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. 2026 Feb 10;15(4):543. doi: 10.3390/plants15040543

Symbiotic Germination in Cattleya purpurata: An Ultrastructural Journey from Fungal Dependence to Autotrophy

Eliana de Medeiros Oliveira 1,2,3,*, Kelly Besen 4, Lucas Camargo dos Santos 4, Mateus Felipe Uller 4, Paulo Emilio Lovato 4, Miguel Pedro Guerra 4, Juliana Lischka Sampaio Mayer 3
Editors: Emilio de Castro Miguel, Maura Da Cunha
PMCID: PMC12944015  PMID: 41754250

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 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.

Keywords: Orchidaceae, symbiotic germination, protocorm, ontogeny, Cattleya purpurata, Tulasnella

1. Introduction

Orchids possess intrinsic morphological and anatomical characteristics that limit their distribution and establishment. Among these, the aggregation of pollen into pollinia, an adaptation to zoophilous cross-pollination, and the production of vast quantities of microscopic, endosperm-less seeds with minimal nutritional reserves in the embryo stand out [1,2]. Consequently, in nature, seed germination and the formation of the protocormo, a structure unique to orchids, obligatorily depend on the nutritional input provided by specific mycorrhizal fungi [3,4]. This initial mycoheterotrophic phase is a critical bottleneck in the orchid life cycle and the establishment of new generations, involving complex cellular interactions that remain underexplored at the ultrastructural level, particularly in epiphytic species.

The literature on orchid mycorrhizal fungi (OMFs) has historically focused on terrestrial species from the Global North [5,6,7,8], while information on tropical epiphytes is comparatively scarcer [9,10,11]. Studies on terrestrial orchids reveal not only a high degree of specificity for fungal partners [12] but also stage-specific mycobionts during development [13]. In contrast, association networks in epiphytic orchids appear more conserved and show stronger evidence of coevolution with their mycobionts, possibly as an adaptation to the more stressful epiphytic habitat [14]. Among OMFs, which constitute an artificial, non-monophyletic group within the Basidiomycota characterized by features such as 90° hyphal branching and monilioid cells [15,16,17,18], the genus Tulasnella is particularly notable for its broad capacity to promote germination across diverse clades within the Orchidaceae family [19,20].

The successful transition from a mycoheterotrophic protocorm to a photosynthetic seedling hinges on finely tuned symbiotic compatibility, which determines nutrient transfer efficiency and ultimately influences seedling survival in natural habitats [21,22,23]. Recent studies are beginning to unravel the metabolic dynamics of this transition, highlighting strategies such as the mobilization of scant embryonic lipid reserves and sophisticated nutrient transfer mechanisms [24,25]. These specific symbiotic dependencies, coupled with broader threats like habitat loss and climate change, render orchids especially vulnerable [26,27].

Cattleya purpurata (Lindl. & Paxton) Van den Berg, an epiphytic orchid endemic to the Brazilian Atlantic Forest, exemplifies this vulnerability. The species faces the common ecological challenges of orchids, compounded by anthropogenic pressures such as illegal collection and habitat destruction, which have led to its classification as a threatened species on the official state lists of both Santa Catarina [28] and São Paulo [29]. A detailed understanding of its symbiotic germination process is therefore not only a question of orchid biology but also a critical step towards developing effective conservation tools, such as the production of resilient symbiotic seedlings for species reintroduction programs.

Given this context, the present study aimed to provide a detailed ultrastructural analysis of symbiotic germination in C. purpurata. Our specific objectives were to: (1) elucidate the spatiotemporal pattern of colonization and cellular interaction between the embryo and protocorm, and its Tulasnella mycobiont; (2) document the sequence of ultrastructural alterations in both symbionts, with a focus on the processes of peloton digestion and the metabolic transition to autotrophy; and (3) generate a protocol for symbiotic protocorm production to provide direct support for the conservation and reintroduction of this species into nature.

2. Materials and Methods

To study the ontogeny of the symbiotic interaction between fungus, embryo, and protocorm, seeds of Cattleya purpurata were surface-sterilized and cultivated on oat-agar culture medium plates, where a mycelial disk of the fungus Tulasnella (CBMAI 3011, from the Coleção Brasileira de Microrganismos do Meio Ambiente e da Indústria (CBMAI) at the Universidade Estadual de Campinas (UNICAMP), Campinas, SP, Brazil) was added.

2.1. Fungal Isolate and Seed Acquisition

2.1.1. Fungal Isolation

Isolates of the fungus Tulasnella sp. were obtained using the seed baiting technique adapted from [30]. Seeds of C. purpurata were placed on moist sponges inside voile bags and installed near the roots of adult plants in their natural habitat for 180 days. Germinated protocorms were collected, surface-disinfected (70% ethanol (Merck, Darmstadt, Germany) for 2 min, followed by 2% NaClO (Sigma-Aldrich, St. Louis, MO, USA) for 5 min), and plated on Potato Dextrose Agar (PDA) (Kasvi, São José dos Pinhais, PR, Brazil), modified Norman-Merlin Medium (MNM) (prepared from ammonium nitrate (Sigma-Aldrich), magnesium sulfate (Sigma-Aldrich), monopotassium phosphate (Sigma-Aldrich), glucose (Sigma-Aldrich), and agar (Kasvi)), and Malt Extract Agar (Kasvi) for the isolation of mycorrhizal fungi. Cultures were incubated in darkness at 23 °C. Emerging mycelium was subcultured and maintained on PDA for the symbiotic germination experiment.

2.1.2. Seed Acquisition and Preparation

Seed capsules were obtained through manual cross-pollination of C. purpurata [31]. After six months, capsules were harvested, and seeds were extracted aseptically in a laminar flow hood. Embryo viability was confirmed using the triphenyl tetrazolium chloride (TTC) test [32]. For germination, seed lots were surface-disinfected (2% NaClO (Sigma-Aldrich) for 5 min), rinsed thoroughly, and suspended in sterilized distilled water.

2.2. Symbiotic Germination Assay

For the symbiotic germination assay, a mycelial disk (2 mm in diameter) of the Tulasnella sp. isolate was added to oat-agar medium (Kasvi). The seed suspension (400 µL) was distributed over the medium surface. A total of 62 plates were incubated in growth chambers (BOD) at 23 °C with a 12 h photoperiod. Over 20 days of co-culture, developing protocorms were collected daily and processed immediately for microscopy analyses.

2.3. Laser Scanning Confocal Microscopy

Materials were fixed in 4% paraformaldehyde (Sigma-Aldrich) in 0.1 M sodium phosphate buffer (pH 7.2), prepared from sodium phosphate salts (Sigma-Aldrich), and stained with acid fuchsin (Sigma-Aldrich) for 1 min. Hyphae visualization was possible due to fuchsin’s red fluorescence (excitation 514 nm, emission 600–640 nm), while plant cell walls were identified by their blue autofluorescence (excitation 405 nm, emission 460–490 nm). Images were acquired using a Leica SP5 laser scanning confocal microscope (Leica Microsystems, Wetzlar, Germany) at the Laboratório Central de Microscopia Eletrônica (LCME), Universidade Federal de Santa Catarina (UFSC), with a z-volume of 40 µm and z-stacks of 3 µm. All images were overlaid (maximum projection) to obtain a composite image showing all fluorescing organisms and structures across the three scanning axes (x, y, and z). Image processing was performed using Leica Microsystems® LAS AF Lite software (4.0) at the Laboratório Multiusuário de Estudos Biológicos (LAMEB), UFSC.

2.4. Light Microscopy and Transmission Electron Microscopy (TEM)

The samples underwent a fixation stage in 3% glutaraldehyde (Electron Microscopy Sciences [EMS], Hatfield, PA, USA) in 0.1 M sodium cacodylate buffer (pH 7.2), prepared from sodium cacodylate (EMS), for 24 h. This was followed by post-fixation in 1% osmium tetroxide (EMS) (2 h) and dehydration in an increasing acetone series (30–100%) (Sigma-Aldrich, St. Louis, MO, USA). They were then embedded in SPURR resin (EMS) and polymerized at 70 °C. Semi-thin sections stained with toluidine blue (Sigma-Aldrich) were analyzed using an Olympus BX41 microscope (Olympus Corporation, Nagano, Japan) at the Núcleo de Estudos da Uva e do Vinho (NEUVIN), Centro de Ciências Agrárias (CCA), UFSC. Ultra-thin sections were contrasted with uranyl acetate (EMS) and lead citrate (EMS) before examination with a JEOL JEM-1011 TEM (Jeol Ltd., Tokyo, Japan) at the LCME, UFSC.

2.5. AI Assistance in Graphical Abstract Preparation

The graphical abstract for this manuscript was partially developed with the assistance of generative artificial intelligence (GenAI) tools, specifically ChatGPT 5.2. This tool was utilized for generating and refining visual elements and descriptive textual components. All AI-generated graphical abstract content was meticulously reviewed and edited, and full responsibility for the final representation lies with the human authors.

3. Results

3.1. Morphology and Ultrastructure of the Non-Germinated Seed

Seeds of C. purpurata exhibited an ovoid shape, enveloped by a thin testa composed of thickened cell walls (Figure 1A). The embryo proper exhibited polarity along the longitudinal axis (Figure 1A). At the basal end, the suspensor region was located, consisting of a few cells with a voluminous central vacuole (Figure 1B). The embryo properly displayed two distinct ultrastructural regions: a more basal portion, with very electron-dense cytoplasms, where cells presented many lipid bodies (spherosomes)—this portion was termed the lipid region throughout the study (Figure 1C)—and, in the more apical portion of the embryo, at the pole opposite the suspensor, cells with a more electron-transparent cytoplasm, containing numerous spherical electron-transparent structures (Figure 1D). This region occupied approximately two-thirds of the total embryo volume, positioned adjacent to the lipid region.

Figure 1.

Figure 1

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Light micrograph (A) and transmission electron micrographs (B–D) of Cattleya purpurata. (A) Overview of the seed, composed of the embryo proper, with suspensor cells (su) at the basal region, and enveloped by the testa (te). Note the cells rich in lipid bodies (asterisk), at the base of the embryo. (B) Detail of suspensor cells (su), with the adjacent testa (te). (C) Cells of the ‘lipid region’ (asterisk) showing very electron-dense content. (D) Cells of the apical region (ap), with cytoplasm packed with electron transparent spherical structures.

3.2. Initial Fungal Colonization and Establishment of Symbiosis

Approximately five days after the start of co-culture, hyphae of the fungus Tulasnella sp., which surrounded the seed, were observed penetrating the embryo exclusively through the suspensor region (Figure 2A,B). The hyphae that colonized the suspensor cells and, subsequently, those of the lipid region (Figure 2C), did not form pelotons, i.e., the morphological structure that defines orchid mycorrhiza, characterized by the coiling of hyphae within the host cell cytoplasm. While the hyphae progressed through the cells of the lipid region, the apical region remained free of fungal colonization, with its cells maintaining the vesiculated cytoplasmic appearance (Figure 2D,E).

Figure 2.

Figure 2

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Confocal microscopy images (A,D), and light micrographs (A–C,E) of initial fungal colonization in Cattleya purpurata. (A) Bright field and conjugated fluorescence in confocal microscopy, shows the embryo inside the testa (arrow), with suspensor cells (S) with hyphae labeled in red. (B) Suspensor cells (S) colonized by hyphae (arrowhead), and hyphae outside the testa (arrow). (C) Suspensor and ‘lipid region’ (rl) cells colonized with hyphae (arrowhead) and enveloped by the testa (arrow). (D) Embryo with hyphae fluorescing red (arrowheads), colonizing the suspensor region (S). The apical region (ap) is free of hyphae, the testa (arrow) fluoresces blue. (E) Embryo showing the apical region with cells not colonized by fungus, the arrow indicates the testa.

Between nine and thirteen days of co-culture, the growth of hyphae through the suspensor and lipid region towards the apical region was observed (Figure 3A). Upon reaching the cells of the apical region, the hyphae began to grow intensely and coil within the cytoplasm, thus forming the pelotons characteristic of orchid mycorrhiza (Figure 3B). The formation of pelotons was accompanied by a significant increase in the volume of the host cells (Figure 3C). At this stage, the rupture of the testa, the release of the embryo, which henceforth is called a protocorm, and the initiation of the first rhizoid formation were also recorded (Figure 3D).

Figure 3.

Figure 3

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Bright field (A,B,D) and fluorescence (A,C,D in confocal microscope), of germinating Cattleya purpurata embryos. (A) Hyphae, in red, passing through the suspensor (su) and reaching the apical region of the embryo, forming pelotons (arrowhead). (B) Embryo whose mycorryhized cells (arrowheads) show an increase in volume, rupture the testa (te). (C) Coiled hyphae (arrowheads), forming the peloton and causing the cell volume to increase. (D) Protocorm forming the first rhizoids (arrows).

3.3. Dynamics of the Fungus-Protocorm Interaction and Peloton Digestion

After 16 days of co-culture, the hyphae remained intact in the cells of the suspensor and ‘basal lipid region’, where they elongated and traversed the cytoplasm without forming pelotons (Figure 4A). In contrast, in the cells of the median and apical region of the protocorm, the hyphae formed pelotons. These mycorrhized ground meristem cells exhibited enlarged nuclei, compared to non-colonized cells (Figure 4B). The hyphae located in the cells of the central portion of the protocorm were present at different stages of interaction. Hyphae were observed ranging from intact, with preserved cell walls, to hyphae in various stages of degradation and digestion (Figure 4B,C). Ultrastructural analysis revealed that the digestive process begins with the darkening (more electron-dense cytoplasm) of the fungal cytoplasm (Figure 4C). Concurrently, autophagic vacuoles containing myelin-like structures called myelin-like bodies appeared inside the hyphae undergoing alteration, formed by membranes that rupture and organize into spirals, reminiscent of the morphology of myelin sheaths in animal cells (Figure 4D). At a more advanced stage, the hyphal compartment, still delimited by the fungal cell wall, becomes completely filled with a tangle of small membranes derived from the degradation of the myelin-like bodies (Figure 4E). The final stage of the digestion process was characterized by the presence of amorphous material, with electron-dense regions forming lines, inside the space previously occupied by the hypha (Figure 4F).

Figure 4.

Figure 4

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Transmission electron micrographs (TEM) showing the development of the fungus-protocorm cell interaction, specifically between Tulasnella sp. and Cattleya purpurata. The hyphae (arrowheads) behave differently if they are in cells of the suspensor pole, where they elongate and traverse the cytoplasm (A). Whereas in cells of the median region of the protocorm (B), the hyphae coil, forming pelotons, and these cells, besides increased volume of cytoplasm, exhibit enlarged nuclei (asterisks). (C) Intact hypha (if) and hypha in the process of digestion (df). (D) Myelin-like body (mb) formed inside a very electron-dense hypha. (E) Hyphal compartment delimited by fungal cell walls (fw), full of degraded membranes (arrows). (F) Advanced stage of hypha digestion (white arrowhead) and fungal cellular debris (black arrowheads).

3.4. Ultrastructural Alterations in the Embryo During Germination and Protocorm Development

In non-germinated seeds, two main cell types were identified in the embryo, besides the suspensor cells: (1) apical region, with cells full of protein bodies and small lipid bodies, with half-membrane and electron-transparent content—some lipids are extracted during the process of dehydration—(Figure 5A); and, (2) the basal region with cells abundant in lipid bodies whose lipids were fixed with osmium tetroxide (electron-dense structures) (Figure 5B).

Figure 5.

Figure 5

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Transmission electron micrographs (TEM) micrographs of embryos and protocorms of Cattleya purpurata. The left column shows the evolution of embryonic cells from the median and apical region, and the right column shows the evolution of cells from the basal region, which contains cells rich in lipid bodies of the embryo. (A) Embryo cells showing abundant lipid bodies distributed throughout the cytoplasm and small nuclei (nu). (B) Most of the cytoplasm is filled with lipid bodies (lb), which appear electron-dense (li), or electron-transparent because some of the lipids were extracted in the dehydration process, and small protein bodies (arrowhead). (C) Cells still show many lipid bodies (lb), some protein bodies (pb) and also amyloplasts with starch grains (am). (D) Cells of the ‘lipid region’ of the embryo, with large lipid bodies (lb) with lipid droplets inside (arrows) and nucleus (nu). (E) Many clear lipid bodies (lb) surrounding the many amyloplasts (am), which in turn surround the nucleus (nu). (F) Cells with lipid bodies (lb) with electron-dense lipid droplets (arrows). (G) Plastid already with grana stacks (*), (asterisks), with organized thylakoid membranes, still with starch grains (am). (H) Mitochondria (mi) surround and encircle lipid droplets (arrows).

Around seven to nine days of co-culture, significant ultrastructural changes began in the embryo. In the cytoplasmic periphery of the cells of the apical and median region, the appearance of plastids containing starch grains was observed (Figure 5C). In parallel, in the cells of the basal region, the lipid bodies, which previously occupied most of the cellular volume, underwent a significant numerical reduction (Figure 5D). This reorganization made the cell nuclei more evident and frequent in the observations (Figure 5D).

With advancing development, between nine and 13 days of co-culture, a proportional increase in the number of amyloplasts was recorded, which were frequently observed clustered around the nuclei (Figure 5E). In the basal region, the degradation of lipid bodies was accompanied by the formation of a large central vacuole (Figure 5F).

Between 16 and 20 days of co-culture, the plastids in the cells evidenced the organization of thylakoid membranes into grana stacks (Figure 5G), indicating the transition from amyloplasts to chloroplasts and the onset of autotrophic metabolism in the protocorms. Additionally, in the cells of the basal lipid region, a high density of mitochondria was observed, many of which were closely associated with the remaining lipid droplets, surrounding them (Figure 5H). This spatial association suggests a strong correlation between lipid mobilization and mitochondrial energy metabolism during the initial development of the protocorm.

4. Discussion

This study elucidated the detailed ontogeny of the symbiosis between seeds and protocorms of Cattleya purpurata and the fungus Tulasnella sp., revealing new ultrastructural and dynamic insights into the development of these distinct partners. Our results demonstrate that during germination, the embryo cells exhibit a pronounced structural and metabolic polarity, not only morphologically but also in terms of nutrient reserves distributed distinctly along the longitudinal axis, an organization similar to that described for some terrestrial orchids [33].

One of the novelties revealed in this work lies in the temporal and spatial dynamics of reserve mobilization. As widely documented, orchid seeds rely primarily on storage lipids (lipid bodies, spherosomes) and protein bodies as their main energy reserves, with an absence or scarcity of carbohydrates [3,34,35]. However, we demonstrate that lipid mobilization in C. purpurata is a gradual process, slowly providing energy to the embryo. Crucially, events essential for germination, such as hyphal penetration, embryonic cell hypertrophy, organelle differentiation, and the formation and digestion of pelotons, occur before or simultaneously with any significant lipid mobilization. This contrasts with the model proposed for the terrestrial orchid Gymnadenia conopsea, where glyoxysome-mediated lipid catabolism is active before fungal infection [24], and with the complex system described in the model plant Arabidopsis thaliana [36]. Our observations provide ultrastructural evidence that a large part of the energy required for the initial momentum of germination and development in C. purpurata derives almost entirely from the mycorrhizal fungus, representing a strategy of immediate symbiotic dependence. The striking association of mitochondria with the remaining lipid droplets in later stages suggests that lipid mobilization, when it occurs, is primarily coupled to mitochondrial dynamics. The absence of glyoxysomes in our findings, together with this intimate mitochondrion-lipid association, corroborates earlier observations in Cattleya aurantiaca, where lipid body degradation also occurred slowly, without detectable glyoxysomes, and was frequently associated with mitochondria [37]. This ultrastructural difference highlights a potential metabolic variability in orchid germination strategies. The dynamics of utilizing fungal energy from the outset, while conserving the meager embryonic lipid reserves, may have evolved as an efficient adaptation to the epiphytic niche of C. purpurata.

One of the most significant findings concerns the process of peloton digestion: our ultrastructural data strongly suggest that the fungus may actively participate in modulating its own digestion. The observed morphological continuum—from intact hyphae to hyphae with electron-dense cytoplasm containing autophagic vacuoles with myelin-like bodies—indicates a process of programmed hyphal senescence preceding complete degradation. The presence of these structures is classically associated in fungi with senescence and cell death [38,39]. This raises a compelling hypothesis: could the fungi be signaling which pelotons should be digested?

Our hypothesis that programmed senescence precedes and regulates peloton digestion points to an active modulation by the fungal symbiont, which goes beyond the common notion of a passive cellular collapse. This interpretation is corroborated by physiological and isotopic data from the orchid Spiranthes sinensis, where [25], using high-resolution stable isotope imaging (SIMS), demonstrated that senescent pelotons receive an active influx of carbon and nitrogen directly from the active fungal hyphae, transforming the aging pelotons into nutritional ‘hot spots’ prior to lysis. This discovery clarifies the function of senescence that our ultrastructural data describe in C. purpurata: it represents a final, regulated stage of nutrient packaging and host reward. By actively directing resources to the unit destined for digestion, the fungus not only participates in modulating its own turnover but also maximizes the efficiency of symbiotic transfer. This active nutrient transfer supports a model of a regulated process where senescence prepares a rich, priority target for the orchid’s digestive response, ensuring a nutritional reward that justifies the host’s energetic cost. If the lifespan of a peloton is a few days [40,41], then the selective digestion of these enriched units could represent a mechanism of symbiotic synchrony, maintaining the vigor of the symbiotic mycelium and regulating the mutualistic balance.

The rapid conversion of proplastids into amyloplasts and then into chloroplasts with organized grana in less than 20 days evidences the remarkable metabolic and morphological plasticity of C. purpurata. The embryo transitions from complete fungal dependence to the establishment of autotrophic potential in a short timeframe, a key factor for post-embryonic success. The intimate association observed between mitochondria and remaining lipid droplets suggests that lipid mobilization, when it finally occurs, is closely coupled to mitochondrial energy metabolism, likely sustaining the protocorm’s accelerated growth.

5. Conclusions and Future Perspectives

This study provides a comprehensive ultrastructural perspective on the symbiotic germination of the epiphytic orchid Cattleya purpurata, revealing new insights into the dynamics of its partnership with Tulasnella sp. We demonstrate that germination follows a strict spatiotemporal pattern, with fungal entry restricted to the suspensor cells and progressive colonization towards the apical embryo region, where peloton formation occurs exclusively.

One of our most significant findings is the evidence for a programmed fungal senescence process that precedes peloton digestion. The ultrastructural continuum observed—from intact hyphae to hyphae with electron-dense cytoplasm containing myelin-like bodies within autophagic vacuoles—strongly suggests an active, regulated participation of the fungus in modulating its own turnover. This supports an emerging paradigm shift from a model of passive host digestion to one of coordinated symbiotic dismantling.

Simultaneously, we documented the remarkable metabolic plasticity of the embryo, which transitions from complete mycoheterotrophy to photosynthetic potential in less than 20 days. This rapid shift was marked by the swift conversion of proplastids to amyloplasts and then to functional chloroplasts with grana, coupled with delayed, mitochondrion-associated mobilization of lipid reserves. This temporal uncoupling suggests a strategy of immediate fungal dependence to conserve the meager embryonic resources, a potentially adaptive trait for the epiphytic niche.

Beyond advancing fundamental knowledge, this work yielded an optimized, reliable protocol for symbiotic seedling production of C. purpurata. The Tulasnella fungal strain that promoted germination and protocorm development is deposited and available in the CBMAI-UNICAMP collection. The resulting mycorrhized plants, presumed to be more resilient to environmental stresses, represent a direct tool for conservation, serving as good candidates for reintroduction programs aimed at restoring vulnerable populations in the Atlantic Forest.

Methodologically, the integrated use of confocal, optical, and transmission electron microscopy proved to be an efficient tool for unraveling complex cellular interactions, setting a benchmark for future studies in plant development and symbioses.

Inspired by the questions that emerged, we propose several future research avenues: (1) refining fixation protocols to preserve lipid bodies for a complete analysis of reserve mobilization; (2) employing advanced techniques like Array Tomography to reconstruct 3D organelle networks and further elucidate embryonic metabolism; (3) investigating the molecular signaling that regulates peloton senescence and digestion—a key finding of this work; and (4) implementing field trials to compare the establishment success of symbiotic versus asymbiotic seedlings in natural habitats, translating our in vitro findings into tangible conservation outcomes for Cattleya species.

Acknowledgments

The authors acknowledge the institutional support of the Universidade Federal de Santa Catarina (UFSC), the Universidade Federal da Bahia (UFBA), and the Universidade Estadual de Campinas (UNICAMP). We extend our gratitude to the technicians of LAMEB-CCB and LCME at UFSC. We especially thank Gerhard Wanner (Ludwig-Maximilians University of Munich) for sharing his knowledge during in-depth discussions on seed germination, ultrastructural aspects of lipid mobilization, and plastid biogenesis. J.L.S.M. acknowledges the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, productivity grant 309175/2023-2) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, grant 2025/01615-6). M.P.G. thanks the Brazilian National Council for Scientific and Technological Development (CNPq, Grant 304522/2023-6). P.E.L. thanks the Brazilian National Council for Scientific and Technological Development (CNPq, Grant 305402/2025-0). During the preparation of this manuscript, the authors used ChatGPT 5.2 for assistance in the generation and refinement of the graphical abstract. The authors have reviewed and edited the output and take full responsibility for the content of this publication.

Author Contributions

Conceptualisation (E.d.M.O., P.E.L., M.P.G., K.B., L.C.d.S., M.F.U. and J.L.S.M.). Methodology (E.d.M.O., K.B., L.C.d.S. and M.F.U.). Morphology and ultrastructure analysis (E.d.M.O., K.B., L.C.d.S. and M.F.U.). Writing—original draft (E.d.M.O., P.E.L., M.P.G., K.B., L.C.d.S., M.F.U. and J.L.S.M.). Supervision (P.E.L., M.P.G. and J.L.S.M.). All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

Additional raw microscopy image datasets, due to their large size, will be available upon reasonable request to the corresponding author.

Conflicts of Interest

The authors declare no conflict of interest.

Funding Statement

This research received no external funding.

Footnotes

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

Additional raw microscopy image datasets, due to their large size, will be available upon reasonable request to the corresponding author.

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