Dp21 camera system

To determine whether Hessian fly larvae disrupt the integrity of epidermal cell wall layer, neutral red (NR) staining of crown tissue was carried out to assess permeability at 3 DAH for 6 plants from each of the accessions TA2452, TA2473, and TA1651 as per the method described in Williams et al. [25 (link)]. The 1st leaf from Hessian fly-infested wheat seedlings was carefully peeled off to avoid wounding during the dissection process and expose the crown tissue (feeding site). Uninfested seedlings were also dissected in the same manner and poked with a 0.2 mm minuten pin prior to staining, as positive controls, to mimic wounding. Tissue samples were soaked in aqueous 0.1% (w/v) NR stain (Sigma-Aldrich, St. Louis, MO) for 10 min, and then washed thoroughly in water. Overall intensity of red staining was scored for all plants according to the scale established in Williams et al. [25 (link)] with a score of 0 indicating no stain and 7 being a completely red crown. Following staining, photomicrographs were taken for representative plants using a DP21 camera system on SZX2 stereomicroscope (Olympus).

The DAB (3,3′-diaminobenzidine) staining method was used to detect H₂O₂ accumulation^{94 (link)} in Bd plants in response to Hessian fly larval attack. The Bd plants were grown and infested as described above. DAB staining solution was prepared by dissolving DAB powder (Sigma-Aldrich, St. Louis, MO) in deionized water (1 mg/mL) and the pH of the solution was adjusted to 3.8 with 0.2 N HCl. The DAB solution was kept in dark by covering with aluminum foil. At 1 DAH, six infested Bd plants were dissected under a microscope by cutting below the root/crown junction and the leaf sheaths were removed to expose the larval feeding sites on the main stem. Four uninfested control Bd plants were dissected in the same manner. Each plant was placed in a 15 mL falcon tube covered with aluminum foil. To each tube 10 mL of DAB solution was added, and vacuum infiltrated for 5 min. Following vacuum infiltration, the tubes were shaken at 100 rpm in a MaxQ 4000 Benchtop Orbital shaker (ThermoFisher Scientific, Waltham, MA) for 18 h at room temperature under dark. The plants were then photographed with a DP21 camera system on SZX2 stereomicroscope (Olympus America Inc., Center Valley, PA).