Differential interference contrast microscope

The development of female gametophyte (course of megasporogenesis and megagametogenesis) was studied from the female flowers collected at (i) 3 and 1 day before anthesis, (ii) on the day of anthesis and (iii) 1 and 3 days after anthesis. The details of anthetic stages of the female flowers have been recorded in our earlier work on the species (Mangla and Tandon 2014 ). For semi-thin sections, the flowers were collected and fixed in Karnovsky's fixative (Karnovsky 1965 ), dehydrated in alcohol series and embedded in glycol methacrylate. Sections were cut using a rotary microtome (AO Spencer, USA) and stained with toluidine blue O (Sigma, pH 4.4; Feder and O'Brien 1968 (link)). For ovule clearing, the flowers were fixed in 3.7% formaldehyde, 5% acetic acid, 50% ethanol (FAA) (Ruzin 1999 ). Ovules were dissected out from the pistils under a stereomicroscope, treated with lactic acid solution (saturated with chloral hydrate) for 24 h. The ovules were then washed three times in 70 % ethanol (5 min each), transferred to the clearing solution (Herr 1971 (link)) and kept for 7 days at 28 °C. The cleared ovules were observed under a differential interference contrast microscope (Carl Zeiss, Germany).

An endogenous peroxidase-dependent 3,3-diaminobenzidine (DAB) assay was used to investigate H₂O₂ production in plants (Thordal-Christensen et al., 2010 (link)). To detect H₂O₂ accumulation, leaflets taken 65 h after On infection were immersed in DAB solution (1 mg/ml, pH 3.8) for 8–12 h until DAB staining was visible at the vein of the top leaflet. The DAB-stained leaflets were fixed and stained with Coomassie Brilliant Blue R250 in methanol (0.6%, w/v), following Li (2005) with minor modifications. Samples were observed under a differential-interference contrast microscope (Carl Zeiss, Germany), and images were acquired with a Color Video Camera equipped with image analysis software (Image-Pro Plus 4.1, Media Cybernetics, L.P.). In each microscopically examined sample, we observed more than 200 primary haustoria and secondary haustoria, and recorded percentages of host cells showing HR. Three biological replicates of microscopic samples of both ShORR-1 silenced and control plants were used in these examinations.