Kremer James M, Sohrabi Reza, Paasch Bradley C, Rhodes David, Thireault Caitlin, Schulze-Lefert Paul, Tiedje James M, He Sheng Yang
MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI, USA.
Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI, USA.
Nat Protoc. 2021 May;16(5):2450-2470. doi: 10.1038/s41596-021-00504-6. Epub 2021 Apr 28.
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.
植物微生物组的复杂结构和功能受多种变量驱动，包括环境、微生物-微生物相互作用和宿主因素。同样，常驻微生物群可影响许多宿主表型。无菌生长系统和可控环境使研究人员能够分离这些变量，标准化方法使全球研究界能够统一实验方案、重复实验并广泛开展合作。我们开发了两种易于构建的基于泥炭的无菌生长平台：流动培养系统（FlowPot system）和无菌培养系统（GnotoPot system）。无菌泥炭适合微生物群定殖，并在有无微生物的情况下支持模式植物拟南芥的生长。流动培养系统独特之处在于，可通过灌溉端口用水、养分和/或微生物群悬液冲洗基质，并且一个网筛固定器可将植物倒置，用于浸蘸或真空渗透实验方案。灌溉端口还便于被动排水，防止根部缺氧。相比之下，无菌培养系统使用园艺行业广泛使用的压缩泥炭球。无菌培养系统的构建步骤较少，所需的用户操作也较少，从而降低了污染风险。两种方案最多需要4天完成，实际操作时间为4-5小时，包括基质和种子灭菌。在本方案中，我们提供了这两种系统的详细组装和接种程序。两种系统都是模块化的，不需要无菌生长室，每个容器成本低于2美元。
