using this system with the stress resistance gene EsDREB from the desert plant Eremosparton songoricum (Litv.) exhibited improved salt tolerance. We thus present an efficient tool for the genetic analysis of DT moss species, paving the way for the development of stress-resistant crop cultivars.