植物科学

The tomato (Solanum lycopersicum) is a widely cultivated crop worldwide that serves as a model system for fruit development studies. Agrobacterium tumefaciens–mediated transformation of tomato has played a central role as a tool for analyzing the function of candidate genes and producing transgenic lines with enhanced resistance to pathogens, tolerance to abiotic stresses, and improved fruit quality traits. Among the many tomato varieties, the miniature dwarf cultivar Micro-Tom (MT) has been increasingly adopted as a model system for tomato research due to its short life cycle, small size, and high transformation efficiency. This protocol outlines a replicable methodology for A. tumefaciens–mediated transformation of Micro-Tom from cotyledon explants, utilizing cost-effective plant growth regulators for shoot regeneration, high transformation rates, reduced regeneration time, and enhanced rooting conditions.

Lipid droplets have emerged as dynamic organelles involved in diverse cellular processes beyond simple lipid storage. In plants and cyanobacteria, growing evidence highlights their importance in stress adaptation and signaling, yet methods to study their structure and purity remain limited. Traditionally, in situ transmission electron microscopy (TEM) has been used to visualize lipid droplets within intact cells. While powerful, this approach cannot easily evaluate isolated lipid droplets or confirm their purity. In this protocol, we describe a rapid method for preparing and visualizing cyanoglobule lipid droplets isolated from cyanobacteria. The isolated droplets are directly processed for TEM using negative staining with uranyl acetate, providing a straightforward and efficient workflow. The procedure can be applied broadly to lipid droplets from diverse organisms, independent of species or cellular origin. This protocol offers a simple, fast, and widely applicable approach to assessing lipid droplets, expanding the toolkit for researchers studying their structure and function.

Roots are essential organs for plants, facilitating water and nutrient uptake from the soil to support growth. Traditional methods for studying root systems, such as rhizoboxes and rhizotrons, have provided valuable insights. However, advanced methods such as fabricated ecosystems (EcoFAB) combined with new generation microscopes now enable a more detailed investigation of the rhizosphere, the microenvironment surrounding roots, allowing a deeper understanding of root tissue, exudates, and plant–soil interactions. This microenvironment can be used to investigate the adaptation of plants to environmental stress (salinity, drought, higher temperatures). Our procedure focuses on establishing standardized protocols for plant growth tailored to the EcoFAB system, which offers a controlled environment to study root dynamics. This work also contributes new insights into the early stages of plant germination, an area currently underexplored in the literature. While numerous studies focus on plant growth or genetic aspects, such as gene induction, the germination phase remains underexplored. We have developed optimized germination protocols for multiple plant species, ensuring uniform seedling size and sufficient development for seamless integration into the EcoFAB system.

In plants, the apoplast contains a diverse set of proteins that underpin mechanisms for maintaining cell homeostasis, cell wall remodeling, cell signaling, and pathogen defense. Apoplast protein composition is highly regulated, primarily through the control of secretory traffic in response to endogenous and environmental factors. Dynamic changes in apoplast proteome facilitate plant survival in a changing climate. Even so, the apoplast proteome profiles in plants remain poorly characterized due to technological limitations. Recent progress in quantitative proteomics has significantly advanced the resolution of proteomic profiling in mammalian systems and has the potential for application in plant systems. In this protocol, we provide a detailed and efficient protocol for tandem mass tag (TMT)-based quantitative analysis of Arabidopsis thaliana secretory proteome to resolve dynamic changes in leaf apoplast proteome profiles. The protocol employs apoplast flush collection followed by protein cleaning using filter-aided sample preparation (FASP), protein digestion, TMT-labeling of peptides, and mass spectrometry (MS) analysis. Subsequent data analysis for peptide detection and quantification uses Proteome Discoverer software (PD) 3.0. Additionally, we have incorporated in silico–generated spectral libraries using PD 3.0, which enables rapid and efficient analysis of proteomic data. Our optimized protocol offers a robust framework for quantitative secretory proteomic analysis in plants, with potential applications in functional proteomics and the study of trafficking systems that impact plant growth, survival, and health.

When plants undergo senescence or experience carbon starvation, leaf cells degrade proteins in the chloroplasts on a massive scale via autophagy, an evolutionarily conserved process in which intracellular components are transported to the vacuole for degradation to facilitate nutrient recycling. Nonetheless, how portions of chloroplasts are released from the main chloroplast body and mobilized to the vacuole remains unclear. Here, we developed a method to observe the autophagic transport of chloroplast proteins in real time using confocal laser-scanning microscopy on transgenic plants expressing fluorescently labeled chloroplast components and autophagy-associated membranes. This protocol enabled us to track changes in chloroplast morphology during chloroplast-targeted autophagy on a timescale of seconds, and it could be adapted to monitor the dynamics of other intracellular processes in plant leaves.

Sheath blight, caused by Rhizoctonia solani, is a major fungal disease of rice that leads to significant yield losses globally. Conventional inoculation methods often fail to achieve consistent and uniform infection, limiting their applicability in antifungal screening studies. This protocol describes a reliable in planta inoculation method for R. solani using mature sclerotia placed at the internodal region of tillering-stage rice seedlings. The procedure includes step-by-step instructions for seed germination, seedling preparation, pathogen culture, artificial inoculation, and post-infection application of antifungal treatments, including botanical compounds such as Ocimum gratissimum essential oil and thymol. Lesion development is monitored and quantified over time, and data are analyzed statistically to evaluate treatment efficacy. The protocol is optimized for reproducibility, scalability, and compatibility with sustainable disease management approaches. It provides a robust platform for evaluating antifungal agents in a biologically relevant and controlled environment.

The rhizosphere, a 2–10 mm region surrounding the root surface, is colonized by numerous microorganisms, known as the rhizosphere microbiome. These microorganisms interact with each other, leading to emergent properties that affect plant fitness. Mapping these interactions is crucial to understanding microbial ecology in the rhizosphere and predicting and manipulating plant health. However, current methods do not capture the chemistry of the rhizosphere environment, and common plant–microbe interaction study setups do not map bacterial interactions in this niche. Additionally, studying bacterial interactions may require the creation of transgenic bacterial lines with markers for antibiotic resistance/fluorescent probes and even isotope labeling. Here, we describe a protocol for both in silico prediction and in vitro validation of bacterial interactions that closely recapitulate the major chemical constituents of the rhizosphere environment using a widely used Murashige & Skoog (MS)-based gnotobiotic plant growth system. We use the auto-fluorescent Pseudomonas, abundantly found in the rhizosphere, to estimate their interactions with other strains, thereby avoiding the need for the creation of transgenic bacterial strains. By combining artificial root exudate medium, plant cultivation medium, and a synthetic bacterial community (SynCom), we first simulate their interactions using genome-scale metabolic models (GSMMs) and then validate these interactions in vitro, using growth assays. We show that the GSMM-predicted interaction scores correlate moderately, yet significantly, with their in vitro validation. Given the complexity of interactions among rhizosphere microbiome members, this reproducible and efficient protocol will allow confident mapping of interactions of fluorescent Pseudomonas with other bacterial strains within the rhizosphere microbiome.

Fusarium crown rot (FCR), mainly caused by Fusarium pseudograminearum, is a devastating soil-borne disease of wheat that results in severe yield and quality reduction. FCR is characterized by stem base necrosis and whitehead development. In recent years, FCR has escalated in both incidence and severity, emerging as a critical threat to global wheat production, particularly within key cultivation zones such as China's Huang-Huai-Hai Plain. The development of resistant cultivars is an effective and environmentally sustainable strategy for FCR disease control. However, the lack of standardized and reproducible inoculation protocols has hindered the accurate assessment and screening of disease-resistant wheat germplasms. To address this limitation, we established a robust FCR inoculation system utilizing F. pseudograminearum propagated on a millet grain substrate, facilitating rapid and reliable evaluation of both host resistance and fungal pathogenicity. Laboratory validation demonstrated high infection efficiency and strong reproducibility of this method.

This protocol outlines a reliable method for the micropropagation of Nicotiana benthamiana using axillary shoot branching. Axillary shoot induction involves stimulating the outgrowth of dormant buds located at the leaf axils, allowing for the development of genetically stable shoots without callus formation or the use of exogenous plant growth regulators. Nodal explants are cultured on MS medium supplemented with kinetin and indole-3-butyric acid (IBA) to induce shoot formation. Isolated shoots are then transferred to hormone-free MS medium for rooting. This method is simple, reproducible, and supports rapid plant multiplication for downstream applications such as agroinfiltration or transient protein expression.

Oomycetes are a predominantly plant-pathogenic group of organisms often considered and managed as fungi; however, due to their evolutionary divergence from true fungi, many conventional fungicides are ineffective against them. Their unique physiological characteristics make them challenging to work with, highlighting the need for a standardized and reproducible procedure for anti-oomycete assays. Previous studies describe methods to obtain sporulation forms in the laboratory, but there remains a disconnect between spore production and the subsequent screening process for potential biological pesticides based on microbial organic extracts. This protocol bridges that gap by providing a complete and reliable workflow from spore production to screening. In this study, we present an efficient in vitro protocol to identify microbial extracts with activity against Phytophthora capsici and Pythium ultimum. The protocol includes a method for obtaining zoospores of P. capsici and oospores of P. ultimum, followed by a simple and rapid screening assay to detect microbial extracts that inhibit the growth of these pathogens. The extracts are dispensed onto plates in two concentrations and allowed to dry. This facilitates pauses in the protocol and allows for storage of the plates until the biological material is ready for the assay. The protocol’s effectiveness has been validated with these two oomycetes, resulting in the identification of active extracts in both cases. Moreover, it can be adapted to other pathogens.