Protoplasts of Gelidium robustum (Rhodophyta)

Abstract

Viable protoplasts were isolated from apices of the agarophyte Gelidium robustum (Gardn.) Hollenb. & Abb. using a combination of commercial cell-wall degrading enzymes and extracellular wall-degrading enzymes isolated from a marine bacterium. The protoplasts were approximately 8-15 /m in diameter, liberated mainly from the surface cell layers and from cells at the distal ends of medullary filaments. The bacterial enzyme alone was not sufficient to liberate significant numbers of protoplasts. Maximum yield was 9 x 105 protoplasts/g tissue (wet wt.). Optimum osmolality occurred between 1750-1950 mOs kg-1 ; yield and viability were severely diminished at osmolalities less than 1350 mOs kg-. Viability, as determined by fluorescein diacetate staining and Evans Blue exclusion 1 hr after removal from the enzyme solution, was approximately 80-95%. Roughly 80% of the cells did not show Calcofluor fluorescence, while 40% stained positively for the presence of sulfated polysaccharides. Cell wall regeneration was observed with inconsistent reproducibility, and no cell division was observed when the protoplasts were placed in culture medium.

Protoplast isolation and regeneration from Gracilaria changii (Gracilariales, Rhodophyta)

“…To date, protoplasts have been successfully isolated from nine genera of red seaweeds, Porphyra (Kito et al 1998;Dipakkore et al 2005), Kappaphycus (Salvador and Serrano 2005), Grateloupia (Chen and Chian 1994), Bangia (Araki et al 1994), Gelidium (Coury et al 1993), Solieria (Pinchetti et al 1993), Palmaria (Liu et al 1992), Chondrus (Zhang 1991) and Gracilaria (Cheney et al 1986;Björk et al 1990;Chu et al 1998;Araki et al 1998). Few reported the success of protoplast regeneration into whole plants especially for the anatomically complex red seaweeds such as Gracilaria species.…”

Analysis of viability and cell types of macroalgal protoplasts using flow cytometry

“…3), or by fluorescence signatures of photosynthetic pigments, as in Gelidium robustum (Coury et al, 1993) or a mixture of P. perforata and Ulva sp. (Fig.…”

“…For cytometric analysis of seaweeds that generally give low protoplast yields (e.g. Gelidium; Coury et aL, 1993), or of preparations of somatic hybridizations where desirable fusion products make up a very small proportion of the entire population, the only essential purification step is a filtration that excludes debris and clumps large enough to clog the flow bp. Smaller debris can easily be excluded from analyses based on low light-scatter and fluorescence signatures.…”

“…To date, the application of FCM to macroalgal protoplasts has been limited to the detection of two cell types in protoplast preparations from Gelidium robusturn (Coury et al, 1993). The present study was undertaken to determine the feasibility of analyzing samples from macroalgal protoplast preparations with respect to identifying cell types and determining protoplast viability in other macroalgae.…”

Obtaining plantlets from apical meristem of the red alga Gelidium sp.

“…(Polne-Fuller et al, 1984;Chen, 1987Chen, , 1989Chen, , 1998Dipakkore et al, 2005). Obtaining plantlets from protoplasts and callus of highly organized red algae including Gelidium is possible (Garcia-Reina et al, 1988Coury et al, 1993;Rajakrishna Kumar et al, 2004). However, mass cultivation of plantlets grown from cell cultures of complexly organized red algae has not been successful.…”