Lumar v12

To determine pollen viability, pollen number, and anther size, mature unopened flower buds with indehiscent anthers (anther stage 12–13) were collected and stained in Alexander stain overnight at 4 °C (22 (link)). Stained flower buds were collected and carefully placed onto a microscope slide. Sepals and petals were removed with the help of a thin needle. Style and filament morphology were photographed using a fluorescent dissecting microscope (Lumar V12, Zeiss). Anthers were collected by cutting the filament and putting them onto a separate slide with a coverslip. The coverslip was gently pressed until individual pollen grains were visible. Anthers were then photographed using a compound microscope (Z1 Imager, Zeiss). Pollen number was quantified using the ImageJ software in four independent biological replicates.

Soil with a pH of 5.6 was used for control experiments. For preparing the alkaline substrate, 20 g kg^–1 of CaCO₃ and 12 g kg^–1 of NaHCO₃ were added to the soil and mixed thoroughly, resulting in a pH of 7.2. Col-0 seeds were stratified for 3 days at 4 °C in the dark before they were sown on the soil. Pots were transferred to a growth chamber and plants were grown at 22 °C/18 °C and 16 h/8 h light/dark regime at a light intensity of 120 μmol m^–2 s^–1. To maintain iron available for plants, 2.0 ml of a 4.4 g L^–1 Fe-Sequestrene (6% Iron Chelate, PlantMedia) solution was added to each pot (21 (link)). Twenty-one days after sowing, pictures of the rosette phenotype were taken. Rosette size was quantified using the ImageJ software. All measurements report the average of four independent biological experiments, each involving five seedlings. For the determination of silique length and seed number, plants were grown until maturation, and the first four developed siliques were collected from the primary inflorescence. Collected siliques were then photographed using a fluorescent dissecting microscope (Lumar V12, Zeiss, Germany). Silique length and seed number per silique were analyzed using the ImageJ software. For seed weight, plants were grown until maturation. Seed weight was determined in four independent biological replicates.

Whole-mount images were captured using a stereomicroscope Lumar V12 equipped with bright field and color camera (Zeiss). Pictures of sections were captured using a digital Axiocam MRc camera on a Leica microscope and processed with AxioVision REL 7.8 and Adobe Photoshop CS4 softwares.

The described embryos were removed from incubation, and the cardiovascular phenotype was observed with a stereomicroscope (Zeiss Lumar, V12) and a CCD camera (AxioCam MRc5) at 72 hpf. Abnormal phenotypes of heart looping were observed under the microscope using Tg (cmlc2:GFP) transgenic embryos for the ventricle and atrium at 48 hpf. Haemoglobin staining with o-dianisidine was performed as previously described55 (link). Impaired circulation was defined by the presence of pooled erythrocytes and a low number of circulating erythrocytes. All phenotypes were judged objectively with comparison to the normal phenotype. The counts at each time point were completed separately by two independent observers and averaged.

Transformed L. japonicus roots were treated with a suspension containing 1 μM chitoheptaose (hepta-N-acetylchitoheptaose; Elicityl, Crolles, France) or 10 μg/mL chitin (Sigma-Aldrich) for 4 h. Control plants were treated with corresponding amounts of sterile water. Finally, harvested roots were subjected to histochemical GUS staining using 5-bromo-4-chloro-3-indol-glucuronide cyclohexylamine salt [39 (link)]. Briefly, L. japonicus roots were transferred to 10-mL tubes, then soaked with GUS staining solution and exposed to a vacuum for 20 min. Samples were incubated at 37 °C for 10 h. Finally, roots were rinsed three times with 10% (v/v) commercial bleach and then washed with sterile water. Roots were observed by a stereo microscope and photographed (Lumar.V12, Zeiss, Oberkochen, Germany). Hairy roots with clearly visible blue coloration in at least some root regions were considered as GUS-positive. Transformation efficiency was estimated based on the ratio of GUS-positive hairy roots to the total number of formed hairy roots.