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Microscopy Techniques in Plant Studies—2nd Edition
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Microscopy Techniques in Plant Studies—2nd Edition

A special issue of Plants (ISSN 2223-7747). This special issue belongs to the section "Plant Development and Morphogenesis".

Deadline for manuscript submissions: closed (31 March 2026) | Viewed by 9518

Special Issue Editors

Dr. Emilio de Castro Miguel

E-Mail Website
Guest Editor
Department of Metallurgical and Materials, Federal University of Ceará, Fortaleza, Brazil
Interests: microscopy; plant ultrastructure; cell biology; nanomaterials
Special Issues, Collections and Topics in MDPI journals
Dr. Maura Da Cunha

E-Mail Website
Guest Editor
Campos dos Goitacazes, Universidade Estadual do Norte Fluminensedisabled, Campos dos Goytacazes, Brazil
Interests: plant science; microscopy
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Plant biology studies include several approaches, ranging from gene expression experiments to the evaluation of plant growth when in contact with nanostructured materials. Undoubtedly, all studies contribute to the accumulation of knowledge in plant physiology, anatomy, and ultrastructure. While many techniques can be used to advance research, microscopy techniques can be applied to solve problems in many fields of plant biology and are extremely versatile.

Recently, the investigation of plants using microscopy has surpassed traditional techniques such as light and scanning electron microscopy and has even focused on previously unapplied techniques, such as Raman microscopy, SIMS microscopy, focused ion beam, and others.

With the aim of strengthening the scientific basis for plant science, we welcome articles that enhance our understanding of all aspects of plant biology and that include microscopic investigation. Review articles are also welcome.

Dr. Emilio de Castro Miguel
Dr. Maura Da Cunha
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 250 words) can be sent to the Editorial Office for assessment.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Plants is an international peer-reviewed open access semimonthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2700 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • plant microscopy
  • plant cell
  • ultrastructure cell wall
  • plant anatomy
  • nanotechnology

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Published Papers (7 papers)

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Research

24 pages, 13240 KB  
Article
Teliosporogenesis of the Peanut Smut Fungus Thecaphora frezzii in Arachis hypogaea: A Correlative Multiscale Microscopy Study
by María Florencia Romero, Orlando F. Popoff, Guillermo J. Seijo and Ana Maria Gonzalez
Plants 2026, 15(5), 841; https://doi.org/10.3390/plants15050841 - 9 Mar 2026
Viewed by 1225
Abstract
The smut fungus Thecaphora frezzii causes severe yield losses in peanuts (Arachis hypogaea) in Argentina. Previous work established its fully intracellular biotrophic progression through subterranean organs and its exclusive sporulation within the seed coat, yet the ontogeny of teliospore formation in [...] Read more.
The smut fungus Thecaphora frezzii causes severe yield losses in peanuts (Arachis hypogaea) in Argentina. Previous work established its fully intracellular biotrophic progression through subterranean organs and its exclusive sporulation within the seed coat, yet the ontogeny of teliospore formation in planta remained unresolved. Here, we applied a pragmatic correlative multiscale microscopy approach based on serial paraffin sections examined by stereomicroscopy, light microscopy, confocal laser scanning microscopy, and scanning electron microscopy, enabling spatial correlation of fungal structures within their tissue context. Using this integrative framework, we characterized the organization and progression of sporogenic structures associated with teliosporogenesis. Teliosporogenesis proved to be tightly synchronized with host tissue context and seed developmental stage, and was consistently preceded by a marked reorganization of sporogenous hyphae into three-dimensional coiled hyphal aggregates embedded in a mucilaginous matrix. These precursors undergo hyphal fragmentation followed by central–peripheral differentiation, whereby a small number of central units enlarge and individualize into teliospore initials while peripheral elements collapse, yielding stable teliospore balls as the final sporogenic product. This developmental sequence defines a distinct ontogenetic pattern not captured by current schemes of sporogenesis, here designated the Teliospore-ball type. Our results clarify the developmental pathways of T. frezzii sporulation in planta and demonstrate how accessible multiscale microscopy can be used to integrate structural information across spatial scales in complex plant–fungus interactions. Full article
(This article belongs to the Special Issue Microscopy Techniques in Plant Studies—2nd Edition)
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26 pages, 6855 KB  
Article
Plant Microtechnique with Resin: Towards Plant Histolomics
by Ivan T. Cerritos-Castro, Araceli Patrón-Soberano and Ana Paulina Barba-de la Rosa
Plants 2026, 15(4), 643; https://doi.org/10.3390/plants15040643 - 19 Feb 2026
Viewed by 807
Abstract
Plant microtechnique involves a sequence of skill-intensive histological procedures that often yield poorly reproducible images and limited quantitative information. Nevertheless, it provides an essential cellular and tissue context needed to understand biological functions. In this work, we present an optimized resin-based microtechnique that [...] Read more.
Plant microtechnique involves a sequence of skill-intensive histological procedures that often yield poorly reproducible images and limited quantitative information. Nevertheless, it provides an essential cellular and tissue context needed to understand biological functions. In this work, we present an optimized resin-based microtechnique that replaces paraffin embedding, incorporates a chemically activated adhesive treatment for glass slides, and develop a trichrome stain for resin sections. All these improvements enhanced section stability and image reproducibility, enabled a broader color palette with sharp contrast of tissues, cells and organelles, and selected ultrastructural features using light microscopy. Based on these preparations, a quantitative micrograph analysis workflow was developed based on image segmentation and feature extraction using MATLAB (R2024a) and Adobe Photoshop (CS6). This approach enables the measurement of a wide range of morphometric and compositional features, generating structured histological datasets that we refer to as plant histolomes. As an illustrative application, this workflow was applied to leaves from several model plants species and integrated multiple anatomical traits into a composite feature, the “C4 Kranz-anatomy level”, enabling quantitative comparison along the C3-C4 anatomical transition. The resin-based microtechnique and the histolomics framework developed in this work provide a robust and reproducible basis for the quantitative plant histology, bridging classical microscopy with a data-driven tissue analysis. Full article
(This article belongs to the Special Issue Microscopy Techniques in Plant Studies—2nd Edition)
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21 pages, 4277 KB  
Article
Microfluidic Interrogation of Chitin-Induced Calcium Oscillations in the Moss Physcomitrium patens
by Vanessa Kamara, James Teague, Kathryn E. Pagano, Luis Vidali and Dirk R. Albrecht
Plants 2026, 15(4), 582; https://doi.org/10.3390/plants15040582 - 12 Feb 2026
Viewed by 688
Abstract
Plants defend against pathogens such as fungi by initiating coordinated structural and chemical responses. Pathogen perception triggers rapid cytosolic calcium influx and calcium oscillations that drive defense gene expression, yet the mechanisms by which these signals encode stressor intensity and propagate systematically remain [...] Read more.
Plants defend against pathogens such as fungi by initiating coordinated structural and chemical responses. Pathogen perception triggers rapid cytosolic calcium influx and calcium oscillations that drive defense gene expression, yet the mechanisms by which these signals encode stressor intensity and propagate systematically remain unclear. Here, we present a microfluidic system to characterize intracellular calcium dynamics in protonemal colonies of the moss Physcomitrium patens (Hedw.) upon precise and reversible exposure to fungal chitin oligosaccharides. Epifluorescent imaging of cells expressing the calcium indicator GCaMP6f revealed a rapid, coordinated calcium response to chitin addition, followed by stereotyped oscillations that subsided quickly upon stimulus removal. We implemented an unbiased image segmentation algorithm using pixel-based k-means clustering to automatically locate regions with specific oscillatory signatures. Calcium dynamics were distinct across adjacent cells, distinguishable by cell type, and significantly modulated by circadian rhythm, adaptation time within the device, and stimulus timing. Cytosolic calcium oscillations, which rose and fell symmetrically within about 60 s, occurred spontaneously during the subjective night and following short adaptation periods. Chitin elicited strong oscillations with increased frequency, amplitude, and duration, and repeated pulses entrained regular, colony-wide oscillations at the stimulation interval. This study complements prior investigations of whole plant and growth tip dynamics and provides a quantitative framework to study calcium signaling in plants, including mechanisms of signal propagation and the role of oscillation frequency on gene expression. Full article
(This article belongs to the Special Issue Microscopy Techniques in Plant Studies—2nd Edition)
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14 pages, 7343 KB  
Article
Symbiotic Germination in Cattleya purpurata: An Ultrastructural Journey from Fungal Dependence to Autotrophy
by Eliana de Medeiros Oliveira, Kelly Besen, Lucas Camargo dos Santos, Mateus Felipe Uller, Paulo Emilio Lovato, Miguel Pedro Guerra and Juliana Lischka Sampaio Mayer
Plants 2026, 15(4), 543; https://doi.org/10.3390/plants15040543 - 10 Feb 2026
Viewed by 1383
Abstract
Orchids depend on mycorrhizal fungi for seed germination, a critical process especially for endangered species such as Cattleya purpurata. This study elucidates the ultrastructural ontogeny of the symbiosis between C. purpurata and the fungus Tulasnella sp. We demonstrate a defined spatiotemporal colonization [...] Read more.
Orchids depend on mycorrhizal fungi for seed germination, a critical process especially for endangered species such as Cattleya purpurata. This study elucidates the ultrastructural ontogeny of the symbiosis between C. purpurata and the fungus Tulasnella sp. We demonstrate a defined spatiotemporal colonization pattern: hyphae penetrate exclusively via suspensor cells, migrate through the basal region of the embryo, and only then colonize the apical region. Upon colonization, the fungus triggers changes in the embryonic cells, including nuclear hypertrophy and peloton formation. Ultrastructural analysis revealed a sequence of fungal degradation, from intact hyphae to senescent hyphae containing myelin-like bodies and an electron-dense cytoplasm, suggesting that programmed senescence precedes peloton digestion. This supports the novel hypothesis of active fungal participation in modulating its own digestion, challenging classical models. Simultaneously, embryonic cells exhibited rapid metabolic conversion, with the transition from proplastids to amyloplasts, and then to chloroplasts in less than 20 days, marking the onset of autotrophy. This integrated morphological study not only expands fundamental knowledge about symbiotic development in orchids but also provides an optimized protocol for producing symbiotic seedlings, offering a direct tool for the reintroduction and conservation of this species. Full article
(This article belongs to the Special Issue Microscopy Techniques in Plant Studies—2nd Edition)
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16 pages, 2391 KB  
Article
Effect of the Cytokinin Type in the Culture Medium on the Ultrastructure of Leaf Chloroplasts and Photosynthetic Pigment Content of In Vitro Apple (Malus x domestica Borkh.) Shoots
by Zsuzsa Máthéné Szigeti, Katalin Solymosi, Richárd Kovásznai-Oláh and Judit Dobránszki
Plants 2026, 15(2), 223; https://doi.org/10.3390/plants15020223 - 11 Jan 2026
Viewed by 542
Abstract
Thidiazuron, 6-benzylaminopurine riboside, and meta-topolin are cytokinins often used in apple tissue cultures. Three different CK-containing Murashige and Skoog media were used during the experiments: medium without CK or media containing 4.5 μM thidiazuron, 4.5 μM 6-benzylaminopurine riboside, or 4.5 μM meta-topolin, respectively. [...] Read more.
Thidiazuron, 6-benzylaminopurine riboside, and meta-topolin are cytokinins often used in apple tissue cultures. Three different CK-containing Murashige and Skoog media were used during the experiments: medium without CK or media containing 4.5 μM thidiazuron, 4.5 μM 6-benzylaminopurine riboside, or 4.5 μM meta-topolin, respectively. Comparative ultrastructural studies across cytokinin types and apple cultivars were lacking. We studied the changes in photosynthetic pigment content of the leaves with absorption spectroscopy and chloroplast structure with light and transmission electron microscopy. At the light microscopy level, large changes were detected in the length and length-to-width ratios of the chloroplasts in the spongy and palisade mesophyll cell sections in 6-benzylaminopurine riboside- and meta-topolin-treated leaves of the McIntosh scion. In the chloroplasts of the McIntosh plants treated with 6-benzylaminopurine riboside and meta-topolin, and Húsvéti rozmaring leaves treated with meta-topolin, the diameter of grana increased. In both cultivars, thidiazuron caused the height of grana to increase. Thidiazuron and 6-benzylaminopurine riboside influenced leaf anatomy both in the Húsvéti rozmaring and McIntosh cultivars. 6-benzylaminopurine riboside and thidiazuron treatments reduced the content of photosynthetic pigments in the in vitro leaves of both cultivars. In contrast, meta-topolin treatment had no significant effect on the chlorophyll content as compared to the control. Differences were observed not only among the effects of cytokinins, but even between the two apple scions examined. In in vitro apple shoot cultures, TOP maintained chloroplast integrity and pigment content, whereas TDZ exerted stress-like effects. Full article
(This article belongs to the Special Issue Microscopy Techniques in Plant Studies—2nd Edition)
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24 pages, 16019 KB  
Article
Localization of Hydrogen Peroxide in Dormant Buds of Resistant and Susceptible Chestnut Cultivars: Changes During Gall Developmental Stages Induced by the Asian Chestnut Gall Wasp (Dryocosmus kuriphilus)
by Başak Müftüoğlu and Cevriye Mert
Plants 2025, 14(14), 2089; https://doi.org/10.3390/plants14142089 - 8 Jul 2025
Viewed by 1003
Abstract
Asian chestnut gall wasp (ACGW) (Dryocosmus kuriphilus Yasumatsu), native to China, is an invasive pest that causes significant economic losses in Castanea species. While some cultivars show full resistance by inhibiting insect development in buds, the underlying defense mechanisms remain unclear. In [...] Read more.
Asian chestnut gall wasp (ACGW) (Dryocosmus kuriphilus Yasumatsu), native to China, is an invasive pest that causes significant economic losses in Castanea species. While some cultivars show full resistance by inhibiting insect development in buds, the underlying defense mechanisms remain unclear. In this study, the accumulation and distribution of hydrogen peroxide (H2O2) were investigated in dormant buds of chestnut cultivars that are resistant and susceptible to D. kuriphilus by using the 3,3′-diaminobenzidine (DAB) staining method. Buds were examined under a stereomicroscope during key stages of pest development, including oviposition, transition from egg to larva, gall induction, and gall development. Baseline levels of H2O2 were detected in all buds; however, these levels varied among cultivars, with resistant cultivars exhibiting lower basal levels. The degree of H2O2 accumulation was found to vary depending on plant–insect interaction, physiological processes, and cultivar-specific traits. Histochemical staining revealed that brown spots indicative of H2O2 accumulation were concentrated in the vascular bundles of leaf primordia and in the apical regions. In resistant hybrid cultivars, the defense response was activated at an earlier stage, while in resistant Castanea sativa Mill. cultivars, the response was delayed but more robust. Although consistently high levels of H2O2 were observed throughout the pest interaction in susceptible cultivars, gall development was not inhibited. During the onset of physiological bud break, increased H2O2 accumulation was observed across all cultivars. This increase was associated with endodormancy in susceptible cultivars and with both defense mechanisms and endodormancy processes in resistant cultivars. These findings highlight the significant role of H2O2 in plant defense responses, while also supporting its function as a multifunctional signaling molecule involved in gall development and the regulation of physiological processes. Full article
(This article belongs to the Special Issue Microscopy Techniques in Plant Studies—2nd Edition)
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14 pages, 2640 KB  
Article
Image-Based Quantitative Analysis of Epidermal Morphology in Wild Potato Leaves
by Ulyana S. Zubairova, Ivan N. Fomin, Kristina A. Koloshina, Alisa I. Barchuk, Tatyana V. Erst, Nadezhda A. Chalaya, Sophia V. Gerasimova and Alexey V. Doroshkov
Plants 2024, 13(21), 3084; https://doi.org/10.3390/plants13213084 - 1 Nov 2024
Cited by 3 | Viewed by 2503
Abstract
The epidermal leaf patterns of plants exhibit remarkable diversity in cell shapes, sizes, and arrangements, driven by environmental interactions that lead to significant adaptive changes even among closely related species. The Solanaceae family, known for its high diversity of adaptive epidermal structures, has [...] Read more.
The epidermal leaf patterns of plants exhibit remarkable diversity in cell shapes, sizes, and arrangements, driven by environmental interactions that lead to significant adaptive changes even among closely related species. The Solanaceae family, known for its high diversity of adaptive epidermal structures, has traditionally been studied using qualitative phenotypic descriptions. To advance this, we developed a workflow combining multi-scale computer vision, image processing, and data analysis to extract digital descriptors for leaf epidermal cell morphology. Applied to nine wild potato species, this workflow quantified key morphological parameters, identifying descriptors for trichomes, stomata, and pavement cells, and revealing interdependencies among these traits. Principal component analysis (PCA) highlighted two main axes, accounting for 45% and 21% of variance, corresponding to features such as guard cell shape, trichome length, stomatal density, and trichome density. These axes aligned well with the historical and geographical origins of the species, separating southern from Central American species, and forming distinct clusters for monophyletic groups. This workflow thus establishes a quantitative foundation for investigating leaf epidermal cell morphology within phylogenetic and geographic contexts. Full article
(This article belongs to the Special Issue Microscopy Techniques in Plant Studies—2nd Edition)
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