Peat-based gnotobiotic plant growth systems for Arabidopsis microbiome research

The complex structure and function of a plant microbiome are driven by many variables, including the environment, microbe-microbe interactions and host factors. Likewise, resident microbiota can influence many host phenotypes. Gnotobiotic growth systems and controlled environments empower researchers to isolate these variables, and standardized methods equip a global research community to harmonize protocols, replicate experiments and collaborate broadly. We developed two easily constructed peat-based gnotobiotic growth platforms: the FlowPot system and the GnotoPot system. Sterile peat is amenable to colonization by microbiota and supports growth of the model plant Arabidopsis thaliana in the presence or absence of microorganisms. The FlowPot system uniquely allows one to flush the substrate with water, nutrients and/or suspensions of microbiota via an irrigation port, and a mesh retainer allows for the inversion of plants for dip or vacuum infiltration protocols. The irrigation port also facilitates passive drainage, preventing root anoxia. In contrast, the GnotoPot system utilizes a compressed peat pellet, widely used in the horticultural industry. GnotoPot construction has fewer steps and requires less user handling, thereby reducing the risk of contamination. Both protocols take up to 4 d to complete with 4-5 h of hands-on time, including substrate and seed sterilization. In this protocol, we provide detailed assembly and inoculation procedures for the two systems. Both systems are modular, do not require a sterile growth chamber, and cost less than US$2 per vessel. This protocol describes two peat-based plant growth systems for microbiome research in Arabidopsis. While both systems support microbe-free plants and input microbiota control, GnotoPots have advantages for throughput and FlowPots for versatility.