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The authors thank ITQB NOVA (NOVA University of Lisbon, Oeiras, Portugal) and GREEN-IT Research Unit for access to greenhouse facilities and supporting infrastructure. The authors also thank Semillas Fit&#243; for kindly providing the tomato seeds used in this study, with special thanks to Dr. Narv&#225;ez (Seed Technologist, Semillas Fit&#243;). This work was supported by FCT - Funda&#231;&#227;o para a Ci&#234;ncia e a Tecnologia, I.P., through Green-it Bioresources for Sustainability R&#38;D Unit (UID/04551/2025, DOI: 10.54499/UID/04551/2025; UID/PRR/04551/2025, DOI: 10.54499/UID/PRR/04551/2025) and LS4FUTURE Associated Laboratory (LA/P/0087/2020, DOI: 10.54499/LA/P/0087/2020).

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	<li>Wassermann B, Cernava T, M&#252;ller H, Berg C, Berg G. Seeds of native alpine plants host unique microbial communities embedded in cross-kingdom networks. Microbiome. 2019;7(1):108.</li>
	<li>Ding JYM, Ho LS, Ibrahim J, Teh CK, Goh KM. Impact of sterilization and chemical fertilizer on the microbiota of oil palm seedlings. Front Microbiol. 2023;14:1091755.</li>
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	<li>Links MG, et al. Simultaneous profiling of seed-associated bacteria and fungi reveals antagonistic interactions between microorganisms within a shared epiphytic microbiome on Triticum and Brassica seeds. New Phytol. 2014;202(2):542-53.</li>
	<li>Compant S, et al. The plant endosphere world&#8212;bacterial life within plants. Environ Microbiol. 2021;23(4):1812-29.</li>
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	<li>Amann RI, Ludwig W, Schleifer KH. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev. 1995;59(1):143-69.</li>
	<li>Yu Y, Chen Z, Xie H, Feng X, Wang Y, Xu P. Overhauling the effect of surface sterilization on analysis of endophytes in tea plants. Front Plant Sci. 2022;13:849658.</li>
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	<li>Niza-Costa M, et al. Geographically disperse, culturable seed-associated microbiota in forage plants of alfalfa (Medicago sativa L.) and pitch clover (Bituminaria bituminosa L.): Characterization of beneficial inherited strains as plant stress-tolerance enhancers. Biology. 2022;11(12):1838.</li>
</ol>

The authors declare that they have no competing financial interests or conflicts of interest related to this work. AI-assisted conceptual illustrations are used in this manuscript.

The results presented here demonstrate that the integration of standardized surface sterilization with controlled seed germination provides a robust framework for the culturomic isolation of seed-associated microbiota. A key outcome of this protocol is the ability to balance effective removal of external contaminants with preservation of endogenous microbial populations. This balance is critical in culture-based studies, where overly stringent sterilization can reduce microbial recovery, while insufficient sterilization ...

Seeds harbor diverse microbial communities composed of bacteria and fungi associated with both internal tissues and external surfaces1,2,3. These microorganisms are increasingly recognized as integral components of plant biology because they can influence plant performance from the earliest developmental stages, including germination, seedling establishment, and responses to biotic and abiotic stress1,2,3. In ecological terms, seeds function not only as propagules for plant reproduction but also as reservoirs of microbial inoculum, making them a critical entry point for understanding how plant-associated microbiomes are initiated and maintained across the plant life cycle1,2,3.
Seed-associated microorganisms may constitute the first microbial inoculum encountered by the emerging plant and can therefore contribute to microbiome assembly during the seed-to-seedling transition1,3,4. This early inoculum may influence the microbial communities that subsequently colonize roots and other plant compartments, with consequences for plant establishment and later plant-microbe interactions1,4. In addition, part of the seed microbiota can be vertically transmitted through reproductive structures such as flowers, ovules, and pollen, allowing microbial populations to persist across plant generations4,5. This intergenerational continuity suggests that some seed-borne microorganisms may consistently contribute to plant fitness and adaptation.
Functionally, seed-associated microorganisms may affect host performance through several mechanisms, including the production of phytohormones, antimicrobial compounds, and other bioactive metabolites that modulate plant development or suppress competitors and pathogens5,6,7. Seed-transmitted microbial populations also appear to coexist through differential resource use and ecological partitioning, suggesting that the seed represents a selective habitat rather than a passive container of microbial diversity7. Collectively, these findings support the idea that seeds host microbial populations that may influence early plant development and represent candidates for downstream functional characterization2,3,5,7.
To investigate seed-associated microorganisms in a functionally informative and experimentally tractable way, it is necessary to recover viable microbial populations from seed material. In this context, microbial isolation provides access to living microorganisms that can subsequently be characterized experimentally and evaluated in downstream assays relevant to plant&#8211;microbe interactions. This is particularly important in seed systems, where seed-associated microorganisms may contribute to early plant establishment and represent candidates for functional screening and future applied use.
The recovery of seed-associated microorganisms is highly sensitive to sample preparation procedures, particularly surface sterilization, tissue processing, and the developmental stage of the sampled material. This sensitivity is especially relevant in seed systems because microbial biomass is often low, and small procedural differences can strongly affect both colony-forming unit counts and isolate composition. Among these variables, surface sterilization is one of the most critical and difficult to optimize. Sterilization is necessary to reduce epiphytic contamination prior to microbial isolation, yet excessive exposure to sterilants can reduce the viability or culturability of endogenous microorganisms, whereas insufficient sterilization may allow surface-associated taxa to dominate subsequent cultures8,9,10. Accordingly, the aim of sterilization is not to maximize chemical exposure, but to apply the mildest treatment that effectively suppresses surface-derived contamination while preserving recoverable internal microorganisms5,11.
Because sterilization efficiency depends on plant species, tissue structure, seed coat properties, and the associated microbial community, protocols should be selected from previously validated methodologies and then adapted cautiously to the system under study8,12,13. In low-biomass materials such as tomato seeds, this balance is particularly important: over-sterilization may sharply reduce microbial recovery, whereas under-sterilization increases the likelihood that fast-growing surface contaminants will dominate the isolates obtained. For this reason, sterilization efficiency should be verified routinely rather than assumed. Plating of the final rinse water provides a practical control for residual external contamination, but this control should always be interpreted together with microbial recovery from seed or seedling homogenates. A protocol that yields sterile rinse controls but little or no recoverable internal microbiota may be unsuitable when the objective is comparative microbial isolation rather than maximal decontamination6,8,10,14.
Meaningful comparisons, therefore, require strong procedural standardization. In low-biomass samples, inconsistencies in washing, transfer, homogenization, dilution, plating, or incubation can alter both total microbial recovery and the apparent representation of individual taxa. This is especially important when comparing dry and germinated material, where methodological variation can easily be mistaken for biological change.
A major limitation of many seed microbiology protocols is that they rely exclusively on dry, non-germinated seeds as starting material. Although this provides a useful baseline for the microbial fraction present before seed activation, it may underestimate microorganisms that are dormant, metabolically inactive, stressed, or initially present at low relative abundance2,15.
In the dry state, seeds represent a physiologically quiescent environment in which both host tissues and associated microorganisms may remain relatively inactive. Consequently, the microbial populations recovered directly from dry seeds may not fully represent those that become more readily recoverable once germination begins.
During germination, seeds undergo water uptake, metabolic reactivation, nutrient mobilization, and tissue reorganization, all of which alter the local environment available to associated microorganisms11,16. These changes can promote microbial growth, alter the relative representation of microbial populations, and facilitate the detection or recovery of taxa that were previously rare or poorly recovered in dry seeds. In addition, radicle emergence creates a new plant compartment that may support microbial proliferation and redistribution, making the seed-to-seedling transition a particularly informative stage for microbiological analysis1,17.
For this reason, parallel processing of dry seeds and germinated seedlings provides a more informative experimental design than analysis of dry seeds alone. Dry seeds establish a baseline for the culturable populations present before activation, whereas germinated material allows comparative assessment of taxa that become more readily recoverable during early development. This approach is particularly useful when the aim is not only to catalog recoverable microorganisms, but also to identify taxa associated with the seed-to-seedling transition and prioritize isolates for downstream characterization (Figure 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/72014/72014fig1.jpg" /> 
Figure 1: Conceptual and procedural overview of the comparative isolation workflow. (A) Conceptual representation of the dry tomato seed condition prior to germination. (B) Conceptual representation of the germinated tomato seed condition during the seed-to-seedling transition. (C) Schematic workflow of the protocol, including seed surface sterilization, rinse-control assessment, direct processing of dry seeds or controlled germination, homogenization of pooled biological replicates, serial dilution and plating, CFU-based quantification, isolate recovery, taxonomic identification, and comparative analysis of dry and germinated material. Critical control points highlighted in the workflow include sterilization efficiency, equivalent biological replicates, and consistent plating and CFU analysis. Panels A and B were prepared as AI-assisted conceptual illustrations for visual representation only. <a href="https://www.jove.com/files/ftp_upload/72014/72014fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Because the protocol is intended to compare microbial recovery from dry and germinated material, strict methodological consistency is essential. All comparisons should be performed using the same seed lot, identical sterilization conditions, the same culture media, and equivalent dilution, plating, and incubation procedures. Without this level of standardization, apparent differences between dry and germinated samples may reflect technical variation rather than biologically meaningful enrichment. This is particularly important in isolation workflows, where the recovered fraction is shaped both by the biological sample and by the selective effects of the experimental procedure itself4,15.
Biological replication is likewise indispensable. Low-abundance taxa may vary among seed batches, and microbial recovery from low-biomass samples is inherently stochastic even under carefully controlled conditions. At least five independent biological replicates per condition are recommended for robust comparison. In this workflow, each biological replicate consists of pooled material from 3&#8211;5 dry seeds or 3&#8211;5 germinated seedlings processed as a single sample. In addition, all manipulations after sterilization should be performed under aseptic conditions to minimize secondary contamination and preserve interpretability of the resulting isolate set.
The duration of germination should also be selected carefully. It must be long enough to permit radicle emergence and early seedling development, but not so prolonged that later plant growth introduces additional ecological complexity unrelated to the immediate seed-to-seedling transition. For tomato, a germination period of up to 5 days under sterile conditions provides a practical window in which the seedling remains closely linked to the seed environment while early microbial activation and redistribution can already occur. Consistent timing across experiments is essential because differences in developmental stage may themselves influence microbial recovery patterns. This workflow is most appropriate for comparative recovery of culturable microbiota from low-biomass seed systems when the aim is to evaluate germination-associated shifts under standardized conditions. It is less suitable for direct estimation of total seed microbiome diversity or for experimental systems in which culture-independent approaches are required to capture the broader microbial community.
The objective of this protocol is to provide a reproducible framework for the isolation of culturable tomato seed-associated microbiota by integrating standardized surface sterilization with controlled seed germination. The method enables comparative analysis of dry seeds and germinated seedlings using equivalent biological replicates, each consisting of pooled material from 3&#8211;5 dry seeds or 3&#8211;5 germinated seedlings, to assess germination-dependent shifts in colony-forming unit (CFU) recovery and taxonomic composition. By minimizing sterilization bias and promoting microbial recovery during the seed-to-seedling transition, this approach facilitates the isolation of microorganisms with potential functional relevance during early seedling development and supports downstream studies of early plant&#8211;microbe interactions.

<table><tbody><tr><td>Agar</td><td>Sigma-Aldrich</td><td>01916-500G</td><td>Component of LB agar medium; 15 g/L.</td></tr><tr><td>Autoclave</td><td>Lab1st&amp;nbsp;</td><td>SV120H</td><td>Used to sterilize instruments/materials or validate aseptic treatment.</td></tr><tr><td>Bacteria DNA extraction kit</td><td>Promocell</td><td>PK-MB708-206-100</td><td>DNA extraction&amp;nbsp;</td></tr><tr><td>Colony counting/recording setup</td><td>OCS</td><td>B079Y5W57N</td><td>Needed to record CFUs, morphotypes, and plate differences; manuscript does not specify counter/model.</td></tr><tr><td>Cryostorage vials</td><td>Sigma-Aldrich</td><td>Z359033</td><td>Implied for storage of glycerol stocks at -80 &amp;deg;C; not named explicitly but needed for preservation step.</td></tr><tr><td>Digital isolate record system</td><td>Not specified in manuscript</td><td>Not applicable</td><td>Used to record isolate metadata: origin, replicate, condition, sterilization treatment, isolation date, plate medium, morphology, identification status.</td></tr><tr><td>Ethanol</td><td>VWR</td><td>83813440</td><td>Used to prepare 70% v/v ethanol solution for seed surface sterilization.</td></tr><tr><td>Filter paper</td><td>Fisher Scientific</td><td>11718772</td><td>Optional alternative germination support.</td></tr><tr><td>Forceps/Tweezers</td><td>Sigma-Aldrich</td><td>930229</td><td>Used to transfer seeds and collect germinated seedlings.</td></tr><tr><td>Genomic DNA extraction kit</td><td>PromoCell</td><td>PK-MB708-206-100</td><td>Used to extract genomic DNA from purified bacterial cultures; exact kit/method not specified.</td></tr><tr><td>Glycerol, sterile 80%</td><td>VWR</td><td>24388364</td><td>Mixed 1:1 v/v with liquid culture for cryostock preparation.</td></tr><tr><td>Laminar flow hood Bio48</td><td>Faster</td><td>LAB-CAP-22</td><td>Required for aseptic seed handling, sterilization transfers, and preparation of sterile materials.</td></tr><tr><td>LB agar medium</td><td>VWR</td><td>846495000</td><td>Standard medium used for comparative culturing; per liter: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, 15 g of agar.</td></tr><tr><td>Microcentrifuge tubes</td><td>Ratiolab</td><td>RATI5615000</td><td>Used for seed sterilization and sample processing.</td></tr><tr><td>MS medium</td><td>Sigma</td><td>M0404-10L</td><td>Mentioned as sterile germination support for axenic germination.</td></tr><tr><td>NZYTaq II 2&amp;times; Master mix</td><td>Nzytech</td><td>MB35803</td><td>PCR mix for amplification</td></tr><tr><td>Parafilm</td><td>Parafilm/Merck</td><td>HS234526A</td><td>Used to seal germination plates while reducing contamination.</td></tr><tr><td>Petri dishes</td><td>Labbox</td><td>PDIP-E9N-500</td><td>Used for germination setup, plating, and handling sterilized seeds.</td></tr><tr><td>Phytoagar</td><td>Duchefa Biochemie</td><td>9002-18-0</td><td>Mentioned as an optional alternative sterile germination support.</td></tr><tr><td>Pipettes set and tips</td><td>Merck</td><td>EP3123000918-1EA</td><td>Used for removing sterilization solutions, washing, dilution, and plating.</td></tr><tr><td>R2A agar medium</td><td>HI-MEDIA</td><td>M1743-500G</td><td>Optional additional medium for extended culturomic workflows.</td></tr><tr><td>Sodium chloride, NaCl</td><td>Fisher Chemical</td><td>10040460</td><td>Used to prepare sterile 0.45% NaCl for homogenization and serial dilutions; also part of LB medium.</td></tr><tr><td>Sodium hypochlorite</td><td>Carlo Erba</td><td>370323</td><td>Used to prepare 20% v/v sodium hypochlorite solution for seed surface sterilization. Corrosive; requires PPE.</td></tr><tr><td>Sterile disposable pestles</td><td>Eppendorf</td><td>EP0030120973</td><td>Used for homogenization of dry seeds and germinated seedlings.</td></tr><tr><td>Vortex mixer</td><td>Fisher Scientific</td><td>15901137</td><td>Used to resuspend microbial cells from homogenates.</td></tr></tbody></table>

isolation of tomato seed-associated microbiota through controlled germination and comparative culturing

Isolation of Tomato Seed-Associated Microbiota Through Controlled Germination and Comparative Culturing

Surface sterilization is a critical yet frequently under-optimized step in studies aiming to isolate seed-associated microorganisms, as excessively harsh treatments can reduce or distort endogenous microbial populations. This protocol presents a reproducible approach for isolating culturable tomato seed-associated microbiota using surface-sterilization conditions based on previously validated methodologies, combined with controlled seed germination. Rather than defining universally optimal sterilization treatments, the approach applies standardized conditions that can be adjusted according to seed characteristics while minimizing bias and preserving endogenous microbial populations.
To enhance recovery of culturable microorganisms associated with the seed-to-seedling transition, the protocol incorporates a controlled germination step prior to microbial isolation. In contrast to conventional approaches based on the maceration of dry, non-germinated seeds, controlled germination promotes microbial activation during the seed-to-seedling transition under sterile conditions. Comparative colony-forming unit (CFU)-based analyses of equivalent biological replicates, each consisting of pooled material from 3&#8211;5 dry seeds or 3&#8211;5 germinated seedlings, revealed a marked shift in the culturable community following germination. Genera that were weakly represented in dry seeds became enriched after germination, whereas other taxa that were more abundant in dry seeds decreased in relative representation, supporting studies of early plant-microbe interactions during seedling establishment.
Across biological replicates, genera including Stutzerimonas, Stenotrophomonas, and Priestia were consistently enriched among isolates recovered from germinated seedlings, whereas Paenibacillus remained abundant across conditions but showed marked shifts in species-level composition. By integrating standardized sterilization conditions with controlled germination and comparative analysis of dry and germinated material, this protocol provides a robust and reproducible framework for the culturomic isolation of seed-associated microbiota with potential functional relevance during early seedling development and supports studies of early plant&#8211;microbe interactions during seedling establishment.

Performance of surface sterilization and control of external contamination 
Surface sterilization using standardized conditions based on previously validated methodologies resulted in effective reduction of external microbial contamination while preserving the recovery of culturable microorganisms from seed-derived material. Plating of the final rinse water showed no detectable colony growth, indicating effective removal of surface-associated microorganisms under the conditions used.
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This protocol combines standardized seed surface sterilization with controlled germination to enable the reproducible isolation of tomato seed-associated microbiota during the seed-to-seedling transition. The approach enhances recovery of culturable microorganisms that are underrepresented in dry seeds and supports comparative culturomic studies during early seedling development.

Surface sterilization is a critical yet frequently under-optimized step in studies aiming to isolate seed-associated microorganisms, as excessively harsh ...

isolation-tomato-seed-associated-microbiota-through-controlled

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Source: Hassan, J. A., et al. High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay. J. Vis. Exp. (2020).This video demonstrates a method to screen for bacterial resistance in different tomato strains using ten-day-old seedlings inoculated with a bacterial suspension. It explains how bacteria invade through stomata, deliver effector proteins to suppress plant immunity, and how plant responses differ between susceptible and resistant...

Seedling Flood Assay for Screening Bacterial Resistance in Tomatoes

Source: Nathoo, N., et al. A Hydroponic Co-cultivation System for Simultaneous and Systematic Analysis of Plant/Microbe Molecular Interactions and Signaling. J. Vis. Exp. (2017).This video demonstrates a hydroponic co-cultivation system for studying plant–bacteria interactions under sterile conditions. Plant seedlings are transferred into a sterile, acidic growth medium, incubated, and inspected for contamination before inoculation with Agrobacterium. The activated bacteria attach to plant...

A Co-Cultivation System to Study the Molecular Responses of Plant Hosts and Interacting Bacteria

Endophytic bacteria colonize plant tissues and promote plant growth.To study their impact on tomato plant growth under salt stress, start with sterile seedlings showing developing leaves and roots.Immerse the roots of the experimental seedling in a suspension of endophytic bacteria; leave the control uninoculated.Transfer both groups into sterilized soil to prevent external microbial interference and incubate in a growth chamber.From the soil, the bacteria enter through transient root openings,...

Enhancing Salt Stress Tolerance in Tomato Plants Through Endophytic Bacterial Colonization

Source: Matthysse, A. G. Adherence of Bacteria to Plant Surfaces Measured in the Laboratory. J. Vis. Exp., (2018)This video demonstrates a protocol for measuring root colonization of tomato seedlings by wild-type and cellulose-deficient pathogenic bacteria. Viable counts of tightly adhering bacteria show greater adhesion and microcolony formation by the wild-type strain, highlighting cellulose’s role in stable root colonization.

Assessing Root Colonization of Tomato Seedlings by a Pathogenic Bacterium Using Viable Cell Counts

An easy method for measuring and characterizing bacterial adhesion to plants, particularly roots and sprouts, is described in this article.

Adherence of Bacteria to Plant Surfaces Measured in the Laboratory

Here, we present a versatile method for tomato root transformation followed by inoculation with Ralstonia solanacearum to perform straightforward genetic analysis for the study of bacterial wilt disease.

Tomato Root Transformation Followed by Inoculation with Ralstonia Solanacearum for Straightforward Genetic Analysis of Bacterial Wilt Disease

The seedling flood assay facilitates rapid screening of wild tomato accessions for the resistance to the Pseudomonas syringae bacterium. This assay, used in conjunction with the seedling bacterial growth assay, can assist in further characterizing the underlying resistance to the bacterium, and can be used to screen mapping populations to determine the genetic basis of resistance.

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

To obtain an axenic insect, its egg surface is sterilized, and the hatched larva is subsequently reared using axenic leaves. This method provides an efficient way for axenic insect preparation without administering antibiotics or developing an artificial diet, which can also be applied to other leaf-eating insects.

Preparing and Rearing Axenic Insects with Tissue Cultured Seedlings for Host-Gut Microbiota Interaction Studies of the Leaf Beetle

This protocol presents a detailed summary of strategies to inoculate plant roots with soil-borne microbes. Exemplified for the fungi Verticillium longisporum and Verticillium dahliae, three different root infection systems are described. Potential applications and possible downstream analyses are highlighted, and advantages or disadvantages are discussed for each system.

Inoculation Strategies to Infect Plant Roots with Soil-Borne Microorganisms

This protocol aims to provide detailed procedures for collecting, fixing, and maintaining mycoheterotrophic plant samples, applying different microscopy techniques such as scanning and transmission electron microscopy, light, confocal, and fluorescence microscopy to study fungal colonization in plants tissues and seeds germinated with mycorrhizal fungi.

Microscopy Techniques for Interpreting Fungal Colonization in Mycoheterotrophic Plants Tissues and Symbiotic Germination of Seeds

The tomato seed is an important model for studying genetics and developmental biology during plant reproduction. This protocol is useful for clearing tomato seeds at different developmental stages to observe the finer embryonic structure.

An Efficient Clearing Protocol for the Study of Seed Development in Tomato (Solanum lycopersicum L.)

iPlantMicro Lab..  This protocol combines standardized seed surface sterilization with controlled germination to enable the reproducible isolation of tomato seed-associated microbiota during the seed-to-seedling transition. The approach enhances recovery of culturable microorganisms that are underrepresented in dry seeds and supports comparative culturomic studies during early seedling development.

Isolation of Tomato Seed-Associated Microbiota Through Controlled Germination and Comparative Culturing

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