DEVELOPMENT OF PROTOPLASTS FROM HOLDFASTS AND VEGETATIVE THALLI OF MONOSTROMA LATISSIMUM (CHLOROPHYTA, MONOSTROMATACAE) FOR ALGAL SEED STOCK

Abstract

The aim of this study was to isolate and cultivate the protoplasts of the green alga Monostroma latissimum Wittrock and subsequently induce them to form algal filaments to act as an algal “seed” stock. Protoplasts of the alga were isolated enzymatically with 4% cellulase Onozuka R‐10 and 2% Macerozyme R‐10. The highest number of protoplasts was obtained on a 50‐rpm shaker with 1.2 M of sorbitol after 6 h of incubation, with a yield of 9 × 106 protoplasts·g−1 of fresh thallus (including holdfast). Protoplasts from both holdfasts and erect thalli usually began to form new cell walls within 5 h after isolation and began to divide from day 6 to day 9 in PES medium; cell clusters, filaments, and/or tubular thalli were formed from day 14 to day 18. For algae collected in March, about 60% of protoplasts isolated from vegetative thalli regenerated to form tubular thalli, and about 45% of protoplasts isolated from holdfasts regenerated to form filaments. However, for algae collected in May, about 1% of protoplasts isolated from vegetative thalli developed directly to form tubular thalli, and 59% of protoplasts regenerated to form cell clusters without the ability to differentiate, whereas protoplasts isolated from holdfasts failed to develop. Regenerated filaments were kept in an incubator for more than 3 years at 24° C under the low irradiance of 66μmol photons·m−2·s−1. After this time, they retained the ability to develop to form tubular thalli under irradiance of 166 and 300 μmol photons·m−2·s−1 at 18°–30° C. Subsequently, these tubular thalli can develop to form leafy thalli after being cultivated at high irradiance of 300 μmol photons·m−2·s−1 and at 18°–22° C. Therefore, the filaments could serve as“seed” stock for algal mass culture.

Seaweed protoplasts: status, biotechnological perspectives and needs

“…The yield, viability and regeneration rate of isolated protoplasts is dependent on several factors, including the enzyme constituents and their concentrations, pH, osmotic conditions and ionic strength of protoplast isolation medium, incubation temperature, physiological state and age of donor plant, and protoplast culture medium and its culture conditions Cheney et al 1986;Bjork et al 1990;Butler et al 1990;Chen 1998;Krishna Kumar et al 1999;Chen and Shih 2000;Matsumura et al 2000;Shikh et al 2005;Reddy et al 2006). Pretreatment of thallus with proteases (protease or papain) or plasmolytic solutions was also carried out prior to enzymatic treatment to obtain enhanced yields of viable protoplasts from some members of Chlorophyta and Rhodophyta (Kawashima et al 1989;Yamaguchi et al 1989;Fujita and Saito 1990;Bjork et al 1992;Amano and Noda 1992;Chen and Chiang 1994a, b;Wakabayashi et al 1999).…”

“…In order to provide continuous seed stock for cultivation of green seaweeds, Chen (1998) and Chen and Shih (2000) developed protoplast-based methods for producing stocks of seedlings (micro thalli) with survival abilities of many years in an incubator while maintaining their potential to develop into leafy thalli of Ulva and Monostroma. Protoplasts from Monostroma and Porphyra have been successfully tested for their seeding and regeneration in laboratory conditions (Reddy et al 2006;Shikh et al 2005).…”

Detection of Epigenetic Variations in the Protoplast-Derived Germlings of Ulva reticulata Using Methylation Sensitive Amplification Polymorphism (MSAP)

“…The differentiation of protoplasts into various morphotypes has been reported for a wide range of seaweeds (Reddy et al 1989; Huang et al 1996; Chen 1998; Krishnakumar et al 1999; Chen and Shih 2000; Rusig and Cosson 2001; Dipakkore et al 2005). This is the first attempt made to understand the developmental polymorphism of seaweed protoplasts from the genome perspective.…”

“…Unlike higher plants, seaweed protoplasts regenerate and differentiate into a full thallus without any amendments of phytohormones to culture medium. Nevertheless, the protoplasts from green seaweeds followed different types of regeneration patterns and gave rise to several phenotypically variable morphotypes such as sporangia, microthalli, saccate (or spherical), tubular (or spindle), irregular, and frondose with various life spans (Reddy et al 1989; Huang et al 1996; Chen 1998; Krishnakumar et al 1999; Chen and Shih 2000; Rusig and Cosson 2001). Also, in the red alga Porphyra , three types of protoplast regeneration patterns, i.e., callus, filamentous, and conchocelis have been described (Polne-Fuller and Gibor 1984; Fujita and Migita 1985; Waaland et al 1990; Dipakkore et al 2005).…”

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

“…seedstock from callus cells initially obtained from protoplasts) has been achieved for green algae (Reddy et al, 1989;Reddy & Fujita, 1991;Chen & Shin, 2000;Rusing & Cosson, 2001), for some brown algae (Ducreux & Kloareg, 1988;Matsumura et al, 2000) and for simply organized red algae such as Porphyra spp. (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).…”