Dic microscope

GUS staining was performed as described earlier (Lucas et al., 2011 (link); Racolta et al., 2014 (link)). The transgenic roots were fixed in 90% acetone for 20 min followed by GUS wash solution containing 100 mM Na₂HPO₄/NaH₂PO₄ (pH7.0), 1 mM K₃Fe(CN)₆, 10 mM Na₂EDTA, and 0.1% (V/V) Triton-100. The roots were incubated in staining solution (GUS wash solution and 2 mM X-Gluc) at 37°C in dark for 1 h to overnight. For sectioning, the roots were placed into a drop of water on the slide and cut into slices as thin as possible by hand sectioning (Lux et al., 2005 (link)). Samples are mounted in HCG (8 chloral hydrate: 3 glycerol: 1 ddH₂O) for analysis under DIC microscope (Nikon). For CS observation (Peirson and Dumbroff, 1969 (link); Schreiber and Franke, 2011 (link); Zelko et al., 2012 (link)), the sections were soaked for 1 h in 0.1% (w/v) solution of berberine (Sigma) in distilled water, washed three times with water, stained for 30 min with an aqueous solution of aniline blue (0.5w/v, Polysciences) and rinsed again. The sections were mounted in 50% glycerol and Casparian strip autofluorescence was detected under UV light (405 nm) with Zeiss LSM880. For A. thaliana, the roots were stained by PI (Propidium iodide) and observed under a Zeiss LSM880 confocal microscopy (PI, 561 nm; GFP, 488 nm).

The habitat, locality information and macro-chemical reactions on fresh basidiomata were recorded. Spore prints were taken for each collection. Colour codes were given using Kornerup and Wanscher (1978) as a guide. Microscopic characters were studied in the dried specimens. The following mounting solutions were used to observe the tissues: 10% aqueous potassium hydroxide (KOH) or 28–30% ammonium hydroxide (NH₄OH) solutions or 1% ammoniacal Congo red solution. The microscopic structures were studied at magnifications of 60× and 100×, photographed with a calibrated Nikon Y-TV55 camera, fitted to a Nikon DIC microscope. A total of 60 basidiospores, 30 basidia, 30 pleurocystidia, 30 cheilocystidia and 30 terminal cells and 30 hyphae for both the pileipellis and stipitipellis were measured. The dimensions of the microscopic features are presented in the following format: (a–) b–c–d (−e), in which c represents the average, b the 5^th percentile, d the 95^th percentile and a and e the minimum and maximum values, respectively. Q, the length/width ratio for the spores, is presented in the same format. All microscopic features were drawn by free hand, using a drawing tube. Faces of Fungi (Jayasiri et al. 2015 (link)) and MycoBank numbers are provided for the new species.