Isolation of protoplasts from tissue fragments of Philippine cultivars of Kappaphycus alvarezii (Solieriaceae, Rhodophyta)

Abstract

Protoplasts were isolated from tissue fragments (<1 mm 2 ) of three Philippine cultivars of Kappaphycus alvarezii: the giant cultivar, cultivar L and Bohol wild type, by enzymatic dissolution of cell walls. Yields of viable protoplasts from young and old thalli (apical, middle, basal segments) were compared at various temperatures, duration of treatment and pH using eight combinations of commercial enzymes (abalone acetone powder and cellulase), and prepared extracts from fresh viscera of abalone (Haliotis asinina) and a terrestrial garden snail. Isolated protoplasts were grown in various culture media, temperatures, photoperiods and irradiance values to determine the conditions that favor germination and growth.Protoplast yields in tissues treated with commercial enzymes and the garden snail extract were lower than those obtained in tissues treated with fresh abalone extracts. Generally, the number of viable protoplasts increased with duration of enzyme treatment at 25 • C with a maximum yield of 8.2 × 10 3 g −1 tissue at 48 h. Yields were consistently higher in all cultivars at pH 6.1. The yields were also high from the middle segments of the giant cultivar (3.7 × 10 3 g −1 tissue) and Bohol wild type (4.5 × 10 3 g −1 tissue) treated with fresh abalone extract, and from basal segments of cultivar L and tissues treated with garden snail extract. The germination rate of protoplasts was highest (39.8%) at 25 • C and 20 µmol photon m −2 s −1 , using a 12:12 light dark photoperiod. The filament was 3.7 mm long by Day 5. These findings are relevant to developing cultures from protoplasts for genetic or strain improvement of K . alvarezii cultivars.Abbreviations: AAP, abalone acetone powder; Cel, cellulase; EP6, enzyme extract 6 (5% AAP + 2% Cel); UPV, University of the Philippines in the Visayas

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.…”

Production and regeneration of protoplasts from Grateloupia turuturu Yamada (Rhodophyta)

“…Even if variations in protoplast yields have been generally attributed to the physiological, biochemical, and seasonal changes of algal material used (Cheney et al 1986;Reddy et al 2006), the effects of the concentration and the composition of enzymes, the incubation period, and ionic and osmotic strengths, which have been reported to influence protoplast yields (Reddy et al 2006), were evaluated to optimize the isolation protocol. A maximum yield of 1.5×10 7 protoplasts g −1 fresh weight was obtained after tissue has been incubated in an enzyme mixture with 2% cellulase Onozuka R-10, 0.5% macerozyme R-10, and 2% crude extract of H. tuberculata for 4 h. Generally, the incubation times in mixture enzyme for producing Rhodophyta protoplasts are variable according to the protocols for a period ranging from 1 to 48 h. Enzyme incubation periods are only 1-1.5 h for Palmaria palmata (Liu et al 1992), 2 h for Porphyra yezoensis (Liu et al 2004), 2 to 4 h for Gracilaria (Cheney et al 1986;Yeong et al 2008), 18 h for G. sparsa and G. filicina (Chen and Chiang 1994), and until 48 h for Kappaphyccus alvarezii (Salvador and Serrano 2005). The protocol of this study was far more effective in extracting protoplasts from meristematic tissue than from medullary thallus tissue.…”

“…Among soluble carbohydrates, mannitol was chosen as the standard osmotic stabilizer to protect the naked cells from bursting. Mannitol has been reported to be a suitable osmoticum for the isolation of protoplasts from red algae (Cheney et al 1986;Björk et al 1990;Liu et al 1992;Chen andChiang 1994, 1995;Araki et al 1998;Salvador and Serrano 2005;Yeong et al 2008). The optimization of mannitol concentration as an osmoticum in enzyme mixture revealed 0.8 M as optimal for the maximum production of protoplasts in G. turuturu.…”

Isolation of protoplasts and ion channel recording of plasma membrane patches and whole-cell recordings of the marine alga, Ulva pertusa (Chlorophyta)

“…To obtain protoplasts from algae, commercial enzymes such as cellulase, macerozyme, abalone powder, sea snail enzyme, papain and agarase have been used singly or in combination to produce protoplasts (Björk et al 1992;Salvador and Serrano 2005). However, methods for algae protoplast preparation are not universally established compared with those for terrestrial plants, due to the unique cell wall structure in algae and the diversity in cell wall composition (Araki et al 1998).…”