MycoPatt method permits an in depth exploration of arbuscular mycorrhizal colonization in roots. Mycorrhizal maps point to fungal expansion and present the real position of each structure as a specific pattern. Mycorrhizal colonization is objectively analyzed at 1%resolution.
Each root segment is automatically converted to a digital matrix and generates an extensive database of colonization parameters. To begin, remove the roots from the refrigerator and place them on a working platform for thawing at room temperature. To perform root clearing, place all roots from one plant in a 30 to 50 milliliter jar, then prepare a 10%sodium hydroxide solution with tap water and pour it into the jar until it completely covers the roots.
Shake the jar vigorously for one to two minutes for homogenous dispersion of clearing solution in the roots. Allow the roots to remain in the clearing solution for 48 hours. For root rinsing, pass the content of the jar through a sieve.
Rinse the roots several times in tap water until the clearing solution is completely removed. Next place the rinsed roots in a clean jar for staining. Then prepare a five to five percent ink vinegar solution with tap water.
Pour it into the jar until it covers the roots and shake it for one to two minutes. After 48 hours for partial destaining, rinse the stained roots in tap water for one to two minutes. Shake the jar vigorously to remove the extra staining solution.
Repeat the procedure until the roots are clear for microscopic evaluation. For root segmentation, place the stained roots on a scaled cutting board. Cut the roots into one centimeter segments.
Choose 15 segments for each variant. Next, spread the roots on a slide for segment preparation, use a laminating pouch to cover the roots and gently crush them, starting from an edge. Use a soft plastic tool to display the roots on the slide.
Carefully remove the laminating pouch and cover the sample with a cover slip. Add water to a corner of the slide with a pipette, and let the water slowly spread on the slide. Remove the extra water with a paper towel.
For microscopic analysis, use a microscope equipped with a good resolution camera. Analyze the slides starting from an extremity. Capture each microscopic field.
Use 10 or 40x magnification for thick roots and 40x for thin roots. After capturing, rename each image with a code that will permit the real post-assemblage of root parts. Use presentation software to design a drawing board for image assemblage, set the width two to three centimeters wider than the image width.
After capturing 15 pictures, add all captured images from one segment in the order of their capture, starting with one to 15, and reconstruct the entire length of the root segment. Next, use the vertical alignment to align the images in the center and ensure that each image follows the previous one. Place a grid of 10 by 150 cells on all pictures to cover the entire root segment.
Additionally, place a 10 by 10 grid on each picture and in every cell of this grid, insert a number from one to six if an arbuscular mycorrhizal or AM structure is visible, or leave blank if no AM structure is present. Add a table for a grid of 10 cells in width and 150 cells in length. Change the table with dimensions to the width of the images, then change the length of the table to comprise all the images.
For scoring mycorrhizal colonization, use the unique number to score each type of structure as described in the mycorrhizal pattern method. Use one for hyphae, two for arbuscules, three for vesicles, four for spores, five for auxiliary cells, and six for entry points. Score each observed mycorrhizal structure from each cell of the previously applied grids.
After scoring, insert all the obtained scores into the MycoPatt spreadsheet. Use the copy paste function to transfer all the scores from the presentation into the first sheet named Raw Data. For the primary analysis, use the third sheet named Parameters to visualize the results as percentages separately in three forms.
Use columns A to K to analyze the horizontal image of colonization, columns M to W for the vertical image of colonization, and columns Y to AI to analyze the transversal colonization for each of the 15 10 by 10 squares and the final average colonization. Next, apply the definitions and formulas specific to each parameter. For production and extraction of mycorrhizal maps, visualize the image obtained from the conversion of mycorrhizal structures code into colors in the second sheet named Graphs.
Then export the resultant image in the graph sheet as an image and use the color code in the legend to analyze mycorrhizal patterns. For mycorrhizal map analysis, identify the most important structures and their assemblage. Then describe the mycorrhizal colonization pattern observed in the analyzed roots, followed by a colonization strategy based on observed structural development in the root, the branching pattern, and arbuscular vesicle development.
This protocol provides the details of mycorrhizal structures with good contrast between mycorrhizal structures and root cells for Zea mays and Festal rubra. The unsuccessful clearing and staining procedures resulted in unclear microscopic images of mycorrhizal structures in Festal rubra and Zea mays. Mycorrhizal colonization patterns allowed a complete exploration of the colonization mechanism, this method provides a deep, small scale exploration of colonization patterns and strategies for Zea mays and Festuca rubra with an additional visual expression of colonization parameters.
In Zea mays highly fluctuating colonization potential depending on the plant's developmental stage was observed, however, in Festuca rubra, the colonization processes occur inside the roots and the development of hyphal networks was correlated with a low development speed of the roots. MycoPatt is suitable for mapping the arbuscular mycorrhizal colonization in any plant. It is also helpful to fully explore the colonization mechanism and assess fungal strategy along the roots.
