Name: microscopy techniques for interpreting fungal colonization in mycoheterotrophic plants tissues and symbiotic germination of seeds
Uploaded: 2022-05-17T14:00:00
Description: Watch this Scientific Journal Video about Microscopy Techniques for Interpreting Fungal Colonization in Mycoheterotrophic Plants Tissues and Symbiotic Germination of Seeds at JoVE.com

The authors thank funding from FAEPEX and FAPESP (2015/26479-6). MPP thanks Capes for his master&#39;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.

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.
<ol>
	<li>When collecting MH spe...

<ol>
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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...

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&#160;(i.e., non-photosynthetic plants that obtain carbon from mycorrhizal fungi4,5),&#160;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&#39; 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&#39; 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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63777/63777fig01.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/63777/63777fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetone</td>
			<td>Sigma-Aldrich</td>
			<td>179124</td>
			<td>(for SEM stubs mounting)</td>
		</tr>
		<tr>
			<td>Agar-agar (AA)</td>
			<td>Sigma-Aldrich</td>
			<td>A1296</td>
			<td>(for seeds germination tests)</td>
		</tr>
		<tr>
			<td>Calcofluor White Stain</td>
			<td>Sigma-Aldrich</td>
			<td>18909</td>
			<td>fluorescent dye (detects cellulose)</td>
		</tr>
		<tr>
			<td>Citrate Buffer Solution, 0.09M pH 4.8</td>
			<td>Sigma-Aldrich</td>
			<td>C2488</td>
			<td>(for toluidine blue O staining)</td>
		</tr>
		<tr>
			<td>Conductive Double-Sided Carbon Tape</td>
			<td>Fisher Scientific</td>
			<td>50-285-81</td>
			<td>(for SEM)</td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>(any model)</td>
		</tr>
		<tr>
			<td>Copper Grids</td>
			<td>Sigma-Aldrich</td>
			<td>G4776</td>
			<td>(for TEM)</td>
		</tr>
		<tr>
			<td>Critical-point dryer</td>
			<td>Balzers</td>
			<td></td>
			<td>(any model)</td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica Biosystems</td>
			<td></td>
			<td>(any model)</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Leica Biosystems</td>
			<td></td>
			<td>(= stereomicroscope, any model)</td>
		</tr>
		<tr>
			<td>Entellan</td>
			<td>Sigma-Aldrich</td>
			<td>107960</td>
			<td>rapid mounting medium for microscopy</td>
		</tr>
		<tr>
			<td>Ethyl alcohol, pure (&#8805;99.5%)</td>
			<td>Sigma-Aldrich</td>
			<td>459836</td>
			<td>(= ethanol, for dehydration processes)</td>
		</tr>
		<tr>
			<td>Formaldehyde solution, 37%</td>
			<td>Sigma-Aldrich</td>
			<td>252549</td>
			<td>(for NBF solution preparation)</td>
		</tr>
		<tr>
			<td>Formalin solution, neutral buffered, 10%</td>
			<td>Sigma-Aldrich</td>
			<td>HT501128</td>
			<td>histological tissue fixative</td>
		</tr>
		<tr>
			<td>Gelatin capsules for TEM</td>
			<td>Fisher Scientific</td>
			<td>50-248-71</td>
			<td>(for resin polymerisation in TEM)</td>
		</tr>
		<tr>
			<td>Gelatin solution, 2% in H2O</td>
			<td>Sigma-Aldrich</td>
			<td>G1393</td>
			<td>(dilute for slides preparation - OCT adherence)</td>
		</tr>
		<tr>
			<td>Glutaraldehyde solution, 25%</td>
			<td>Sigma-Aldrich</td>
			<td>G6257</td>
			<td>(for Karnovsky&#8217;s solution preparation)</td>
		</tr>
		<tr>
			<td>HistoResin</td>
			<td>Leica Biosystems</td>
			<td>14702231731</td>
			<td>glycol methacrylate (GMA) embedding kit</td>
		</tr>
		<tr>
			<td>Iodine</td>
			<td>Sigma-Aldrich</td>
			<td>207772</td>
			<td>(for Lugol solution preparation)</td>
		</tr>
		<tr>
			<td>Lead(II) nitrate</td>
			<td>Sigma-Aldrich</td>
			<td>228621</td>
			<td>Pb(NO3)2 (for TEM contrast staining)</td>
		</tr>
		<tr>
			<td>Light Microscope</td>
			<td>Olympus</td>
			<td></td>
			<td>(any model)</td>
		</tr>
		<tr>
			<td>LR White acrylic resin</td>
			<td>Sigma-Aldrich</td>
			<td>L9774</td>
			<td>hydrophilic acrylic resin for TEM</td>
		</tr>
		<tr>
			<td>Lugol solution</td>
			<td>Sigma-Aldrich</td>
			<td>62650</td>
			<td>(for staining)</td>
		</tr>
		<tr>
			<td>Metal stubs for specimen mounts</td>
			<td>Rave Scientific</td>
			<td></td>
			<td>(for SEM, different models)</td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Leica Biosystems</td>
			<td></td>
			<td>manual rotary microtome or other model</td>
		</tr>
		<tr>
			<td>Oatmeal agar (OMA)</td>
			<td>Millipore</td>
			<td>O3506</td>
			<td>(for seeds germination tests)</td>
		</tr>
		<tr>
			<td>OCT Compound, Tissue-Tek</td>
			<td>Sakura Finetek USA</td>
			<td>4583</td>
			<td>embedding medium for frozen tissues</td>
		</tr>
		<tr>
			<td>Osmium tetroxide</td>
			<td>Sigma-Aldrich</td>
			<td>201030</td>
			<td>OsO4 (for TEM postfixation)</td>
		</tr>
		<tr>
			<td>Parafilm M</td>
			<td>Sigma-Aldrich</td>
			<td>P7793</td>
			<td>sealing thermoplastic film</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>158127</td>
			<td>(for Karnovsky&#8217;s solution preparation)</td>
		</tr>
		<tr>
			<td>Poly-L-lysine solution, 0.1% in H2O</td>
			<td>Sigma-Aldrich</td>
			<td>P8920</td>
			<td>(for slides preparation - OCT adherence)</td>
		</tr>
		<tr>
			<td>Poly-Prep Slides</td>
			<td>Sigma-Aldrich</td>
			<td>P0425</td>
			<td>poly-L-lysine coated glass slides</td>
		</tr>
		<tr>
			<td>Polyethylene Molding Cup Trays</td>
			<td>Polysciences</td>
			<td>17177A-3</td>
			<td>(6x8x5 mm, for embbeding samples in GMA resin)</td>
		</tr>
		<tr>
			<td>Polyethylene Molding Cup Trays</td>
			<td>Polysciences</td>
			<td>17177C-3</td>
			<td>(13x19x5 mm, for embbeding samples in GMA resin)</td>
		</tr>
		<tr>
			<td>Potassium iodide</td>
			<td>Sigma-Aldrich</td>
			<td>221945</td>
			<td>(for Lugol solution preparation)</td>
		</tr>
		<tr>
			<td>Potato Dextrose Agar (PDA)</td>
			<td>Millipore</td>
			<td>70139</td>
			<td>(for seeds germination tests)</td>
		</tr>
		<tr>
			<td>Scanning Electron Microscope</td>
			<td>Jeol</td>
			<td></td>
			<td>(any model)</td>
		</tr>
		<tr>
			<td>Silane [(3-Aminopropyl)triethoxysilane]</td>
			<td>Sigma-Aldrich</td>
			<td>A3648</td>
			<td>(for slides preparation - OCT adherence)</td>
		</tr>
		<tr>
			<td>Silane-Prep Slides</td>
			<td>Sigma-Aldrich</td>
			<td>S4651</td>
			<td>glass slides coated with silane</td>
		</tr>
		<tr>
			<td>Silica gel orange, granular</td>
			<td>Supelco</td>
			<td>10087</td>
			<td>(for dessicating processes)</td>
		</tr>
		<tr>
			<td>Sodium cacodylate trihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C0250</td>
			<td>(for glutaraldehyde-sodium cacodylate buffer)</td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>S5881</td>
			<td>NaOH (for Karnovsky&#8217;s solution preparation and TEM contrast staining)</td>
		</tr>
		<tr>
			<td>Sodium hypochlorite solution</td>
			<td>Sigma-Aldrich</td>
			<td>425044</td>
			<td>NaClO (for seeds surface disinfection)</td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic, anhydrous</td>
			<td>Sigma-Aldrich</td>
			<td>71640</td>
			<td>Na2HPO4 (for NBF solution and PB preparation)</td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic monohydrate</td>
			<td>Sigma-Aldrich</td>
			<td>S9638</td>
			<td>NaH2PO4&#183;H2O (for NBF and PB)</td>
		</tr>
		<tr>
			<td>Sputter coater</td>
			<td>Balzers</td>
			<td></td>
			<td>(any model)</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S0389</td>
			<td>C12H22O11 (for cryoprotection and germination test)</td>
		</tr>
		<tr>
			<td>Sudan III</td>
			<td>Sigma-Aldrich</td>
			<td>S4131</td>
			<td>(for staining)</td>
		</tr>
		<tr>
			<td>Sudan IV</td>
			<td>Sigma-Aldrich</td>
			<td>198102</td>
			<td>(for staining)</td>
		</tr>
		<tr>
			<td>Sudan Black B</td>
			<td>Sigma-Aldrich</td>
			<td>199664</td>
			<td>(for staining)</td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td></td>
			<td></td>
			<td>(3 mL, any brand, for TEM contrast staining)</td>
		</tr>
		<tr>
			<td>Syringe Filter Unit, Millex-GV 0.22 &#181;m</td>
			<td>Millipore</td>
			<td>SLGV033R</td>
			<td>PVDF, 33 mm, gamma sterilized (for TEM contrast staining)</td>
		</tr>
		<tr>
			<td>Tek Bond Super Glue 793</td>
			<td>Tek Bond Saint-Gobain</td>
			<td>78072720018</td>
			<td>liquid cyanoacrylate adhesive, medium viscosity</td>
		</tr>
		<tr>
			<td>Toluidine Blue O</td>
			<td>Sigma-Aldrich</td>
			<td>T3260</td>
			<td>(for staining)</td>
		</tr>
		<tr>
			<td>Transmission Electron Microscope</td>
			<td>Jeol</td>
			<td></td>
			<td>(any model)</td>
		</tr>
		<tr>
			<td>Triphenyltetrazolium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>T8877</td>
			<td>(for the tetrazolium test in seeds germination)</td>
		</tr>
		<tr>
			<td>Trisodium citrate dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>S1804</td>
			<td>Na3(C6H5O7)&#183;2H2O (for TEM contrast staining)</td>
		</tr>
		<tr>
			<td>Ultramicrotome</td>
			<td>Leica Biosystems</td>
			<td></td>
			<td>(any model)</td>
		</tr>
		<tr>
			<td>Uranyl acetate</td>
			<td>Fisher Scientific</td>
			<td>18-607-645</td>
			<td>UO2(CH3COO)2 (for TEM contrast staining)</td>
		</tr>
		<tr>
			<td>Vacuum pump</td>
			<td></td>
			<td></td>
			<td>(any model)</td>
		</tr>
		<tr>
			<td>Wheat Germ Agglutinin, Alexa Fluor 488 Conjugate</td>
			<td>TermoFisher Scientific</td>
			<td>W11261</td>
			<td>fluorescent dye-conjugated lectin (detects sialic acid and N-acetylglucosaminyl residues)</td>
		</tr>
	</tbody>
</table>

microscopy techniques for interpreting fungal colonization in mycoheterotrophic plants tissues and symbiotic germination of seeds

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.

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&#160;such traits in the samples after chemical fixation (Figure 4). Figure 4C-F represents adequately fixed samples under light microscopy. Following the fixation procedures de...

Watch this Scientific Journal Video about Microscopy Techniques for Interpreting Fungal Colonization in Mycoheterotrophic Plants Tissues and Symbiotic Germination of Seeds at JoVE.com

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

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 ...

The protocol presents diverse microscopy techniques applied to understand the fungal colonization in mycoheterotrophic plants, from collecting to preparing samples in detail and include essential steps. Such techniques can be applied to various mycoheterotrophic plants, and even to a plant's materials other than mycoheterotrophics. This method is mainly related to structural botany and could also provide insights into mycorrhizal interactions, physiology, reproductive biology, evolution, and ecology of mycoheterotrophic plants.To begin, collect the mycoheterotrophic plants by exploring the soil around the plant base, taking care not to damage the underground organs. Also, avoid pulling the plants from the ground to prevent disconnecting the aerial organs from the subterranean ones. Carefully, dig around aerial structures using a gardening trowel while exploring the subterranean organs like roots, stems, rhizomes and storage organs without damaging these structures.Remove soil particles to preserve fragile structures and delicately wash these organs with tap water to rinse off the remaining soil particles before fixing the samples. Mycoheterotrophic plants associated with leaf litter demand extra attention. Hence, carefully collect the delicate organs connected to the decomposing material through their hyphae without pulling them from the connected structures.Preserve structures with such connections and collect the litter for analysis. For transmission electron microscopy analysis, section the 3 to 4-millimeter-thick samples inside a drop of glutaraldehyde sodium cacodylate buffer into smaller sections of 1 to 2-millimeter thickness. Discard the cut edges outside the drop.Ensure conducting the fixation process at the collection site immediately after the collection of plants. Immediately transfer the sections to a collection tube with a volume of fixative more than 10 times greater than the volume of the samples as it is an additive fixative. For surface analysis of the superficial hyphae in the organs, especially in subterranean ones and those in contact with leaf litter.Observe fresh or fixed material under the dissecting microscope at a 7.5x or above magnification. Search for the areas of interest guided by the superficial hyphae and the rhizomorphs. Select the samples containing areas of superficial rhizomorphs as these can be sectioned to visualize pelotons and hyphae coils within cortical cells in roots and stems.Prepare 0.2 milligrams per milliliter of wheat germ agglutinin fluorochrome conjugate and 1%calcofluor white solutions in 0.1 molar phosphate buffer as described in the text manuscript. Now, incubate the sections on glass slides by properly covering them with wheat germ agglutinin fluorochrome conjugate solution for 30 minutes. After the incubation, wash the sections with 0.1 molar phosphate buffer and incubate them in the calcofluor solution as a mounting medium.Next, place cover slips on the slides and observe it under a confocal or fluorescence light microscope using the indicated filters. After fixing the samples, performing dehydration, and storing it in 70%ethanol, expose any desired surface for scanning electron microscopy analysis by using a sharp and new razor blade to make cuts with a one-way movement. If necessary, use a stereo microscope to select the samples and consider the metal stubs area while determining the sample sizes.Further, dehydrate the samples in an ethanolic series. Maintain small and delicate samples for 30 minutes in each concentration and larger and denser samples for 1 hour. Next, fold small envelopes using tissue paper to organize samples for the next steps.Larger samples can be handled without an envelope. Label the envelopes using a pencil and keep a log of the samples. Carefully, place the samples inside the envelopes using a paintbrush.Now, keep these samples in absolute ethanol. Immediately proceed with critical point drying by operating a critical point dryer according to standard operating procedures. For this, place the samples in absolute ethanol in a sample holder and place it inside the critical point dryer's pressure chamber.The intermediate fluid dissolves into the transition fluid at the critical carbon dioxide point, thus drying the samples. As reabsorption of atmospheric humidity can destroy the samples, store them in a desiccation container immediately after critical point drying. Before mounting the samples on metal stubs, put the gloves on to manipulate the stubs.Immerse them in acetone for 5 minutes to eliminate any fat and let them dry. Under a stereo microscope, fix the samples on the stub using a conductive double-sided carbon adhesive tape and position them. The site from above is the only possible perspective in scanning electron microscopy images.To manipulate the samples, use fine point tweezers. The sample part touched by the tweezers is usually damaged. So be careful and try touching parts positioned away from the areas of interest.Maintain the stubs with samples in a sealed Petri dish with silica gel. Next, proceed with sputter coating to deposit a layer of gold or platinum on the surface of the samples in a low-pressure atmosphere of inert gas by following the standard operating procedures. The coating thickness depends on the sample's topography and is usually between 15 to 40 nanometers.Finally, maintain the coated stubs in a sealed Petri dish with silica gel retaining humidity. The samples can be stored this way for weeks. Use a scanning electron microscope to analyze these samples.An electron beam strikes the in vacuo sample during scanning electron microscopy and the signal emission from such interaction is interpreted as images. Ensure that the solutions and materials used in the symbiotic germination of seeds are sterile. to avoid contamination.Start by autoclaving them for 20 minutes at 121 degrees Celsius. In a laminar airflow hood, superficially disinfect the fruits and seeds by immersing them in a sodium hypochlorite solution containing 2%active chlorine. Slim and fragile seeds can be immersed in a 1:1 diluted sodium hypochlorite solution.After disinfection recuperate the seeds by filtering them using xerographic fabric. Wash the fruits and seeds thrice in autoclaved distilled water to remove the hypochlorite solution before proceeding with the germination tests. If necessary, store them in filter paper envelopes inside glass flasks with silica gel at 4 degrees Celsius and hermetically close the flasks and seal them with cling film, then evaluate the effectiveness of the washing process by transferring some drops of water from the last wash to potato dextrose agar.For symbiotic germination of orchid seeds, incubate the seeds over 1 to 2-centimeter autoclaved filter paper discs placed in Petri dishes containing oatmeal agar culture medium. Now, inoculate the center of the Petri dish with the fragment of culture medium containing mycelium from the chosen isolated fungus. Seal the Petri dishes with cling film and incubate them in the dark at around 25 degrees Celsius or room temperature, depending on the fungal growth.Prepare some dishes with seeds and without fungal inoculation as a negative control for the germination test. Analyze the germination results weekly by collecting quantitative and qualitative data and photographing protocorms and seedlings. The rhizomorphs can be easily recognized usually as dark, shoestring-like structures.Freehand or other methods of obtaining sections thicker than 10 micrometers can better evince pelotons and provide more representative images of fungal patterns of colonization. Freehand sections can also be suitable for hyphae analysis in higher amplification. Although, details are better achieved in thinner sections.The 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. Artifacts by autofluorescence can be seen in glycol methacrylate resin sections.These artifacts are usually related to fluorochrome concentration and can be avoided by washing the samples a greater number of times with the buffer. The freehand section of the root shows internal and external hyphae. The same organ can be seen by scanning electron microscopy with an abundance of rhizomorphs and individual hyphae on its surface.Most orchids species germinate within some weeks after being infected by the inoculated fungus or until nearly more than a month. Light in scanning electron microscopy can be applied to flowers, fruits, and seeds to investigate the reproductive biology or mycoheterotrophic plants. Symbiotic germination of seeds can also be tested in autotrophic orchids.

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