DEVELOPMENTAL STUDIES IN PORPHYRA (BANGIOPHYCEAE). II. CHARACTERIZATIO OF THE MAJOR LECTIN BINDING GLYCOPROTEINS IN DIFFERENTIATED REGIONS OF THE PORPHYRA PERFORATA THALLUS1

Detergent soluble extracts of differentiated regions of the Porphyra perforata J. Ag. thallus (holdfast, rhizoidal, vegetative and reproductive cells) were fractionated on sodium dodecyl sulfate polyacrylamide gels. Glycoproteins were identified by their lectin affinity. Extracts from all areas of the thallus contained glycoproteins, but the staining patterns were different for each region with each of the lectins tested: concanavalin A, Ulex europeaus agglutinin, Ricinus communis agglutinin, soybean agglutinin and peanut agglutinin. These data indicate that the morphologically distinct regions of the thallus also differ biochemically. Analysis of the lectin blots revealed the presence of tissue-specific glycoproteins in the five thallus areas. Such unique glycoproteins could be used as markers of differentiation in this species.     

Protoplasts of Gelidium robustum (Rhodophyta)

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 µm 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 × 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^–1. Viability, as determined by flurorescein 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.Dedicated to the memory of Professor Michael Neushul.     

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

The ability to rapidly distinguish viable sub-populations of cells within populations of macroalgal protoplast isolations was demonstrated using flow cytometry. Viable protoplasts from Ulva sp. and Porphyra perforata J. Ag. were distinguished from non-viable protoplasts based on differential fluorescein accumulation. The identities of cortical and epidermal protoplasts from Macrocystis pyrifera (L.) C. Ag. were inferred based on light-scattering and chlorophyll a autofluorescence. Three cell types could be distinguished among protoplasts released from thalli of P. perforata based on chlorophyll a and phycoerythrin autofluorescence. Mixed protoplast populations of Ulva sp. and P. perforata were also discernable based on relative chlorophyll a and phycoerythrin autofluorescence. The ability to screen heterogenous protoplast populations rapidly, combined with the cell sorting capabilities of many flow cytometers, should prove valuable for seaweed biotechnology.     

DEVELOPMENTAL STUDIES IN PORPHYRA. I. BLADE DIFFERENTIATION IN PORPHYRA PERFORATA AS EXPRESSED BY MORPHOLOGY,ENZYMATIC DIGESTION,AND PROTOPLAST REGENERATION1

Four areas containing different cell morphologies were mapped on Porphyra blades and five different cell types (i.e. tapered with long extensions, large and vacuolated, vegetative and dividing, and reproductive: males and females) were identified in them. Tissues from these areas were dissociated, and protoplasts and single cells were isolated from the dissociated tissue of each distinct region. Regeneration rates of the isolated cells and protoplasts (isolates) varied depending on their morphological type. Regeneration rates were lowest in cultured isolates from the area just above the holdfast (ca. 1 %) and increased gradually to over 80% in isolates from areas of vegetative and reproductive regions away from the holdfast. Four distinct morphological patterns were observed among the regenerating plants. Cells isolated from vegetative areas developed into leafy plants while in liquid culture, and into calli when grown on solid medium. Isolates from reproductive areas developed into either a long thin or short thick filamentous plant. Those from ripe patches of carposporangia developed into thin conchocelis filaments, while isolates from non-differentiated cells bordering the ripe reproductive patches developed into thick filaments resembling the morphology of conchosporangial branches. The blade of Porphyra appears simple as it consists of a single cell layer; however, it is complex both morphologically and physiologically.     

A NOVEL TECHNIQUE FOR PREPARATION OF AXENIC CULTURES OF SYMBIODINIUM (PYRROPHYTA) THROUGH SELECTIVE DIGESTION BY AMOEBAE1

The marine amoeba Trichosphaerium Am-I-7 was used as a tool for preparing unialgal axenic cultures of nondigestible Symbiodinium and Porphyridium species. The resistance of these unicellular algae to the amoebal digestive enzymes, and the differential digestion of bacteria, protozoans, and other algae, resulted in cleansed cells of Symbiodinium and Porphyridium that remained in the amoebal food vacuoles. During multiple fission, the amoeba evacuated its food vacuoles and released the trapped and intact algae, which were then successfully cultured. This method of cleaning was especially useful with algal species that were sensitive to antibiotics or other germicidal agents.     

LITHIUM CHLORIDE EXTRACTION OF DNA FROM THE SEAWEED PORPHYRA PERFORATA (RHODOPHYTA)1

Lithium chloride facilitates the softening of cell walls resulting in a simple, quick (2 h) method for DNA extraction from the red seaweed Porphyra perforata J. Agardh. A 5-min treatment of tissues in Lid at 55°C extracts DNA that is relatively free of the viscous polysaccharides and proteins that are usually coextracted in large amounts from cell walls and cytoplasm. This protocol does not require grinding of tissues, hydroxyapatite binding, cetyl trimethyl ammonium bromide treatments, enzymatic treatments, phenol extraction, or CsCl-gradient centrifugation. The resulting DNA is of sufficient quality to be used as a template for polymerase chain reaction amplification.     

DEVELOPMENTAL STUDIES IN PORPHYRA (RHODOPHYCEAE). III. EFFECT OF CULTURE CONDITIONS ON WALL REGENERATION AND DIFFERENTIATION OF PROTOPLASTS1

Protoplasts isolated from thalli of four Porphyra species regenerated successfully into differentiated plantlets. The efficiency of protoplast isolation and the developmental patterns of the regenerating protoplasts depended on the type of tissues from which they were isolated. However, culture conditions greatly influenced the patterns of development at the cellular and organismal levels. Sorbitol, nitrogen, and agar concentration in the medium controlled rates of cell division, thickening of cell walls, development of rhizoids, and formation of calluses or differentiated blades. Agitation disturbed the attachment of the protoplasts to a substrate. Cells in agitated cultures produced suspensions of single cells and non-polarized small calluses. Calluses which developed from protoplasts survived in storage for over two years. The stored calluses, and cells and protoplasts that were isolated from them, were subcultured successfully. We forsee extensive use of Porphyra cell suspensions for strain selection and vegetative propagation of cultivars. This technology, which makes vegetative cloning of selected Porphyra plants possible, may eliminate the need for cultivation and storage of the conchocelis phase. Protoplasts are also being used as tools for studies in genetic engineering of these commercial species.