Production and seeding of protoplasts of Porphyra okhaensis (Bangiales, Rhodophyta) in laboratory culture

Abstract

High yields of viable protoplasts were produced from Porphyra okhaensis H. Joshi, Oza & Tewari following two-step enzymatic digestion (protease pretreatment and cell wall polysaccharides-degrading enzyme treatment) of the thallus. Pretreatment of the tissues with 1% Protease P6 at 20 ± 1 • C for 30 min prior to digestion with cell wall polysaccharide-degrading enzymes increased the protoplast yield two fold compared to tissues that were digested with polysaccharide-degrading enzyme mixture. The polysaccharide-degrading enzymes employed for protoplast isolation from P. okhaensis were Cellulase Onozuka R-10, Macerozyme R-10, abalone acetone powder and agarase. Suitable pH, temperature and duration of enzyme treatment for optimal production of viable protoplasts were pH 6, 20 ± 1 • C and 3 h, respectively. Mannitol (0.8 M) was found to be an excellent osmotic stabilizer. When the tissue of P. okhaensis pretreated with 1% protease solution was digested with commercial enzyme mixture consisting of 2% Cellulase Onozuka R-10, 2% Macerozyme R-10, 1% abalone acetone powder, 50 units of agarase and 0.8 M mannitol in 1% NaCl (adjusted to pH 6.0 with 25 mM MES buffer) with gentle agitation for 3 h at 20 ± 1 • C, 23.2 ± 0.24 × 10 6 protoplasts g −1 fresh wt. were obtained. The regeneration rate of protoplasts isolated in the present study was found to be 79%. Protoplasts that regenerated cell walls underwent regular cell divisions and developed into leafy gametophytic thallus in the laboratory cultures. Further, the seeding of nylon threads with partially developed protoplasts of P. okhaensis was successful in the laboratory conditions and germlings as long as 3-4 cm were obtained from such seeded threads in one month period in aerated cultures.

Differential response of varying salinity and temperature on zoospore induction, regeneration and daily growth rate in Ulva fasciata (Chlorophyta, Ulvales)

“…The medium was replenished once every 3 days. The regeneration rate was determined by counting the cells that showed and did not show division after 10 days of incubation under five random microscopic fields in each dish under an inverted microscope as described by Dipakkore et al (2005). In addition, observations were made on each day to follow the germination pattern and thallus ontogeny of the zoospores in cultures incubated at 25°C temperature and 30 psu salinity up to 25-30 days.…”

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

“…Protoplasts serve as excellent experimental materials for research ranging from basic biochemical and morphogenetic studies (Cocking 2000) to expressed sequence tag (EST) analysis (Collén et al 2006) and genetic transformation (Dipakkore et al 2005). Over the last two decades, protocols for successful isolation and regeneration of commercial seaweeds have been published (Cheney et al 1986;Kumar et al 1999;Salvador and Serrano 2005;Dipakkore et al 2005). However, parameters for both isolation and regeneration are very much speciesspecific and depend on various factors ranging from complexity of thallus anatomy, cell wall composition, age and environmental history of the plant.…”

“…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.…”

Reproductive Processes in Red Algal Genus Gracilaria and Impact of Climate Change

“…About 5,800 distinct species have been described in this class (Brodie and Zuccarello, 2007) with a few having immense commercial potential. Some red algal species are edible (Dippakore et al, 2005;Wang et al, 2008), while others are used for the extraction of industrially important phycocolloids such as agar and carrageenan (Zemke-White and Ohno, 1999;Meena et al, 2007). Although in most of the cases, naturally occurring biomass has been harvested for industrial use, the cultivation practice is considerably on the rise for the last couple of decades to meet the surging global market demand (Hanisak, 1998;Ganesan et al, 2006;Subba Rao and Mantri, 2006).…”

“…Considerable progress has been made in the cultivation of Porphyra since then and an average of 400,000 t (wet wt.) Porphyra per year is being produced with a market value of over US$1,500 million (Dippakore et al, 2005).…”