Microscopic observation of giant cells was performed as described by Ji et al. [23 (link)]. The experiment was repeated twice, with 10 galls in each replicate. Each 2-week-old plant was inoculated with 100 J2 s, and root galls were collected at 7 dpi. After fixation in 1× PIPES buffer overnight, root galls were dehydrated in several ethanol dilutions and embedded with Technovit 7100 for 2 weeks. The embedded gall tissues were sectioned into 10-μm slices with a CryoStar NX50 Cryostat (Thermo Fisher Scientific, MA, USA) and stained with 0.05% toluidine blue for 5 min. Microscopic observations were performed using a IX83 research inverted microscope (Olympus Optical Company, Tokyo, Japan) at 40 magnification.
Callose deposition was examined according to Millet et al. [33 (link)]. Rice plants were maintained in 0.04% Si or in control conditions for 2 weeks and inoculated with 100 J2 s each. Ten root galls from each treatment were fixed in an ethanol acetic acid solution overnight and then dehydrated in ethanol dilutions. The root galls were stained with 0.01% aniline blue solution. Callose deposition was examined under UV light using an Eclipse Ti epifluorescence microscope (Nikon Tec. Corporation, Tokyo, Japan) and quantified using ImageJ software.
Callose deposition was examined according to Millet et al. [33 (link)]. Rice plants were maintained in 0.04% Si or in control conditions for 2 weeks and inoculated with 100 J2 s each. Ten root galls from each treatment were fixed in an ethanol acetic acid solution overnight and then dehydrated in ethanol dilutions. The root galls were stained with 0.01% aniline blue solution. Callose deposition was examined under UV light using an Eclipse Ti epifluorescence microscope (Nikon Tec. Corporation, Tokyo, Japan) and quantified using ImageJ software.
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