Development of PEG-mediated genetic transformation and gene editing system of Bryum argenteum as an abiotic stress tolerance model plant

Abstract

Bryum argenteumis a fascinating, cosmopolitan, and versatile moss species that thrives in various disturbed environments. Because of its comprehensive tolerance to the desiccation, high UV and extreme temperatures, so it is emerging as a model moss for studying the molecular mechanisms underlying the responses of plants to abiotic stresses. However, due to the lack of basic tools such as gene transformation, targeted genome modi cation, molecular mechanisms underlying the survival of B. argenteum in different environments are poorly understood. Here, we reported the protonema of B. argenteum can survive up to 95.42% water loss. The genome size of B. argenteum is approximately 313 Mb by kmer analysis, smaller than reported 700 Mb. Then, we established a simple method for protonema induction and an e cient protoplast isolation and regeneration protocol for B. argenteum. Moreover, a PEG-mediated B. argenteum protoplast transient transfection and stable transformation system was developed. Two homologues of ABI3(ABA-INSENSITIVE 3) gene were cloned from B. argenteum. This genetic transformation system was further optimized for CRISPR/Cas9 system for targeted delivery of the BaABI3A and BaABI3B gene into B. argenteum protoplasts, which triggered mutagenesis at the target in about 2%-5% of the regenerated plants. Isolated ABI3A and ABI3B mutants are all sensitive to desiccation, which suggested BaABI3A and BaABI3B play redundant roles in desiccation stress. Our results provide a rapid and simple approach for molecular genetics in B. argenteum. The results of this study will help to understand the molecular mechanisms of plant extreme environmental adaptation. Key messageTo establish a sterile culture system and protoplast regeneration system for Bryum argenteum, and to establish and apply CRISPR/Cas9 system in Bryum argenteum