A freeze fracture analysis of the surface of embryonic and non-embryogenic suspension cells of Daucus carota

Abstract

Cell surface ultrastructure and exocytosis was studied during one batch cycle in cells of an embryogenic suspension culture of carrot, by means of freeze fracture electron microscopy of samples ultra-rapidly frozen by plunging in liquid propane. The culture of proliferating cells contains three cell types: (i) pro-embryogenic masses (PEMs) attached to (ii) clusters of isodiametric cells and (ii) elongated single cells, the latter deriving from the clusters, as long as the synthetic auxin 2,4-dichlorophenoxy acetic acid (2,4-D) is present in the culture medium. Differences between the three cell types occur in cell wall texture, number of plasmodesmata and number of exocytosis configurations, but not in the number of plasma membrane rosettes. Wall texture is ordered in embryogenic cells, but unordered in elongated, non-dividing cells. Embryogenic cells adhere more closely together and have a higher number of plasmodesmata. Exocytosis configurations occur as small pores, slit-shaped membrane fusion sites with a maximum length of 220 nm and, most abundantly, depressions (in the protoplasmic fracture face), bulges (in the exoplasmic fracture face) and horseshoe-shaped configurations (in both fracture faces), the latter three are 160 ± 20 nm in diameter. Morphology and size of exocytosis configurations are similar and do not change in time in the three cell types, but density differs in time and among cell types. From 1 to 14 days after subculturing the density of the 160 nm exocytosis configurations drops considerably in elongated cells, slightly but with statistical significance in cluster cells and hardly in PEMs.