Plant Science

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.

Synthetic trans-acting small interfering RNAs (syn-tasiRNAs) are 21-nucleotide small RNAs designed to induce highly specific and efficient gene silencing in plants. Traditional approaches rely on the transgenic expression of ~1 kb TAS precursors, which limits their use in non-model species, under strict GMO regulations, and in size-constrained expression or delivery systems. This protocol describes a rapid workflow for the design, assembly, and delivery of syn-tasiRNAs derived from much shorter precursors, referred to as minimal precursors. The pipeline includes in silico design of highly specific syn-tasiRNA sequences, cloning of minimal precursors into plant expression or potato virus X (PVX)-based viral vectors through Golden Gate or Gibson assembly, and delivery to plants through Agrobacterium-mediated expression or by spraying crude extracts containing recombinant PVX expressing the minimal precursors. These methodologies make syn-tasiRNA-based tools more accessible and broadly applicable for plant research and biotechnology across diverse species and experimental contexts.

Banana (Musa spp.) is a globally important horticultural crop that faces significant challenges from pests and diseases, which threaten yield and long-term sustainability. The efficient production of clean, disease-free planting material is essential for both commercial plantations and small-holder systems. This paper presents a rapid and reproducible protocol for direct plant regeneration from immature male inflorescences of banana. The method involves surface sterilization of immature male flowers, longitudinal dissection, and culture on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BAP), enabling direct shoot regeneration from floral meristems without an intermediate regenerable callus phase. This approach offers several advantages over traditional embryogenic cell suspension (ECS) methods, including simplified sterilization, high regeneration efficiency, and scalability. The protocol was successfully applied to multiple banana cultivars, including Cavendish (AAA) and Lady Finger (AAB), achieving 100% shoot regeneration efficiency with plantlet production within 6–8 months. This protocol provides a reliable and efficient alternative for rapid mass propagation of banana plants, supporting sustainable production and research applications.

Protein isolation combined with two-dimensional electrophoresis (2-DE) is a powerful technique for analyzing complex protein mixtures, enabling the simultaneous separation of thousands of proteins. This method involves two distinct steps: isoelectric focusing (IEF), which separates proteins based on their isoelectric points (pI), and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), which separates proteins by their relative molecular weights. However, the success of 2-DE is highly dependent on the quality of the starting material. Isolating proteins from plant mature roots is challenging due to interfering compounds and a thick, lignin-rich cell wall. Bacterial proteins and metabolites further complicate extraction in legumes, which form symbiotic relationships with bacteria. Endogenous proteases can degrade proteins, and microbial contaminants may co-purify with plant proteins. Therefore, comparing extraction methods is essential to minimize contaminants, maximize yield, and preserve protein integrity. In this study, we compare two protein isolation techniques for lupine roots and optimize a protein precipitation protocol to enhance the yield for downstream proteomic analyses. The effectiveness of each method was evaluated based on the quality and resolution of 2-DE gel images. The optimized protocol provides a reliable platform for comparative proteomics and functional studies of lupine root responses to stress, e.g., drought or salinity, and symbiotic interactions with bacteria.