Fig. 8

Nuclei isolation from protoplasts. a After the protoplast isolation and incubation in nuclei isolation buffer (NIB), the crude mixture of nuclei and cell debris (green solution) is placed on a sucrose gradient. b DAPI staining of the crude solution shows the nuclei (large arrow) attached to the chloroplasts (small arrow). c After the first centrifugation, a top green liquid layer is visible and does not contain any nuclei as determined after staining with DAPI. d A green fraction, between the 0.625 and the 1.25 M sucrose layers is visible, and after DAPI staining shows the presence of the nuclei (large arrow), although cell debris are also present (small arrow) (e). f After the second centrifugation in sucrose gradient, the interface between the 0.625 and 1.25 M sucrose layers contains the pure nuclei fraction (arrow), visible after DAPI staining. g Three layers were observed: a top green layer containing chlorophyll, a white middle layer containing the nuclei, and a third layer in the interface between the 1.25 and 2.5 M sucrose containing cell wall debris and cells that did not release protoplasts (h). Scale bars: 50 µm