Figure 1

Isolation of the two-celled proembryos and protoplasts of the apical and basal cells in tobacco. (a) Freshly isolated two-celled proembryos with cell wall (arrows). (b) Two attached protoplasts derived from cell wall-digested two-celled proembryo. The small (arrows) and large protoplasts (arrowheads) are derived from the apical and basal cells of the two-celled proembryos, respectively. (c) Small living protoplasts from the apical cells. (d) Fluorescent image of the same protoplasts in (c), stained with FDA. (e) Large living protoplasts from the basal cells. (f) Fluorescent image of the same protoplasts in (e), stained with FDA. (g) One fertilized embryo sac with a two-celled proembryo (arrow) and a lot of endosperm cells. Inset is a magnification of the two-celled proembryo in embryo sac. (h-j) The detailed isolation process of a pair of protoplasts from a two-celled proembryo in an individual droplet. (k) Cell size of the apical and basal cells, and diameter of the protoplast from the in vivo two-celled proembryos. Bars in (k) show the mean ± standard error (SE), and "n" means the number of measured cells. Bar = 30 μm in Figure a-g; Bar = 15 μm in Figure h and inset; Bar = 10 μm in Figure i and j. **Significant difference at P < 0.01.