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Life without a cell membrane: regeneration of protoplasts from disintegrated cells of the marine green alga Bryopsis plumosa

Department of Biology, Kongju National University, Kongjushi, Chungnam 314-701, Korea

View larger version (119K): [in a new window]   Fig. 1. The sequential process of sub-protoplast regeneration from disintegrated cells of Bryopsis plumosa. (A) Vegetative plant with distichous branches. (B) Protoplasm comes out from the wounded cell and spreads in seawater. (C) Aggregation of the extruded cell organelles in seawater. (D) Regenerated sub-protoplast with a primary envelope 20 minutes after wounding. (E) The secondary lipid-based membrane inside the primary envelope 12 hours after wounding (pe, primary envelope; sm, secondary membrane). Bars, 10 µm.

View larger version (83K): [in a new window]   Fig. 2. Time-lapse photography of early sub-protoplast regeneration. Bar, 10 µm.

View larger version (22K): [in a new window]   Fig. 3. The size distribution and survival rate of the regenerated sub-protoplasts. Size distribution (line) was studied 1 hour after wounding. Survival rate according to the size of sub-protoplasts (bars) was calculated using the equation %=(number of cells after 24 hours/number of cells after 1 hour)x100.

View larger version (60K): [in a new window]   Fig. 4. DAPI staining and Calcofluor White staining of the regenerated sub-protoplasts. (A) Nuclei stained with DAPI, fluorescence blue. (B) Calcofluor White staining shows accumulation of cellulose on the surface of the primary envelope at 6 hours after wounding. Bars, 10 µm.

View larger version (84K): [in a new window]   Fig. 5. Nile Red staining (orange colour) of lipid and membranes at various times during the regeneration of sub-protoplast. (A) Active movement of lipid material towards the interior of the protoplasmic mass occurred during the first few minutes. (B) The swirling movement of the cell organelles stopped and lipid material was localized to the centre of the protoplasmic mass. (C) The sub-protoplast became spherical and covered with primary envelope, but the envelope is not stained at this stage. (D) The lipid material begins to be incorporated into the envelope at 1 hour after wounding. (E-F) Strong labelling of the primary envelope and secondary membrane. Bars, 10 µm.

View larger version (39K): [in a new window]   Fig. 6. Fluorescein diacetate staining in the regenerated sub-protoplast. (A-D) Confocal laser scanning micrographs of the sub-protoplast regeneration taken at various times post-wounding. Artificial colours indicate an increase of fluorescence intensity inside the sub-protoplast over time. (A'-D') Quantitative analysis of the fluorescence intensities, measured along a line through the central portion of the sub-protoplast (D, distance; Sfi, subtracted fluorescence intensity). Bar, 10 µm.