Embryo isolation and seed clearing

For embryo isolation, seeds were first treated in enzyme solution (1% cellulase R10, 0.5% macerozyme R10, 10.5% mannitol, and 3 mM MES, pH 5.5) for 30 min in the dark. Then droplets of the seed suspension were gently grinded by a flat-headed glass rod. After grinding several droplets of 10.5% mannitol were added for releasing and washing embryo sacs. Living embryos could be further isolated by a second enzymatic maceration procedure followed by a brief micromanipulation.

For seed clearing, whole seeds were collected in a 2ml-centrifuge tube and were fixed by 50% methanol and 10% acetic acid at 4 °C for 12-h and then subjected to an overnight treatment of 1% SDS and 0.2 M NaOH at room temperature. Thereafter, the seeds were rinsed in water, followed by an incubation in 2.5% NaClO for 1-h, and then rinsed again. Next, the seeds were transferred into 1% periodic acid for 1-h at room temperature and in 80% ethanol for 10 min at 80 °C. Subsequently, the seeds were transferred back to the same fixative and incubated for 1-h before the seeds were rinsed again with water. After washing, the seeds were incubated in Schiff reagent with propidium iodide (PI) (P4170; Sigma) (100 mM sodium metabisulphite and 0.15 M HCl; PI to a final concentration of 0.1 mg/ml) for about 3-h. The samples were stored in a chloral hydrate solution (4 g chloral hydrate, 1 ml glycerol, and 2 ml water) and kept overnight at room temperature. Next, the chloral hydrate was replaced by Hoyer’s solution (30 g gum arabic, 200 g chloral hydrate, 20 g glycerol, and 50 ml water). The seeds were kept in the Hoyer’s solution for at least 10 days before observation under a confocal microscope.