Regeneration of plantlets fromEnteromorpha (Ulvales, Chlorophyta) protoplasts in axenic culture

Abstract

High yields of protoplasts have been obtained from vegetative thalli of three species of Enteromorpha by enzymatic degradation of the cell wall. Several commercial and crude enzymes prepared from the digestive system and hepatopancrease of abalone and top-shell were tested at different concentrations and combinations to evaluate the yield. Commercial enzymes in combination with either abalone or top-shell crude enzymes, consistently produced a high yield of protoplasts from all three species. High regeneration rate (85-95 %) occurred in the protoplasts cultured at a density greater than 1.72 x 103 cells cm-2 at 20 and 25 °C . Light intensities tested in the present study did not affect protoplast wall formation and regeneration. Protoplasts, after regenerating the cell wall, followed different types of developmental patterns under identical culture conditions. In one type some cells underwent repeated cell divisions and formed a round and oval shaped hollow thallus with a single layer of cells. In the second type many cells underwent one or two cell divisions (occasionally no division) and soon matured and discharged many motile spores, which on germination grew into normal plantlets. In the third type some cells divided irregularly to form a mass of callus-like cells (except E. prolifera). Culture medium supplemented with either mannitol, sorbitol, dextrose, saccharose or NaCl at higher concentrations (> 0.4 M) inhibited cell division and further differentiation in all species.

Seaweed protoplasts: status, biotechnological perspectives and needs

“…Unlike higher plants and their protoplasts, all cells and protoplasts of seaweeds, with their simple morphological and anatomical thallus structure, regenerate and differentiate directly into a complete thallus, in many cases even without the addition of any growth regulators to the culture medium. Maximum regeneration rate (>90%) of protoplasts in Ulva and Enteromorpha was observed in cultures grown at 20°C and 25°C, while at 30°C there was a lack of differentiation of fronds from the rhizoidal system which grew prominently (Reddy and Fujita 1991). The continued presence of osmoticum >0.4 M in culture medium hampered cell division and further growth in both Ulva and Porphyra .…”

DEVELOPMENT OF PROTOPLASTS OFULVA FASCIATA(CHLOROPHYTA) FOR ALGAL SEED STOCK

“…Protoplasts of U. fasciata were isolated enzymatically with 4% cellulase Onozuka R‐10 and 2% Macerozyme R‐10 (Yakult Honsha Co. Ltd., Tokyo, Japan) in 10 mL of 1.2 M sorbitol solution. All isolation procedures were carried out in the dark (Marchant and Fowke 1977, Cheney et al 1986, Reddy and Fujita 1991) to promote the sinking of isolated protoplasts (Liu et al 1992) at 24° C. The yield of freshly isolated protoplasts was counted with a hemacytometer (Bright‐Line, improved Neubauer, 0.1 mm deep) under a light microscope (Zeiss, Axioskop; Zeiss, Jena, Germany). The experiments in this study were performed at least three times.…”

Induction of fast-growing and morphologically different strains through intergeneric protoplast fusions of Ulva and Enteromorpha (Ulvales, Chlorophyta)

“…Protoplasts from U. pertusa and E. prolifera were prepared separately according to the previous methods (Reddy etal., 1989;Reddy & Fujita, 1991). After isolation, protoplasts were recovered from the debris by filtration through 30 tim mesh and centrifuged.…”

“…Fujita and Saito (1990) also recently demonstrated intraspecific protoplast fusion of Porphyra by electrofusion method. In earlier studies, we have reported the development of suitable techniques for protoplast preparation, culture and regeneration of Ulva and Enteromorpha (Reddy & Fujita, 1991) in axenic culture. In this study we report the intergeneric electrofusion and regeneration of fusion products of protoplasts from Ulva pertusa with Enteromorpha prolifera, including:…”