Fresh ovules were dissected from siliques using forceps and mounted in Hoyer’s solution (chloral hydrate:glycerol:water, 8:1:2 (w/v/v)) for 30 min to 6–8 h depending on the embryo developmental stage (Chen et al., 2015 (link)). Then, the cleared ovules were observed and photographed with differential interference contrast microscopy (Olympus TH4-200 equipped with a CCD of a SPOT digital microscope camera).
To observing endosperm cellularization, we used a reported method (Liu et al., 2014b (link)) modified as followed. The fresh siliques were harvested and fixed in 4% glutaraldehyde in PBS (pH 7.0). After being vacuumed until all pods were sunk in the fixative, the material was placed into fresh fixative and fixed overnight at room temperature. Next, the samples were dehydrated and rehydrated by a series of graded alcohols for 20 min for each gradient. Finally the ovules were dissected from the rehydrated siliques using forceps, and mounted onto the slides with Hoyer’s solution until the tissue was cleared, then observed with 488 nm excitation under a confocal laser scanning microscope (Olympus FluoView FV1000).
To observing endosperm cellularization, we used a reported method (Liu et al., 2014b (link)) modified as followed. The fresh siliques were harvested and fixed in 4% glutaraldehyde in PBS (pH 7.0). After being vacuumed until all pods were sunk in the fixative, the material was placed into fresh fixative and fixed overnight at room temperature. Next, the samples were dehydrated and rehydrated by a series of graded alcohols for 20 min for each gradient. Finally the ovules were dissected from the rehydrated siliques using forceps, and mounted onto the slides with Hoyer’s solution until the tissue was cleared, then observed with 488 nm excitation under a confocal laser scanning microscope (Olympus FluoView FV1000).
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