Mechanical isolation and ultrastructural characterization of viable egg cells in Plumbago zeylanica

A protocol for isolating viable eggs in Plumbago zeylanica by mechanical dissection is reported. The optimum solution for isolation was 0.8 M mannitol + 10 mM MOPS + 10 mM CaCl2, (pH 4.5-5.0) with an osmolality of 860-940 mmol/kg. Eggs retain their viability for at least 24 h. Isolated eggs were true protoplasts without cell walls and could tolerate osmolality of 437 mmol/kg to 965 mmol/kg. Observation of the isolated eggs using transmission electron microscopy indicated that they were well preserved and reflected the ultrastructure of physiologically active cells, displaying features similar to those of in vivo egg cells. Notable differences include the absence of a filiform apparatus and the accumulation of dense particles in the plastids, which was most conspicuous in egg cells that were damaged during isolation.