5 bromo 4 chloro 3 indolyl beta d glucuronic acid

Anthers containing premeiotic microspores to mature pollen were isolated from wheat plants identified to contain the TaMs1::gusplus cassette. Histochemical GUS activity was detected using 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid (Gold Biotechnology, Inc). The samples were incubated in a 1 mM X-Gluc solution in 100 mM sodium phosphate at pH 7.0, 10 mM sodium ethylenediaminetetraacetate, 2 mM FeK₃(CN)₆, 2 mM K₄Fe(CN)₆ and 0.1% Triton X-100. After vacuum infiltration at 2600 Pa for 20 min, the samples were incubated overnight at 37 °C. Anther samples were then immersed in a fixative solution of 4% sucrose, 1× PBS, 4% paraformaldehyde and 0.25% glutaraldehyde, at 4 °C overnight. The samples were then dehydrated in ethanol series of increasing concentrations (30, 50, 70, 85, 90, 95 and 100%). Tissues were embedded in Technovit® resin, then sectioned on a microtome to a thickness of 8–14 µm, counterstained with ruthenium red and DPX mounted (Sigma, St. Louis, MO) on glass slides. The sections were observed using a LEICA ASLMD laser dissection microscope coupled with a CCD camera (The University of Adelaide microscopy).

The construct pTOOL36-TaMs1::gusplus [16 (link)] was transformed into wheat (cv. Fielder) using Agrobacterium tumefaciens according to Ainur et al., 2014 [35 (link)]. GUS activity in transgenic lines from leaves, roots and anthers containing microspores at pre-meiosis to maturity were analysed by histochemical staining using 5-Bromo-4-chloro-3-indolyl-beta-D-glucuronic acid (Gold Biotechnology, Inc). Samples were incubated in a 1 mM X-Gluc solution in 100 mM sodium phosphate, pH 7.0, 10 mM sodium ethylenediaminetetraacetate, 2 mM FeK₃(CN)₆, 2 mM K₄Fe(CN)₆ and 0.1% Triton X-100. After vacuum infiltration at 2600 Pa for 20 min, samples were incubated 72 h at 37 °C.
Samples were incubated in fixative solution of 4% sucrose, 1x PBS, 4% paraformaldehyde, 0.25% glutaraldehyde, at 4 °C overnight. Samples were subsequently dehydrated in an ethanol series of increasing concentration (30, 50, 70, 85, 90, 95 and 100%). Tissues were then embedded in Technovit® resin, microtome sectioned at 8–14 μm, counter-stained with ruthenium red and then mounted in DPX solution (Sigma, St. Louis, MO). Sections were observed using standard light microscopy on a LEICA DM1000 microscope coupled with a CCD camera. The precipitated product from the β-glucuronidase reaction appears blue under bright field and pink under dark field.

Arabidopsis roots infected with H. schachtii and M. incognita were excised 9 dpi. Histochemical staining for GUS expression was performed at 37 °C for 4 h using X-Gluc solution [(0.1 M NaH₂PO₄, 10 mM EDTA, 0.5 mM each of K₃Fe(CN)₆ and K₄F₂(CN)_6, 3H₂O, 0.1% Triton X-100 and 1 mg/mL 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid (cyclohexylammonium salt) (Gold Biotechnology, St. Louis, MO, USA)], and then mounted onto glass slides. Samples were examined using a Nikon SMZ 800 stereo microscope, and images were captured with a SPOT 2 digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI, USA).