Effects of Mps1 Inhibition on PEM Morphology

The analyses of PEM morphology were performed by measuring the area and size of embryogenic cells and suspensor cells. For both analyses, samples were collected before (0) and after 6, 15, 21, and 27 days of incubation without (control) or with (10 μM) Mps1 inhibitor, which showed cellular growth inhibition in the SCV analyses. Samples were collected and prepared on slides, followed by examination under an Axioplan light microscope (Carl Zeiss, Jena, Germany) equipped with an AxioCam MRC5 digital camera (Carl Zeiss). After the images were obtained, area and size were measured using AxioVision LE software, version 4.8 (Carl Zeiss).

The area measurements were performed from PEMs, from the group of embryogenic-type cells that form the embryonal head, and from the suspensor-type cells. For these analyses, for each treatment and each incubation time, three slides were prepared, and at least ten images of PEMs were obtained.

For the cell size analyses, the PEMs were treated with cellulase (Fluka Analytical, Buchs, Switzerland) 0.1% for 3 h to dissociate the embryogenic and suspensor cells of PEM. As embryogenic-type cells are isodiametric, the size was measured based on the diameter, and as suspensor-type cells are elliptic and elongated, the size was measured using the length and width (at the middle of the cell). For these analyses, for each treatment and each incubation time, three slides were prepared, and fifty images from each cell type (embryogenic or suspensor) were obtained.