Skiada Vasiliki, Papadopoulou Kalliope K
Laboratory of Plant and Environmental Biotechnology, Department of Biochemistry and Biotechnology, University of Thessaly, Biopolis, Larissa, Greece.
Bio Protoc. 2025 Feb 20;15(4):e5186. doi: 10.21769/BioProtoc.5186.
In nature, filamentous fungi interact with plants. These fungi are characterized by rapid growth in numerous substrates and under minimal nutrient requirements. Investigating the interaction of these fungi with their plant hosts under controlled conditions is of importance for many researchers aiming to proceed with molecular or microscopical investigations of their favorite plant-fungus interaction system. The speed of growth of these fungi complicates transferring plant-fungal interaction systems in laboratory conditions. The issue is more complicated when monoxenic conditions are desired, to ensure that only two members (a fungus and a plant) are present in the system under study. Here, two simple closed systems for investigating plant-filamentous fungi associations under laboratory, monoxenic conditions are described, along with their limitations. The plant and fungal growth conditions, methods for sampling, staining, sectioning, and subsequent microscopical imaging of colonized plant tissues with affordable, common laboratory tools are described. Key features • Setting up closed systems for microscopical observations of plant-filamentous fungi (emphasis on model legumes-) associations and temporal in vivo observations of the association(s). • Preparing root samples for microscopical observations: staining, sectioning, and mounting on microscopical slides. • Using low-cost equipment for performing microscopical observations and imaging. • Using fluorescence microscopy: provision of common fluorophores to highlight specific plant and fungal tissues, compartments, and structures.
在自然界中，丝状真菌与植物相互作用。这些真菌的特点是能在多种基质中快速生长，且对营养需求极低。对于许多旨在对其感兴趣的植物 - 真菌相互作用系统进行分子或显微镜研究的研究人员来说，在可控条件下研究这些真菌与其植物宿主之间的相互作用至关重要。这些真菌的生长速度使得在实验室条件下转移植物 - 真菌相互作用系统变得复杂。当需要单菌培养条件以确保所研究的系统中仅存在两个成员（一种真菌和一种植物）时，问题会更加复杂。在此，描述了两种用于在实验室单菌培养条件下研究植物 - 丝状真菌关联的简单封闭系统及其局限性。还描述了植物和真菌的生长条件、采样方法、染色、切片以及随后使用经济实惠的普通实验室工具对定殖植物组织进行显微镜成像的方法。关键特性 • 建立用于显微镜观察植物 - 丝状真菌（重点是模式豆科植物）关联以及对该关联进行体内实时观察的封闭系统。 • 制备用于显微镜观察的根样本：染色、切片并装片到显微镜载玻片上。 • 使用低成本设备进行显微镜观察和成像。 • 使用荧光显微镜：提供常见的荧光团以突出特定的植物和真菌组织、区室及结构。
