Szx2 illt

At the beginning of flowering, three buds were collected from the bottom of the main inflorescence of five plants randomly, resulting in 15 buds for each replication. In total, 58,860 buds (4 environments × 327 lines × 3 replications × 15 buds) were sampled. The sampled buds were fixed in FAA, of which 10 randomly selected buds were dissected for further observation. The calyx and petals of sampled bud were removed by a dissecting needle and taken out of the ovary. The ovaries were kept in 2 ml Eppendorf tubes containing 90% alcohol solution twice and then washed with ddH₂O. The trichloroacetaldehyde hydrate solutions were added in a small amount to submerge the sample and make the ovaries transparent. The ovaries were kept in a transparent solution for 12 h to 3 days’ maximum and then transferred to glass slides under the microscope (SZX2-ILLT, Olympus Corporation, Japan). The ovule numbers were counted manually following previously described methods (Yang et al., 2017; Ali, 2018 ; Yu et al., 2020 (link)).

Fertilized eggs were incubated in a constant-temperature incubator maintained at 37 °C and 40%–60% humidity for seven days. Gentle suction was applied to the hole located at the broad end of the egg to create a false air sac directly over the chick chorioallantoic membrane (CAM), and a 1–2 cm² segment was immediately removed from the eggshell. A round gelatin sponge (5 mm × 5 mm) saturated with PSS solution (100 or 200 μg/egg) or saline was placed into the area between the pre-existing vessels, and the embryos were further incubated for 48 h. The zones of neovascularization under and around the gelatin sponge were photographed under a stereomicroscope (SZX2-ILLT, OLYMPUS). The data obtained were analyzed and quantified using ImageJ software (ImageJ 1.8.0, Rawak Software Inc., Stuttgart, Germany).

The horizontal and vertical diameters of the S. paniculata seeds (20 grains) were measured using a vernier caliper with a precision of 0.01mm. while the 1000-seed mass was determined using an electronic balance with a sensitivity of 0.001 g. The viability of fresh S. paniculata seeds was assessed by staining them with 1% TTC (2,3,5-triphenyltetrazolium chloride) in the dark at 25°C for 24 hours. Seed viability was confirmed using stereo-microscopy (SZX2-ILLT; Olympus Corporation, Tokyo, Japan), and seeds were considered viable when both the embryo and endosperm exhibited a red stain. Stereo-microscopy was employed to observe the external morphology and internal structural characteristics of the seeds.

Mussel (Mytilus galloprovincialis) juveniles (0.5 cm in diameter) were sampled in intertidal pools during low neap tides, at Memória Beach, North Portugal (41°13’59” N; 8°43’28” W) and appropriately transported to the laboratory. Mussel plantigrade larvae (0.5–2 mm) were screened among the mussel aggregates in a binocular magnifier (Olympus SZX2-ILLT, Tokyo, Japan), isolated with filtered seawater and gently washed to remove adhered organic particles immediately before the bioassays. The selected M. galloprovincialis plantigrade larvae were further screened for typical explored behaviour before bioassays. The antisettlement bioassays were performed using 5 larvae per replicate in 24-well microplates with 4 replicates per condition during 15 h, at 18 ± 1 °C, in the darkness [30 (link)]. Test solutions were prepared in filtered seawater and obtained by serial dilution (2, 4, 8, 16 and 32 µM) of the compounds stock solution (3.65 mM) in DMSO. A negative control (0.01% DMSO) and a positive control (5 μM CuSO₄) were included in all bioassays. After exposure, the antisettlement activity was determined by the presence/absence of efficiently attached byssal threads produced by each individual larva for all the conditions tested, and the semi-maximum response concentration that inhibited 50% of larval settlement (EC₅₀) and the median lethal dose (LC₅₀) was determined, if applicable.

The morphological response of XEGI50 to V. dahliae was observed under a laser microscope (Olympus SZX2-ILLT, Japan) at different magnifications. Bacteria were incubated in ISP₂ medium. V. dahliae was grown on PDA medium. A 6-day-old mycelial disk (5 mm) was placed at the center of a 7 cm modified culture PDA plate. The bacteria were placed at four corners on the bacterial lawn at four equidistant points of 2.5 cm from the plate periphery. All plates were wrapped with parafilm, incubated at 28 ± 2°C for 6 days, and observed for the inhibition of the pathogen. Plates with pathogenic fungi alone served as control.

M. galloprovincialis juvenile (0.5 cm shell length approximately) aggregates were collected from mussel beds from the intertidal rocky shore, during low neap tides at Memória beach, Matosinhos, Portugal (41°13′59″N; 8°43′28″W), and immediately transported to the laboratory. M. galloprovincialis plantigrade post-larvae (0.5–2 mm) were screened among the small mussel aggregates in a binocular magnifier (Olympus SZX2-ILLT), gently washed with filtered seawater to remove organic debris and sand particles and isolated in a petri dish with filtered seawater immediately before the bioassays.

The sexual structures were observed under a light microscope Axio Imager A2 ( Zeiss, Jena, Germany) and a stereo microscope SZX2-ILLT (Olympus, Tokyo, Japan). The structures were also paraffin sectioned or stained with calcofluor white (50 μg/mL) and detected under a fluorescence microscope Axio Imager A2 (Zeiss, Jena, Germany) or a laser scanning confocal fluorescence microscope LSM880 (Zeiss, Jena, Germany). Cryoelectronic scanning electron microscopy Regulus 8100 (Hitachi, Tokyo, Japan) and transmission electron microscopy H7650 (Hitachi, Tokyo, Japan) were also used to analyze the ultrastructure of the sexual offspring.