PROTOPLAST ISOLATION AND CELL DIVISION IN THE AGAR‐PRODUCING SEAWEED GRACILARIA (RHODOPHYTA)¹

Abstract

Methods were developed for the isolation of large numbers of healthy protoplasts from two species of the agarophyte Gracilaria; G. tikvahiae McLachlan and G. lemaneiformis (Bory) Weber‐van Bosse. This is the first report of protoplast isolation and cell division in a commercially important, phycocolloid‐producing red seaweed, as well as for a member of the Florideophycidae. The optimal enzyme composition for cell wall digestion and protoplast viability consisted of 3% Onozuka R‐10, 3% Macerozyme R‐10, 1% agarase and 0.5% Pectolyase Y‐ 23 dissolved in a 60% seawater osmoticum containing 1.0 M mannitol. The complete removal of the cell wall was confirmed by several different methods, including electron microscopic examination, and the absence of Calcofluor White (for cellulose) and TBO (for sulfated polysaccharide) staining. Spontaneous protoplast fusion was observed on several occasions. Protoplast viability was dependent upon the strain and age of the parent material, as well as the mannitol concentration of the enzyme osmoticum. Cell wall regeneration generally occurred in 2‐6 days; cell division in 5‐10 days. Protoplast‐produced cell masses up to the 16‐32 cell stage have been grown in culture. However, efforts to regenerate whole plants have been unsuccessful to date.

Microorganisms as digestors of seaweed cell walls

“…This may indicate that the stainable layer became covered or was changed as differentiation proceded (unpublished observations). Walls of few algae have been dissolved by commercially available enzymes (Miller et al, 1979;Tang, 1982;Saga & Sakai, 1984;Polne-Fuller & Gibor, 1984;Cheney, 1986). Crude enzyme extracted, from entrails of herbivorous snails were useful for dissolving (or in other cases, only softening) walls of only a few brown and red seaweeds (Liu et al, 1984;Polne-Fuller & Gibor, 1984;Fisher, 1986).…”

Protoplasts of Gelidium robustum (Rhodophyta)

“…In the past ten years, protocols for isolation of protoplasts from commercially important red seaweeds such as Porphyra (Polne-Fuller & Gibor, 1984), Gracilaria (Cheney et al, 1986), Gigartina (Gross, 1990), Chondrus (Le Gall etal., 1990), Kappaphycus (E. Zablackis, pers. comm.…”

Parasexual fusion products in green algae: Enteromorpha and Ulvaria (Ulvales, Chlorophyta)

“…Protoplast isolation and hybridization research in seaweeds is a comparatively new field and continues to lag behind in the application of techniques developed for land plants and unicellular algae (Berliner, 1983 ;Cheney, 1986 ;Cheney et al, 1986) . Although protoplast fusion has been reported in several diverse seaweed taxa (Primke et al, 1978 ;Zhang, 1983 ;Cheney, 1986 ;Saga et al, 1986 ;Polne-Fuller & Gibor, 1987), evidence for heterokaryon formation and regeneration appears to be limited to Porphyra yezoensis Ueda (Fujita & Migita, 1987) .…”