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.
Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching mycoheterotrophic plants (i.e., plants that obtain carbon from fungi), remarkable aspects of their structural adaptations, the patterns of tissue colonization by fungi, and the morphoanatomy of subterranean organs can enlighten their developmental strategies and their relationships with hyphae, the source of nutrients. Another important role of symbiotic fungi is related to the germination of orchid seeds; all Orchidaceae species are mycoheterotrophic during germination and seedling stage (initial mycoheterotrophy), even the ones that photosynthesize in adult stages. Due to the lack of nutritional reserves in orchid seeds, fungal symbionts are essential to provide substrates and enable germination. Analyzing germination stages by structural perspectives can also answer important questions regarding the fungi interaction with the seeds. Different imaging techniques can be applied to unveil fungi endophytes in plant tissues, as are proposed in this article. Freehand and thin sections of plant organs can be stained and then observed using light microscopy. A fluorochrome conjugated to wheat germ agglutinin can be applied to the fungi and co-incubated with Calcofluor White to highlight plant cell walls in confocal microscopy. In addition, the methodologies of scanning and transmission electron microscopy are detailed for mycoheterotrophic orchids, and the possibilities of applying such protocols in related plants is explored. Symbiotic germination of orchid seeds (i.e., in the presence of mycorrhizal fungi) is described in the protocol in detail, along with possibilities of preparing the structures obtained from different stages of germination for analyses with light, confocal, and electron microscopy.
Structural research in botany, covering plant morphology and anatomy, is basic in understanding the whole organism1,2, and provides indispensable perspectives to integrate and contribute to knowledge regarding the ecology, physiology, development, and evolution of plants3. Methods in plant morphology and anatomy currently comprise protocols, equipment, and knowledge developed recently as well as more than a century ago2. The continuous execution and adaptation of classical methods (e.g., light microscopy) along with more recent techniques (e.g., confocal microscopy, X-ray microtomography) have the same essential basis: theoretical knowledge enabling the development of a methodology.
The main tool in plant anatomy and morphology is the image. Despite the misconception that such analyses are simple observations, giving space to subjective interpretations2, analyzing and understanding images in this area requires knowledge of the methods applied (the equipment, type of analysis, methodological procedures), cell components, histochemistry, and the plant body (tissue organization and function, ontogeny, morphological adaptations). Interpreting the images obtained via a variety of methods can lead to correlating form and function, deciphering the chemical composition of a structure, corroborating in describing taxa, understanding infections by phytopathogens, and other such assessments.
When researching mycoheterotrophic (MH) plants (i.e., non-photosynthetic plants that obtain carbon from mycorrhizal fungi4,5), remarkable aspects of their structural adaptations, the patterns of tissue colonization by fungi, and the morphoanatomy of subterranean organs can enlighten their development strategies and relationships with hyphae, which are the source of nutrients. The subterranean organs of MH plants usually show important adaptations related to their association with soil fungi, hence it is essential to perform these anatomical and morphological investigations6. MH species' aerial organs should not be ignored, as endophytes can be also present in these tissues, even if they are not mycorrhizal fungi (personal observations, not published yet).
Besides the well-established essentiality of mycorrhizal fungi association with MH species during their entire life cycle7, every orchid species, even the autotrophic ones, have an initial obligate mycoheterotrophic stage in natural environments. It occurs because the orchids' embryo is undifferentiated and lacks an endosperm or cotyledons, thus being incapable of developing and establishing itself in natural environments without the nutritional support of fungal partners4,8. Considering that, symbiotic germination protocols can be applied not only to MH species but also to photosynthesizing orchids, aiming to investigate orchid-fungus specificity in germination and protocorm development, a vastly applied methodology in initiatives for the conservation of threatened species9,10,11.
In this methods assembly, we describe important steps involved in collecting, fixing, and storing MH plant samples for anatomical studies (section 1), surface analysis and sample selection (section 2), sectioning methods (freehand: section 3, microtomy: section 4, cryomicrotomy: section 5), staining and mounting (section 6), fluorescence and confocal microscopy of fungal endophytes (section 7), scanning electron microscopy (section 8), and transmission electron microscopy (section 9). Additionally, we describe a symbiotic germination method for orchid seeds (MH and autotrophic, section 10), as the imaging methods previously mentioned can be successfully applied to analyze fungal colonization of seeds, protocorms, and seedlings in the germination process.
Figure 1: Schematic summarization of imaging methods. The schematics provide indications of protocol steps in which they are detailed. Abbreviations: GMA = glycol methacrylate, OCT = optimal cutting temperature compound, SEM = scanning electron microscopy. Please click here to view a larger version of this figure.
The microscopy techniques described here in detail (Figure 1) are preceded by the following essential steps: collecting, fixing, dehydrating, embedding, and sectioning samples. As the steps are variable (Figure 1) depending on the chosen technique(s), it is important to think ahead, considering the fixatives to be prepared and transported to the collection site, how the samples must be prepared before fixing, the dehydration processes to be used (section 1), and different embedding possibilities and sectioning methods (sections 4, 5, and 9). Figure 1 summarizes sequentially all the steps required for each microscopy technique thoroughly described below.
1. Collecting, fixing, and maintaining samples
NOTE: Fully MH plants can usually be found in the dark forest understory12,13, mainly in humid and litter-abundant areas, whereas partially MH plants can be found in more open forests12,13. MH plants usually have well-developed subterranean organs in a variety of shapes and sizes.
10% neutral buffered formalin (NBF)14 | |
step 1 | add 10 mL of 37-40% formaldehyde solution in 80 mL of distilled water |
step 2 | add 0.4 g of sodium phosphate monobasic monohydrate (NaH2PO4·H2O) to the solution |
step 3 | add 0.65 g of sodium phosphate dibasic, anhydrous (Na2HPO4) |
step 4 | make up the volume to 100 mL |
Table 1: 10% neutral buffered formalin recipe14.
Karnovsky's solution (modified15) | |
step 1 | in 20 mL of distilled water at 60-70 °C |
step 2 | add 0.8 g of paraformaldehyde (to obtain 4% w/v), stirring |
step 3 | add 1-4 drops of 40% NaOH and stir until the solution becomes clear |
step 4 | cool it and add 30 mL of 0.2 M phosphate buffer pH 7.2 (Table 3) |
step 5 | dilute 25% glutaraldehyde in 0.1 M PB (pH 7.2) to obtain 1% glutaraldehyde (final volume: ~60 mL) |
step 6 | add 1% glutaraldehyde (step 5) to the solution obtained in step 4 until making up to 100 mL of fixative |
Table 2: Karnovsky's solution recipe (modified15).
0.2 M phosphate buffer (PB) pH 7.2 | |
step 1 | add 14.196 g of sodium phosphate dibasic, anhydrous (Na2HPO4) to 400 mL of distilled water |
step 2 | add 13.8 g of sodium phosphate monobasic monohydrate (NaH2PO4·H2O) |
step 3 | stir until the solution is clear |
step 4 | adjust the final volume to 500 mL with distilled water |
step 5 | adjust pH to 7.2 |
step 6 | for a 0.1 M PB, dilute 1:1 |
Table 3: 0.2 M phosphate buffer recipe.
3% glutaraldehyde 0.2 M sodium cacodylate buffer (modified16) | |
step 1 | 0.2 M cacodylate buffer: add 4.28 g of sodium cacodylate trihydrate in 100 mL of distilled water |
step 2 | adjust pH to 7.2 |
step 3 | add 12 mL of 25% glutaraldehyde in 25 mL of the solution in step 2 (0.2 M cacodylate buffer pH 7.2) |
step 4 | make up the volume to 100 mL with distilled water |
Table 4: 3% glutaraldehyde 0.2 M sodium cacodylate buffer recipe (modified16).
2. Surface analysis of organs in fixed and non-fixed material
3. Freehand sections of plant organs
NOTE: Freehand sections of plant organs can be challenging, especially for small and thin structures. However, these sections of tissues with fungal endophytes can, in some cases, better evince hyphae and other features in comparison with thin sections.
4. Embedding plant samples in resin and sectioning
5. Freezing plant samples and sectioning with a cryostat
NOTE: The essential consideration in cryosectioning biological tissue is to reduce damage due to ice crystal formation when freezing samples. Cryoprotection is usually done by infusing chemically inert solutions such as glycerol or sucrose19,20.
6. Staining plant sections and endophytes for light microscopy
NOTE: Many types of stains can be used for plant sections. It is challenging to differentially stain endophytic fungi and plant tissues. Although not a staining procedure, a method for marking fungi structures is presented in section 7 (fluorescence with a wheat germ agglutinin conjugate). Freehand sections (explained in section 3), resin sections (section 4), and cryosections (section 5) can be stained, although phenol and alcohol-based stains are challenging for these samples as GMA resin and OCT lose adherence to the slide in these cases.
7. Application of a fluorochrome conjugated to wheat germ agglutinin in fluorescence and confocal microscopy
NOTE: This method can be applied to freehand sections (explained in section 3), resin sections (section 4), and cryosections (section 5). Cryosections can be adequate for confocal microscopy purposes, as thicker samples can be provided when compared to resin sections, but not as thick as freehand ones. A fluorochrome conjugated to wheat germ agglutinin (WGA, see Table of Materials) is applied to fungal imaging in fluorescence microscopy26. A confocal microscope is not essential, although it can provide clear three-dimensional images of plant structures27.
8. Scanning electron microscopy of plant organs
9. Transmission electron microscopy
lead citrate solution (for TEM contrast staining) | |
step 1 | surround a beaker with tinfoil |
step 2 | dissolve 0.266 g of lead nitrate [Pb(NO3)2] in 6 mL of recently boiled and cooled distilled water |
step 3 | agitate for 2 min |
step 4 | add 0.352 g of trisodium citrate [Na3(C6H5O7).2H2O] (the solution must acquire a milky appearance) |
step 5 | agitate for 15 min, seal the beaker with tinfoil and transfer the solution to a 10 mL beaker |
step 6 | add 1.6 mL of 1N NaOH and 2.4 mL of distilled water (the solution must be translucent) |
step 7 | if necessary, adjust the pH close to 12 |
Table 5: Lead citrate solution recipe.
Figure 2: Contrast staining scheme with lead citrate and uranyl acetate solutions. (A) Prepare the Petri dishes, one turned upside-down (in the center) with thermoplastic film so drops can be placed above it, inside a wider one. NaOH pellets are places around the central dish. (B) Uranyl acetate drops are placed in the circles with the letter U, and lead citrate drops in the circles marked L. DW indicates drops of distilled water. The grids are stained sequentially in the column, so five grids can be stained simultaneously as represented. Please click here to view a larger version of this figure.
10. Symbiotic germination of orchid seeds
Figure 3: Schematic summarization of symbiotic germination of seeds methodology. The schematics provide indications of detailed steps in the protocol. Abbreviations: OMA = oatmeal agar, PDA = potato dextrose agar. Please click here to view a larger version of this figure.
Germination stage | Description |
0 | No germination |
1 | Swelling of the embryo |
2 | Testa rupture |
3 | Absorbent hairs develop |
4 | Stem projection develops |
5 | Protecting scales (bracts) develop |
6 | First roots develop |
Table 6: Description of protocorm developmental stages applied to periodic analyses of germination tests. Modified from stages described in Otero et al.36.
Following the essential stages of fixing plant tissue yields cellular structures as similar as possible to the living state, considering the morphology, volume, and spatial organization of cellular components and tissues16. Observe such traits in the samples after chemical fixation (Figure 4). Figure 4C–F represents adequately fixed samples under light microscopy. Following the fixation procedures described and acquiring familiarity with the samples structure help analyze fixation success.
Figure 4: Superficial analysis and sections from filiform roots of the MH orchid Wullschlaegelia aphylla (Sw.) Rchb.f. (A and B) Rhizomorphs in filiform root surface. (C and D) Freehand sections not stained, evincing pelotons in cortical cells. (E and F) Thin sections stained with toluidine blue O. Abbreviations: en = endodermis, ep = epidermis, ct = cortex, hy = hypha, pc = parenchymatous cell, pe = phloem elements, pl = peloton, rfl = root (filiform), rm = rhizomorph, vc = vascular cylinder, xe = xylem element. Scale bars: A = 2 mm; B = 500 µm; C and E = 200 µm; D = 100 µm; F = 20 µm. Please click here to view a larger version of this figure.
Note in Figure 4C,D the regularity of structures and absence of damaged areas in a freehand section of a sample fixed by 10% NBF. The cellular volume is preserved, resembling living tissues. Comparing with a freehand-sectioned fresh organ is also important in recognizing well-fixed samples. In Figure 4E,F, sections from samples embedded in GMA resin and fixed by 10% NBF were stained with toluidine blue O. Note the well-preserved structures of cell walls, without distortions or damaged areas, showing very similar traits as in a freehand-sectioned sample (Figure 4C,D).
When analyzing the surface of subterranean organs, the presence of rhizomorphs indicates hyphae colonizing internal tissues. Rhizomorphs are vegetative structures composed of an aggregate of highly differentiated hyphae and formed by a few species of fungi, mainly saprotrophic that decompose wood37,38. The rhizomorphs can be easily recognized, usually as dark shoestring-like structures37, seen in Figure 4A,B and Figure 7D. Searching for these fungal structures facilitates sample selection to observe the pattern of fungal colonization in plant organs. In Figure 4C,D, the sections were obtained in areas with superficial rhizomorphs, whereas in Figure 4E, a section of the same organ without selection of such criterium is shown. Individualized hyphae can also be identified under a dissecting microscope with a perceptive observation.
Figure 5: Freehand sections of Uleiorchis sp. storage structure. (A) Section under a dissecting microscope. (B) Pelotons under a light microscope, concentrated in a region of the cortex of the organ. (C and D) Hyphae details of the pelotons analyzed. Abbreviations: hy = hypha, pc = parenchymatous cell, pl = peloton, s = septa. Scale bars: A = 2 mm; B = 500 µm; C and D = 50 µm. Please click here to view a larger version of this figure.
As previously explained, freehand sectioning can be preferred in comparison to thin sectioning depending on the aim. Freehand or other methods of obtaining thicker sections (more than 10 µm thick) can better evince pelotons and provide more representative images of fungal patterns of colonization (for instance, Figure 4C,D and Figure 5A,B). Freehand sections can also be suitable for hyphae analysis in higher amplification, as demonstrated in Figure 5C,D, although details are better achieved in thinner sections, as in Figure 7A, from a sample embedded in GMA resin. Some details of plant cell structures are adequately observed in thin sections, for instance, Figure 4F. Mounting is also an important step, as better-quality images can depend on the mounting medium. GMA resin slides can be mounted in water or glycerin, although a commercial mounting medium (see Table of Materials) will improve the final image as it fills imperfections from the sectioning process. In Figure 6C, imperfections can be seen (arrowheads) in a GMA resin section, as the slide was mounted with water.
Figure 6: Sections of fusiform roots of W. aphylla stained with Lugol solution. (A) Freehand section stained with toluidine blue O and Lugol. (B) Stained only with toluidine blue O. (C) Thin section in GMA resin stained with lactophenol cotton blue and Lugol, arrowheads: imperfections from blade irregularities. (D) Freehand section stained only with Lugol. Abbreviations: cw = cell wall, pc = parenchymatous cell, sg = starch grains, vc = vascular cylinder. Scale bars: A = 200 µm; B and C = 100 µm; D = 50 µm. Please click here to view a larger version of this figure.
The main benefit in using toluidine blue O (more adequate results in thin sections) is the important metachromatic properties of this stain, meaning it acquires different colors depending on the cellular component it binds to and functions as a polychromatic stain suitable to differentiate different compositions of cell walls23. In Figure 4F, secondary cell walls in xylem elements can be easily identified by the light color toluidine acquires. Meanwhile phloem elements, composed only of primary cell walls, are identified by their thinner and darker cell walls. Another important stain, mainly considering MH plants, is Lugol solution, as starch grains are easily identified when stained by it. Sections are represented in Figure 6A–D: freehand section stained with toluidine blue O and Lugol in Figure 6A and only toluidine blue O in Figure 6B; thin section in GMA resin stained with lactophenol cotton blue and Lugol in Figure 6C; freehand section stained only with Lugol in Figure 6D.
Incubation of freehand (Figure 7C), GMA resin (Figure 7B), or OCT sections with WGA-fluorochrome conjugate and Calcofluor White can enhance the structures of the fungal cell wall and plant cell wall, respectively. It is an important method of confirming hyphae, as WGA conjugate has specificity to N-acetylglucosaminyl residues, present in the cell wall of fungi. Figure 7A demonstrates a section of floral stem stained with toluidine blue O, while Figure 7B is from the same organ and confirms the structures seen in Figure 7A are hyphae. Artifacts by autofluorescence can be seen in GMA resin sections (arrowheads, Figure 7B). These artifacts are usually related to fluorochrome concentration and can be avoided by washing the samples more number of times with the buffer. In Figure 7C, a freehand section of root is shown, with internal and external hyphae. The same organ can be seen by SEM in Figure 7D, with an abundance of rhizomorphs and individual hyphae on its surface. Adequate SEM micrographs have good contrast between shades of grey, so tridimensionality can be seen and well interpreted. Choose representative images and avoid misleading ones (further reading: tips for choosing electron micrographs32).
Figure 7: Sections from the floral stem and filiform roots of W. aphylla and superficial analysis of filiform root by SEM. (A) Thin section of floral stem in GMA resin, stained with toluidine blue O. (B) Thin section of floral stem in GMA resin incubated with WGA-fluorochrome conjugate + Calcofluor White, arrowheads: artifacts by autofluorescence. (C) Freehand section of filiform root incubated with WGA-fluorochrome conjugate + Calcofluor White. (D) Scanning electron micrograph of the filiform root surface. Abbreviations: cw = cell wall, hy = hyphae, pc = parenchymatous cell, pl = peloton, rfl = root (filiform), rm = rhizomorph. Scale bars: A, C, and D = 100 µm; B = 20 µm. Please click here to view a larger version of this figure.
Fruits from different orchid species were immersed in sodium hypochlorite with 2% active chlorine for 15 min, to guarantee complete superficial disinfection of the organs (Figure 8A). Seeds with more rigid seed coats as from Vanilla sp. (Figure 8A) and Pogoniopsis schenckii (Figure 9B–D) were immersed in the same solution for 10 min (Figure 8C), whereas slimmer ones, as from Laelia sp. and Cattleya sp., maintained for 7 min (Figure 9A). Transferring an aliquot of water from the last wash confirmed the effectiveness of the disinfection process before proceeding to the germination tests, considering both durations of immersion described.
Figure 8: Superficial disinfection of fruits and seeds of Vanilla panifolia, a photosynthetic orchid species. (A) Fruit immersion in sodium hypochlorite with active chlorine. (B) Fruit longitudinally sectioned. (C) Seeds filtered using serigraphic fabric after immersion in sodium hypochlorite, ready to be sown or stored in silica gel. Please click here to view a larger version of this figure.
Sowing the seeds over filter paper discs assure there is enough humidity and oxygen for germination and embryo development (Figure 9A–D) without being completely submerged under the superficial water layer from the culture medium. Some fungal isolates can grow vigorously over the seeds. The medium containing 2 g/L of crushed oat flakes provides better control of fungal growth, improving the visualization and analysis of seeds (Figure 9B). After ca. 50-60 days of isolate inoculation, the medium must be renewed, so the fungus remains active. This can be done by transferring the seeds to a new OMA medium of the same formulation. The seeds can be transferred with the filter paper, facilitating their transference without damaging protocorms' delicate structures, besides keeping them in the original position without compromising the counting fields previously established.
Figure 9: Protocol of symbiotic germination. (A) Seeds arranged over filter paper in OMA medium. (B) Petri dishes with seeds and an inoculated fungus (potentially mycorrhizal), incubated for ca. 21 days. (C and D) Symbiotic germination dishes with different fungal isolates. Please click here to view a larger version of this figure.
Most orchid species germinate within some weeks after being infected by the inoculated fungus or until nearly more than a month (Figure 10). Seeds that do not germinate within 3 months will probably not germinate unless adjusting the methodology. In such cases, consider possibilities such as seed dormancy or the fungal isolate not being mycorrhizal. Some species need specific protocols to break dormancy, others simply present a high specificity to certain mycorrhizal partners, different from the ones chosen for the symbiotic germination test (data not published).
Figure 10: Pogoniopsis schenckii seeds, an achlorophyllous and MH orchid, in OMA medium with fungal inoculate39 capable of stimulating germination. (A–D) Not germinated seeds and protocorms in different developmental stages. Abbreviations: ng = no germination, pt = protocorm, ri = ruptured integument, ts = turgid seed. Scale bars = 200 µm. Please click here to view a larger version of this figure.
In the first few weeks after inoculating the isolate, the dishes are evaluated, as it usually takes 7-14 days until the hyphae achieve the seeds. Consider this period, as it is preponderant to start registering the development of the embryos. The subtle changes during germination stages can only be detected under a dissecting microscope, considering the structures are so minute. Some species protocorms must be analyzed under a light microscope to identify absorbent hairs and tell them apart from hyphae. GI analysis can provide more plausibly comparable results, generating a representation of collected data and conferring more weight to values corresponding to more advanced germination stages. The final values can range between zero and six (or according to the last stage defined). Different statistical tests can be applied to GI analyses (e.g., ANOVA, significance level), depending on the researcher's questions and demands when carrying out symbiotic germination tests.
Image analyses in plant anatomy and morphology have an important potential to fulfil objectives and help understand the relationships between mycoheterotrophic plants and their indispensable fungal endophytes, as demonstrated by studies of subterranean organs6,40, structural analyses of symbiotic germination of seeds39, and aerial and reproductive structures41. Structural botany, despite having lost its prestige and space to other areas of plant sciences in the last decade1, is still prominent in answering questions and helping to unveil novelties and essential plant traits related to development, ecology, physiology, and evolution. These methods are collectively a basis for structural studies of plants, considering important aspects of MH plant analysis.
Essential information is provided to carefully collect MH plants, as otherwise the well-developed underground organs can be damaged and not completely collected. Remarkable adaptations of these structures6 and the intimate contact with fungi from soil, connected to autotrophic plants42 or decomposing leaf litter40 are to be considered when collecting and preserving the underground structures. The essential steps of sample fixation need to be adequately followed, regarding correct preparation of fixatives and time issues, minimum time required between collecting and fixing, and minimum time required in the fixative before proceeding to storage. Structural analysis of well-preserved samples depends on the fixation process, and the most common and best-preserving fixatives applied to study plant anatomy and electron microscopy are described in the protocols section. Other fixatives14,16,25 can also be applied.
As previously stated, the process of using chemical fixatives is decisive and can be evaluated when obtaining sample images. In LM, cellular structures should be as similar as possible in comparison with living tissues16. The volume, morphology, and spatial disposition of identifiable cellular components should resemble as much as possible to the fresh tissues (non-fixed). In TEM, well-preserved tissues can be evinced when the tonoplast is visible, with smooth contours, and not pulled away from the cytoplasm16. The plasma membrane cannot be detached and shrunk away from the cell wall. Samples for TEM must be collected as thinly as possible and inside a drop of fixative, as explained in the protocol section. The use of buffers associated with fixing agents provides important conditions of osmolarity and ionic composition to the samples being fixed, avoiding as many changes as possible in cellular structure and ultrastructure16. In SEM, one major concern is with isotonic fixatives and preparing them with buffers, so there are no considerable changes in sample volume (swelling or shrinkage)16.
Many different embedding methods for LM are available to obtain sections for various purposes. Considering staining and fluorescent-dyes incubation, two important methodologies are presented to analyze MH plants organs. The ones described above (freehand sections, GMA resin, and OCT embedding) are among the most common and simple, providing methodological freedom and suitability to many types of analysis. The conjugate WGA-fluorochrome has considerable potential in MH plants studies, as it is currently more applied to fungal pathogens28,29,30 and scarcely used with MH plants fungal endophytes. The basic and essential steps for scanning and transmission electron microscopy are detailed, as these techniques can greatly contribute to the structural analysis of plants. SEM and TEM literature is rich, and further reading32,33,34,43 is recommended if other types of electron microscopy analyses are to be tested.
Regarding symbiotic germination procedures, it is possible to conduct fruit asepsis before dehiscence, without the necessity of disinfecting the seeds directly. However, seeds from fruits already open or colonized by endophytes (already described for MH species39,41) must be directly disinfected. An important remark: time and conditions of storage and asepsis processes can reduce the viability of the seeds from some species as observed while performing these experiments. Tetrazolium reagent confers a color ranging from light to dark red to embryos from viable seeds. Embryos from non-viable seeds remain with their natural color. Seeds with heavily pigmented teguments or rigid seed coats may need a pre-treatment before the germination test. It is recommended to perform scarification of their tegument, enabling the visualization of the embryo35.
Some potential mycorrhizal fungal isolates from tropical orchids, for instance, Ceratobasidium species, have a vigorous and accelerated growth in a nutrient-rich culture medium. During the treatment, it is possible that the isolates completely cover the seeds when they grow, making data collection difficult or even impossible. The commonly used protocol containing 4 g/L of crushed oat flakes9 prevented data collection in symbiotic germination of P. schenckii, demanding an adequation to 2 g/L of crushed oat flakes39. Using approximately 2.5 g/L of oat flakes to compose OMA medium in symbiotic germination appears to be satisfactory in limiting the growth of more vigorous fungal isolates (unpublished data).
Limitations can arise from the methods described, some of which can be effectively overcome by adapting the procedures or applying other methods. As discussed before, GMA embedding is only effective for sectioning up to 8 µm thick. However, OCT embedding provides different thickness sections, including thicker ones (e.g., 10-20 µm). Staining only fungi structures in plant tissues is not easily conducted, although WGA-fluorochrome is an important and effective conjugate fluorochrome that marks fungal cell walls, although it is expensive. Other cost limitations can arise in SEM and TEM methods, as the high costs and essentiality of equipment make such analyses not trivial and usual to every research group. The symbiotic germination of seeds tests, although simple and less expensive, demand mycorrhizal fungi to inoculate the seeds and careful procedures avoiding contaminations.
The authors have nothing to disclose.
The authors thank funding from FAEPEX and FAPESP (2015/26479-6). MPP thanks Capes for his master's degree scholarship (process 88887.600591/2021-00) and CNPq. JLSM thanks CNPq for productivity grants (303664/2020-7). The authors also thank the access to equipment and assistance provided by LME (Laboratory of Electron Microscopy – IB/Unicamp), INFABiC (National Institute of Science and Technology on Photonics Applied to Cell Biology – Unicamp), and LaBiVasc (Laboratory of Vascular Biology – DBEF/IB/Unicamp); LAMEB (UFSC) and Eliana de Medeiros Oliveira (UFSC) for contributions to cryoprotection protocol; LME for contributions to TEM protocol.
Acetone | Sigma-Aldrich | 179124 | (for SEM stubs mounting) |
Agar-agar (AA) | Sigma-Aldrich | A1296 | (for seeds germination tests) |
Calcofluor White Stain | Sigma-Aldrich | 18909 | fluorescent dye (detects cellulose) |
Citrate Buffer Solution, 0.09M pH 4.8 | Sigma-Aldrich | C2488 | (for toluidine blue O staining) |
Conductive Double-Sided Carbon Tape | Fisher Scientific | 50-285-81 | (for SEM) |
Confocal Microscope | Zeiss | (any model) | |
Copper Grids | Sigma-Aldrich | G4776 | (for TEM) |
Critical-point dryer | Balzers | (any model) | |
Cryostat | Leica Biosystems | (any model) | |
Dissecting microscope | Leica Biosystems | (= stereomicroscope, any model) | |
Entellan | Sigma-Aldrich | 107960 | rapid mounting medium for microscopy |
Ethyl alcohol, pure (≥99.5%) | Sigma-Aldrich | 459836 | (= ethanol, for dehydration processes) |
Formaldehyde solution, 37% | Sigma-Aldrich | 252549 | (for NBF solution preparation) |
Formalin solution, neutral buffered, 10% | Sigma-Aldrich | HT501128 | histological tissue fixative |
Gelatin capsules for TEM | Fisher Scientific | 50-248-71 | (for resin polymerisation in TEM) |
Gelatin solution, 2% in H2O | Sigma-Aldrich | G1393 | (dilute for slides preparation – OCT adherence) |
Glutaraldehyde solution, 25% | Sigma-Aldrich | G6257 | (for Karnovsky’s solution preparation) |
HistoResin | Leica Biosystems | 14702231731 | glycol methacrylate (GMA) embedding kit |
Iodine | Sigma-Aldrich | 207772 | (for Lugol solution preparation) |
Lead(II) nitrate | Sigma-Aldrich | 228621 | Pb(NO3)2 (for TEM contrast staining) |
Light Microscope | Olympus | (any model) | |
LR White acrylic resin | Sigma-Aldrich | L9774 | hydrophilic acrylic resin for TEM |
Lugol solution | Sigma-Aldrich | 62650 | (for staining) |
Metal stubs for specimen mounts | Rave Scientific | (for SEM, different models) | |
Microtome | Leica Biosystems | manual rotary microtome or other model | |
Oatmeal agar (OMA) | Millipore | O3506 | (for seeds germination tests) |
OCT Compound, Tissue-Tek | Sakura Finetek USA | 4583 | embedding medium for frozen tissues |
Osmium tetroxide | Sigma-Aldrich | 201030 | OsO4 (for TEM postfixation) |
Parafilm M | Sigma-Aldrich | P7793 | sealing thermoplastic film |
Paraformaldehyde | Sigma-Aldrich | 158127 | (for Karnovsky’s solution preparation) |
Poly-L-lysine solution, 0.1% in H2O | Sigma-Aldrich | P8920 | (for slides preparation – OCT adherence) |
Poly-Prep Slides | Sigma-Aldrich | P0425 | poly-L-lysine coated glass slides |
Polyethylene Molding Cup Trays | Polysciences | 17177A-3 | (6x8x5 mm, for embbeding samples in GMA resin) |
Polyethylene Molding Cup Trays | Polysciences | 17177C-3 | (13x19x5 mm, for embbeding samples in GMA resin) |
Potassium iodide | Sigma-Aldrich | 221945 | (for Lugol solution preparation) |
Potato Dextrose Agar (PDA) | Millipore | 70139 | (for seeds germination tests) |
Scanning Electron Microscope | Jeol | (any model) | |
Silane [(3-Aminopropyl)triethoxysilane] | Sigma-Aldrich | A3648 | (for slides preparation – OCT adherence) |
Silane-Prep Slides | Sigma-Aldrich | S4651 | glass slides coated with silane |
Silica gel orange, granular | Supelco | 10087 | (for dessicating processes) |
Sodium cacodylate trihydrate | Sigma-Aldrich | C0250 | (for glutaraldehyde-sodium cacodylate buffer) |
Sodium hydroxide | Sigma-Aldrich | S5881 | NaOH (for Karnovsky’s solution preparation and TEM contrast staining) |
Sodium hypochlorite solution | Sigma-Aldrich | 425044 | NaClO (for seeds surface disinfection) |
Sodium phosphate dibasic, anhydrous | Sigma-Aldrich | 71640 | Na2HPO4 (for NBF solution and PB preparation) |
Sodium phosphate monobasic monohydrate | Sigma-Aldrich | S9638 | NaH2PO4·H2O (for NBF and PB) |
Sputter coater | Balzers | (any model) | |
Sucrose | Sigma-Aldrich | S0389 | C12H22O11 (for cryoprotection and germination test) |
Sudan III | Sigma-Aldrich | S4131 | (for staining) |
Sudan IV | Sigma-Aldrich | 198102 | (for staining) |
Sudan Black B | Sigma-Aldrich | 199664 | (for staining) |
Syringe | (3 mL, any brand, for TEM contrast staining) | ||
Syringe Filter Unit, Millex-GV 0.22 µm | Millipore | SLGV033R | PVDF, 33 mm, gamma sterilized (for TEM contrast staining) |
Tek Bond Super Glue 793 | Tek Bond Saint-Gobain | 78072720018 | liquid cyanoacrylate adhesive, medium viscosity |
Toluidine Blue O | Sigma-Aldrich | T3260 | (for staining) |
Transmission Electron Microscope | Jeol | (any model) | |
Triphenyltetrazolium chloride | Sigma-Aldrich | T8877 | (for the tetrazolium test in seeds germination) |
Trisodium citrate dihydrate | Sigma-Aldrich | S1804 | Na3(C6H5O7)·2H2O (for TEM contrast staining) |
Ultramicrotome | Leica Biosystems | (any model) | |
Uranyl acetate | Fisher Scientific | 18-607-645 | UO2(CH3COO)2 (for TEM contrast staining) |
Vacuum pump | (any model) | ||
Wheat Germ Agglutinin, Alexa Fluor 488 Conjugate | TermoFisher Scientific | W11261 | fluorescent dye-conjugated lectin (detects sialic acid and N-acetylglucosaminyl residues) |