PROTOPLASTS OF GELIDIUM-ROBUSTUM (RHODOPHYTA)

Viable protoplasts were isolated from apices of the agarophyte Gelidiu m robustum (Gardn.) Hollenb. & Abb. using a combination of commercial cell-wall degrading enzymes and extracellular wall-degrading enzymes i solated from a marine bacterium. The protoplasts were approximately 8- 15 mum in diameter, liberated mainly from the surface cell layers and from cells at the distal ends of medullary filaments. The bacterial en zyme alone was not sufficient to liberate significant numbers of proto plasts. Maximum yield was 9 x 10(5) protoplasts/g tissue (wet wt.). Op timum osmolality occurred between 1750-1950 mOs kg-1; yield and viabil ity were severely diminished at osmolalities less than 1350 mOs kg-1. Viability, as determined by fluorescein diacetate staining and Evans B lue exclusion 1 hr after removal from the enzyme solution, was approxi mately 80-95%. Roughly 80% of the cells did not show Calcofluor fluore scence, while 40% stained positively for the presence of sulfated poly saccharides. Cell wall regeneration was observed with inconsistent rep roducibility, and no cell division was observed when the protoplasts w ere placed in culture medium.