Three-dimensional ultrastructure of generative cell mitosis in the pollen tube of Nicotiana tabacum.

Generative cell mitosis was examined in stylar-grown pollen tubes of Nicotiana tabacum using serial sectioning, transmission electron microscopy and computer-assisted reconstruction. Before mitosis, the generative cell has a cage-like organization of cytoplasmic microtubules. The mitotic spindle forms when the cytoplasmic microtubules reduce in frequency and kinetochore microtubules form in an area delimited by sheets of endoplasmic reticulum; no preprophase band of microtubules is observed. At metaphase, 21 pairs of kinetochores are distributed unevenly along the length and depth of the cell without the formation of a strictly planar metaphase plate. The metaphase spindle is highly oblique, with diffuse subpoles distributed along the sides of the cell, colocalized with endoplasmic reticulum lamellae. From these dispersed subpoles the kinetochore bundles emanate, closely associated with tubular endoplasmic reticulum. Anaphase consists of three principal processes: convergence of diffused mitotic poles, shortening of the kinetochore bundles, and the elongation of the spindle by an average of nearly 50%. At mid-anaphase, a phragmoplast begins to form, mainly by the assembly of new microtubules at the equatorial area, which form as a cluster of numerous short microtubules. Cytokinesis is essentially conventional, with centrifugal cell plate formation. Cytoplasmic microtubules are restored in the newly formed "brother" sperm cells in a distribution similar to that in the generative cell but fewer in number.