J Appl Phycol (2008) 20:619–632DOI 10.1007/s10811-007-9237-9 Seaweed protoplasts: status, biotechnological perspectives and needs C. R. K. Reddy & Manoj K. Gupta & Vaibhav A. Mantri & Bhavanath Jha Received: 22 April 2007 / Revised and Accepted: 23 July 2007 / Published online: 19 September 2007 # Springer Science + Business Media B.V. 2007 Abstract Protoplasts are living plant cells without cell walls which offer a unique uniform single cell system that facilitates several aspects of modern biotechnology, including genetic transformation and metabolic engineering. Extraction of cell wall lytic enzymes from different phycophages and microbial sources has greatly improved protoplast isolation and their yield from a number of anatomically more complex species of brown and red seaweeds which earlier remained recalcitrant. Recently, recombinant cell wall lytic enzymes were also produced and evaluated with native ones for their potential abilities in producing viable protoplasts from Laminaria. Reliable procedures are now available to isolate and culture protoplasts from diverse groups of seaweeds. To date, there are 89 species belonging to 36 genera of green, red and brown seaweeds from which successful protoplast isolation and regeneration has been reported. Of the total species studied for protoplasts, most belonged to Rhodophyta with 41 species (13 genera) followed by Chlorophyta and Phaeophyta with 24 species each belonging to 5 and 18 genera, respectively. Regeneration of protoplast-to-plant system is available for a large number of species, with extensive literature relating to their culture methods and morphogenesis. In the context of plant genetic manipulation, somatic hybridization by protoplast fusion has been accomplished in a number of economically important species with various levels of success. Protoplasts have also been used for studying foreign gene expression in Porphyra and Ulva. Isolated protoplasts are also exploited in numerous miscellaneous studies involving membrane function, cell structure, C. R. K. Reddy (*) : M. K. Gupta : V. A. Mantri : B. Jha Discipline of Marine Biotechnology and Ecology, Central Salt and Marine Chemicals Research Institute, Bhavnagar 364 002, India e-mail:
[email protected] bio-chemical synthesis of cell walls etc. This article briefly reviews the status of various developments in seaweed protoplasts research and their potentials in genetic improvement of seaweeds, along with needs that must to be fulfilled for effective realization of the objectives envisaged for protoplast research. Keywords Cell wall lytic enzymes . Marine macroalgae . Protoplast isolation . Protoplast fusion . Somatic hybridization . Selection of transformants Introduction Protoplasts are naked living plant cells devoid of cell walls which provide a unique uniform cell system underpinning several aspects of modern biotechnology. Recently, due to public antagonism, especially in Europe, to recombinant DNA technologies (GM crops) there has been renewed interest in exploiting protoplasts in somatic hybridization, cybridization, protoclonal variation studies, proteomics and metabolomics in higher plants (Davey et al. 2005). Interestingly, it was the observation of natural enzymatic degradation of cell walls during fruit ripening that stimulated interest in investigations on preparation of protoplasts using cell wall lytic enzymes in higher plants. Treatment of either cells or tissues with specific cell wall lytic enzymes results in total removal of their rigid and complex polysaccharide cell walls. Although protoplasts isolation from macrophytic benthic marine algae was reported as early as 1970 using mechanical methods (Tatewaki and Nagata 1970; Enomoto and Hirose 1972; Kobayashi 1975), the success in producing a large number of viable protoplasts became possible only after the development of an enzymatic method by Millner et al. DO09237; No of Pages 620 (1979) for Enteromorpha intestinalis (Linnaeus) Nees. Since then, considerable efforts have been made to isolate and culture protoplasts from a wide variety of multicellular marine macrophytic algae (see reviews by Polne-Fuller and Gibor 1987; Evans and Butler 1988; Butler et al. 1990; Reddy 1991; Reddy et al. 1994; Aguirre-Lipperheide et al. 1995). Nevertheless, regeneration of protoplasts to complete thalli, especially from anatomically complex seaweeds as well as giant kelps, is a relatively recent development (Sawabe et al. 1997; Matsumura et al. 2000). Thus, protoplasts can now be prepared and regenerated routinely from a diverse group of seaweed species with ease and success. This article briefly reviews the status of various developments in seaweed protoplasts research and their potential in genetic improvement of seaweeds, along with needs that must to be fulfilled for effective realization of the objectives envisaged for protoplast research. Enzymes used for protoplast isolation Seaweed protoplasts are prepared mainly by employing enzymatic methods. The enzymatic methods require a fair understanding of chemical composition of cell walls. The seaweed cell walls are structurally complex, diverse in chemical composition and differ from land plant cell walls by the abundance of amorphous matrix components as compared to the crystalline skeletal components (Kloareg and Quatrano 1988). As in higher plants, the most common skeletal polysaccharide is cellulose, a linear polymer of Dglucose with a β-1,4 linkages (Kreger 1962). The cellulose content of cell walls in Phaeophyta and Rhodophyta ranges from 1 to 8% of the dry weight of thallus (Black 1950; Ross 1953) and in some Chlorophyta members contain up to 70% (Frei and Preston 1961). In contrast, the members of Caulerpales and Dichotomosiphonales contain skeletal polysaccharides based on β-1,3 xylans, while for members of Codiales and Dasycladales they are based on β-1,4 mannans in place of the most common cellulose framework (Frei and Preston 1961; Mackie and Preston 1968). Similarly, the matrix polysaccharides of seaweeds show great structural variability with significant eco-physiological and commercial interest. In Chlorophyta, the matrix polysaccharides are either xylogalactoarabinans or glucuronoxylorhamnans with varying sulphate contents (Percival and Young 1971; Medcalf et al. 1972). In Rhodophyta, mainly linear sulphated galactans are found composed of two regularly repeated galactose units alternatively linked by β(1–4) and α(1–3) linkages or D-galactose alternatively linked by β(1–4) and α(1–3) linkages. The major matrix component in brown seaweeds (10–45% of the thallus dry wt) is a polyuronide made up of two β-1,4-D-mannuronic acid and α-1,4-L-guluronic acid. J Appl Phycol (2008) 20:619–632 Thus, seaweeds, unlike higher plants, exhibit a variety and complexity in their cell wall composition and, accordingly, a combination of specific enzymes are needed to digest the cell walls completely to isolate protoplasts from different groups. Protoplasts from green seaweeds can be prepared using commercially available cellulases alone or in combination with macerozyme, whereas brown and red seaweeds require alginase and agarase/carragenase, respectively, in addition to commercial cellulases. The latter groups of enzymes are not commercially available or are cost prohibitive. The most common sources of noncellulolytic seaweed cell wall degrading enzymes are either herbivorous marine invertebrates which feed on them or culture filtrates of marine bacteria (Table 1). Most recently, Inoue et al. (2007) cloned for the first time cDNA encoding abalone alginate lyase (HdAly) from hepatopancreas of Abalone in to baculovirus, and confirmed production of recombinant enzyme (rHdAly) following infection with Sf9 insect cell system. The purified enzyme thus produced was as effective in digesting the cell wall matrix as native HdAly. Further, the protoplast yield obtained from Laminaria japonica using rHdAly was also comparable with those yields obtained using native HdAly. The advantage of using recombinant enzymes for protoplast preparation is manifold. First recombinant enzymes are pure and free from several contaminant enzymes, like lipases, proteases and nucleases that are reported to have deleterious effect on protoplast viability. Secondly, the enzyme activity is consistent, free from variations and can be produced at will throughout the year. Protoplast isolation, culture and regeneration 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 (Polne-Fuller and Gibor 1984; 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). The protoplast yields have also been reported to vary with pH, osmoticum type and its concentration in protoplast J Appl Phycol (2008) 20:619–632 621 Table 1 Enzymes isolated from Phycophages and micro-organism for seaweeds protoplast isolation Enzymes Source Reference Agarase Alteromonas sp. Aplysia dactylomela Arthrobacter Aspergillus niger; A.tamari Chaetomium globosum Cytophaga diffluens Delesseria sanguinea Littorina littorea; L. striata Pseudomonas atlantica Pleurotus ostreatus Rhizopus arrhizus Stachybotrys atra Trichoderma viride Uronema marinum Alginovibrio aquatilis Alteromonas sp. H-4 Aplysia dactylomela; A. depilans; A. juliana Diadema antillarum Dolabella auricula Haliotis tuberculata Littorina striata Patella vulgata Pseudomonas alginovora Pseudoalteromonas sp. Sphingomonas sp. Strongylocentrous intermedius Turbo coronatus Vibrio sp. Strain-510 Pseudomonas carrageenovra Delesseria sanguinea Alteromonas sp. ND137 Aplysia juliana Chaetomium globosum Pseudomonas sp. P-1; P.sp. ND 137 Trichoderma viride Aplysia juliana Aeromonas sp. F-25 Acinetobacter sp. Alteromonas sp. ND137 Aspergillus nidulans-051 biA1 Flavobacterium sp. Haliotis iris Pseudomonas sp. ND 137 Vibrio sp. AP-2 Aspergillus niger Chaetomium globosum Pleurotus ostreatus Trichoderma viride Acinetobacter sp. Alcaligenes sp. XY-234 Alteromonas sp. ND137 Aspergillus niger Chaetomium globosum Flavobacterium sp. Pleurotus ostreatus Pseudomonas sp. ND 137; Pseudomonas sp. PT-5 Trichoderma viride Vibrio sp. AX-4 Quatrano and Caldwell 1978 Gomez-Pinchetti and Garcia Reina 1993 Quatrano and Caldwell 1978 Nikolaeva et al. 1999; Patel 1979 Nikolaeva et al. 1999 Nikolaeva et al. 1999 Le Gall et al. 1990 Nikolaeva et al. 1999; Gomez-Pinchetti and Garcia Reina 1993 Chen et al. 1994 Nikolaeva et al. 1999 Payton and Roberts 1979 Nikolaeva et al. 1999 Nikolaeva et al. 1999 Nikolaeva et al. 1999 Stevens and Levin 1979 Sawabe et al. 1997 Gomez-Pinchetti et al. 1989; Boyen et al. 1990a; Wakabayashi et al. 1999 Gomez-Pinchetti et al. 1989 Nisizawa et al. 1968 Boyen et al. 1990a Gomez-Pinchetti et al. 1989 Ducreux et al. 1987 Boyen et al. 1990b Cited from Takamistu et al. 2006 Wong et al. 2000 Saga and Sakai 1984 Tang 1982 Hu et al. 2006 Le Gall et al. 1990; Smith and Bidwell 1989 Le Gall et al. 1990 Yamasaki et al. 1998 Wakabayashi et al. 1999 Nikolaeva et al. 1999 Fujita and Migita 1987; Yukihisa and Yuto 2006 Nikolaeva et al. 1999 Wakabayashi et al. 1999 Araki et al. 1987 Yamasaki et al. 1998 Yamasaki et al. 1998 Packer 1994 Yamasaki et al. 1998 Packer 1994 Yukihisa and Yuto 2006 Araki et al. 1987 Nikolaeva et al. 1999 Nikolaeva et al. 1999 Nikolaeva et al. 1999 Nikolaeva et al. 1999 Yamasaki et al. 1998 Araki et al. 1998 Yamasaki et al. 1998 Nikolaeva et al. 1999 Nikolaeva et al. 1999 Yamasaki et al. 1998 Nikolaeva et al. 1999 Yukihisa and Yuto 2006; Yamaura et al. 1990 Nikolaeva et al. 1999 Araki et al. 1987 Alginate lyases Carrageenase I Carrageenase II Cellulase Laminarinase Mannase Porphyranase Protease Xylanase 622 isolation solution. At higher pH (>7), the initial protoplast release in both Ulva and Enteromorpha was delayed and subsequent yield was less than that obtained for pH 5 and 6 (Reddy, unpublished data). Butler et al. (1990) observed decreased protoplast yields with increased pH values for Laminaria and attributed the low yields to the decreased activity of cellulase. Although mannitol or sorbitol is commonly used as osmotic stabilizer in protoplast isolation and culture solutions, the inorganic osmoticum, NaCl, gave improved yield of protoplasts in Laminaria (Butler et al. 1989). Recently, Uppalapati and Fujita (2002) and Reddy et al. (2006) further simplified the protoplast isolation method by significantly reducing the ionic strength of protoplast isolation medium. These studies revealed that the simple dissolution of cell wall lytic enzymes (2% Cellulase Onozuka R-10) in low ionic medium (1% NaCl in deionised water) provided substantially high yields of viable protoplasts in shorter durations in different green seaweeds than the one prepared with normal seawater. Such enzyme preparations in low ionic strength medium together with macerozyme (2%), abalone acetone powder (1%) and agarase (50 units), also provided high yields of protoplasts when tested for Porphyra pretreated with 1% protease (Shikh et al. 2005). To date, there are 89 species belonging to 36 genera of green, red and brown seaweeds from which successful protoplast isolation and regeneration has been reported (Table 2). Of the total species studied for protoplasts, most belonged to Rhodophyta with 41 species (13 genera) followed by Chlorophyta and Phaeophyta with 24 species each belonging to 5 and 18 genera, respectively. Moreover, the species for which protoplast technology has been developed are primarily either edible or of phycocolloid importance. Many of these studies are developmental and mainly aimed at development of fundamental techniques for successful protoplast isolation and regeneration (rarely for foreign gene expression). Seaweed protoplasts are potentially totipotent. When given the correct chemical and physical stimuli, each protoplast is capable of regenerating a new wall and undergoing repeated mitotic cell division to produce a complete plant. Algal cells and protoplasts are most frequently cultured in a medium which is used to culture the same whole intact thallus. 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 J Appl Phycol (2008) 20:619–632 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 (Reddy et al. 1989; Polne-Fuller and Gibor 1990). The list of species capable of regeneration into complete plant from protoplast is steadily increasing. The plant regeneration from protoplasts of most anatomically complex seaweeds, like Laminaria, Undaria and Gracilaria, has been achieved recently. It is anticipated that the cellular biotechnology, which has been progressing tardily due to failure of regeneration of protoplasts in many seaweed species, may gain momentum and can now be effectively applied for economic gains of seaweed resources. Protoplasts as seed stock for cultivation Several studies have attempted to diversify the application of protoplasts for as many applications as possible. 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). The protoplast-seeded nylon threads contained homogenous establishment of seedlings throughout its surface, due to secondary seeding of spores released from protoplast-derived sporangia developed during regeneration stages of protoplasts. As a result, threads seeded with protoplasts of M. oxyspermum always had a dense growth of germlings derived from both primary and secondary seeding (Reddy et al. 2006). Biochemical studies involving protoplasts Protoplasts have been used for physiological studies in higher plants and are potentially invaluable tools for similar studies in seaweeds. Protoplasts may have advantages over intact tissue due to absence of anionic cell wall which in turn promotes the easier transport of inorganic ions across the membrane. Protoplasts have been used to investigate the mechanism of inorganic carbon uptake in Chondrus crispus (Smith and Bidwell 1989), Ulva rigida (Bjork et al. 1992; Haglund et al. 1992), Gracilaria tenuestipitata (Haglund et al. 1992), and it has been reported that the involvement of an extra-cellular carbonic anhydrase localized either in the cell wall or outside of plasma membrane J Appl Phycol (2008) 20:619–632 623 Table 2 Seaweed species from which protoplast isolation and regeneration have been accomplished Species Chlorophyta Enteromorpha bulbosa E. compressa E. flexuosa E. intestinalis E. linza E. prolifera E. tubulosa Ulva angusta U. conglobata U. fasciata U. lactuca (wild) U. lactuca (mutant) U. pertusa U. pertusa (mutant) U. reticulata U. rigida Monostroma angicava M. latissimum M. nitidum M. oxyspermum M. zostericola Chaetomorpha aerea Bryopsis plumosa Phaeophyta Cladosiphon okamuranus Calpomenia bullosa Dictyopteris undulata D. prolifera Dictyota dichotoma Durvillaea potatorum Ecklonia radiata Ectocarpus siliculosus Eisenia bycyclis Endarachne binghamiae Fucus distichus F. serratus F. vesiculosus Laminaria japonica L. digitata L. saccharina Macrocystis pyrifera M. angustifolia Padina arborescens Status of success Reference PI PR PR PI PR PI PR BS PR PR PR PR PR BS BS PI PR PR PI BS PR PR PI PR PI&GS PR PR PI PR PI BS PR PI BS BS Yamaguchi et al. 1989 Chou and Lu 1989; Reddy and Fujita 1991; Uppalapati and Fujita 2002; Reddy et al. 2006 Reddy et al. 2006 Millner et al. 1979; Saga et al. 1986 Rusig and Cosson 2001 Saga 1984 Fujita and Migita 1985; Chou and Lu 1989; Reddy and Fujita 1991;Uppalapati and Fujita 2002 Amano and Noda 1992 Kawashima et al. 1989; Reddy and Fujita 1991; Uppalapati and Fujita 2002 Reddy et al. 2006 Polne-Fuller and Gibor 1987 Reddy and Fujita 1989; Uppalapati and Fujita 2002; Reddy et al. 2006 Reddy et al. 1989; Chen and Shih 2000; Uppalapati and Fujita 2002; Reddy et al. 2006 Beer and Bjork 1994 Amano and Noda 1992 Reddy et al. 2006; Yamaguchi et al. 1989 Chou and Lu 1989; Reddy et al. 2006 Zhang 1983; Fujimura et al. 1989; Reddy et al. 1989; Uchida et al. 1992; Uppalapati and Fujita 2002 Saga 1984; Yamaguchi et al. 1989 Fujimura and Kajiwara 1990; Amano and Noda 1992 Fujita and Migita 1985; Reddy et al. 1989; Uppalapati and Fujita 2002 Reddy et al. 2006 Bjork et al. 1992 Reddy et al. 2006 Corzo et al. 1995 Zhang 1983; Saga and Kudo 1989 Chen and Chiang 1994a, b Chen 1998 Fujita and Migita 1985; Uppalapati and Fujita 2002 Yamaguchi et al. 1989 Amano and Noda 1992 Krishna Kumar et al. 1999; Uppalapati and Fujita 2002; Reddy et al. 2006 Saga 1984; Yamaguchi et al. 1989 Klotchkova et al. 2003 Kim et al. 2005 PR Uchida and Arima 1992 PI PI PI PI PI PI PR PI PI PR PI PI PI CW BS CW PI PR BS CW PI PI Yamaguchi et al. 1989 Kajiwara et al. 1988 Kajiwara et al. 1988 Kajiwara et al. 1988 Kevekordes et al. 1993 Kevekordes et al. 1993 Kuhlenkamp and Muller 1994 Yamaguchi et al. 1989; Wakabayashi et al. 1999 Wakabayashi et al. 1999 Kloareg and Quatrano 1987 Mussio and Rusig 2006 Mussio and Rusig 2006 Saga and Sakai 1984; Sawabe et al. 1997 Butler et al. 1990 Rodde and Larsen 1997 Butler et al. 1990 Benet et al. 1994 Benet et al. 1997 Rodde and Larsen 1997 Saga et al. 1986; Kloareg et al. 1989; Polne-Fuller et al. 1990 Kevekordes et al. 1993 Yamaguchi et al. 1989 624 J Appl Phycol (2008) 20:619–632 Table 2 (continued) Species Status of success Reference Pilayella littoralis Sargassum muticum Scytosiphon lomentaria Sphacelaria sp. Undaria pinnatifida Rhodophyta Acrosorium polyneurum Bangia atropurpurea Chondrus crispus PR PI PI PR PI Mejjad et al. 1992 Saga et al. 1986; Polne-Fuller et al. 1990 Yamaguchi et al. 1989 Ducreux and Kloareg 1988; Ducreux et al. 1987; Rusig et al. 1994 Fujita and Migita 1985; Tokuda and Kawashima 1988; Wakabayashi et al. 1999 PI PI BS PI PI PI PR PI PI PI CW PI PI PI PI PR PI PI&CW PI CW PI PI PI PI BS PR PI PI PS PI Yamaguchi et al. 1989 Araki et al. 1994 Smith and Bidwell 1989 Le Gall et al. 1990 Coury et al. 1993 Yan and Wang 1993 Cheney 1990 Chou and Lu 1989 Yamaguchi et al. 1989 Chou and Lu 1989 Cheney et al. 1986 Bjork et al. 1990 Chou and Lu 1989 Bjork et al. 1990 Chou and Lu 1989; Bjork et al. 1990 Cheney et al. 1986; Cheney 1990 Bjork et al. 1990; Araki et al. 1998 Mollet et al. 1995 Chen and Chiang 1994a, b Chen and Chiang 1995 Yamaguchi et al. 1989; Chen and Chiang 1994a, b Yamaguchi et al. 1989 Chou and Lu 1989 Zablackis et al. 1993 Zablackis et al. 1993 Salvador and Serrano 2005 Balestri et al. 1989 Liu et al. 1992; Nikolaeva et al. 1999 Gall et al. 2004 Balestri et al. 1989 PI PI PI PI PR PR PR PR PI PI PR PR PI CW PI PR PI PI PR PI PI Packer 1994 Chou and Lu 1989 Chou and Lu 1989; Gall et al. 1993 Gall et al. 1993 Polne-Fuller et al. 1984 Chen 1987 Chen et al. 1988 Waaland et al. 1990 Fujita and Saito 1990 Shikh et al. 2005 Polne-Fuller et al. 1984, 1990; Saga et al. 1986 Fujita and Saito 1990 Fujita and Saito 1990 Tang 1982 Fujita and Saito 1990 Araki et al. 1987 Song and Chung 1988 Fujita and Saito 1990 Saga and Sakai 1984; Fujita and Migita 1985; Araki et al. 1987; Yamaguchi et al. 1989 Fujita and Saito 1990 Gomez-Pinchetti and Garcia Reina 1993 Gelidium robustum Gracialria asiatica G. chilensis G. chorda G. filicina G. gigas G. lemaneiformis G. G. G. G. G. salicornia sordida tenuestipitata tikvahiae verrucosa Grateloupia sparsa G. filicina G. turuturu Halymenia formosa Kappaphycus alvarezii Laurencia obtusa Palmaria palmata Plocamium cartilagineum Porphyra.sp (wild) P. angusta P. crispata P. dentata P. lanceolata P. leucosticta P. linearis P. nereocystis P. okamurae P. okhaensis P. perforata P. pseudolinearis P. seriata P. suborbiculata P. tenera P. tenuipedalis P. yezoensis Solieria filiformis BS, Biochemical study; CW, cell wall formation; PI, protoplast isolation; PR, plant regeneration; GS, genetic study J Appl Phycol (2008) 20:619–632 is responsible for uptake of HCO3− by activating CO2 transporter at low CO2 concentration which in turn increases the efficiency of transporter by providing a faster conversion of HCO3− to CO2. Reciprocally, several studies have attempted to measure the oxygen evolution rate to evaluate whether the isolated protoplasts are in the same physiological state as cells in an intact plant. Protoplasts of Enteromorpha intestinalis had a slightly higher rate of oxygen evolution than thallus fragments (Millner et al. 1979), while protoplasts of Ulva rigida (Bjork et al. 1992), Macrocystis pyrifera (Davison and Polne-Fuller 1990) and Laminaria saccharina (Benet et al. 1994) retained about 65%, 40%, 100% oxygen evolution, respectively, for at least one day. The proteins pattern in protoplast and thalli of some species of green, red and brown seaweed showed variation, although amino acid composition remained the same (Amano and Noda 1992). Fujimura and Kajiwara (1990) were the first to report the production of biflavour compounds from regenerating protoplast of Ulva pertusa. The bioflavours were found to be derivatives of long chain aldehydes that were responsible for typical seaweed odor. Seceration of é-carrageenan was detected in culture media from the protoplasts of Kappaphycus alvarezii var. tambalang after 24 h of culture in the absence of K+ ions (Zablackis et al. 1993). Most recently, Kim et al. (2005) isolated and characterized a novel lectin (Bryohealin) exerting a characteristic activity in the cellular wound healing process during protoplast formation from Bryopsis plumosa, that appeared to have quite different amino acid composition (about 6%) to that of higher plants (less than 1%) and have a molecular mass of 53.8 kDa. Thus, protoplasts provide unique opportunities to study various fundamental aspects of cell wall biochemical studies which are otherwise impossible with normal intact cells of thallus. Protoplast fusion and somatic hybridization Fusing protoplasts of different origins together to produce novel somatic hybrids provides new opportunities to study somatic cell genetics as well as new approaches to genetic manipulation of plants. Table 3 shows the status of protoplast fusion accomplished for seaweeds. The first report of protoplast fusion and fusion product regeneration between two color morphs of Porphyra was that of Fujita and Migita (1987) using PEG method. Although they were able to produce several green and red chimeric thalli, the genetic nature of somatic hybrids has not been reported. Since then, several attempts have been made to accomplish interspecific and intergeneric protoplast fusions in several algae employ- 625 ing both PEG and electrofusion methods (Reddy and Fujita 1989; Cheney 1990; Fujita and Saito 1990; Reddy et al. 1992; Cheney and Kurtzman 1992; Dai et al. 1993; Mizukami et al. 1995). In most cases, the frequency of heterokaryon formation and subsequent recovery of somatic hybrids or cybrids following the protoplast fusions was very low. In order to circumvent this problem, some researchers used freshly released spores as protoplasts to develop hybrid plants. Kapraun (1989, 1990) demonstrated the regeneration of intraspecific-parasexual hybrids from fusion of zoospores of Enteromorpha and Ulvaria. The cytological and karyological studies of germlings regenerated from presumptive fusion products of Enteromorpha and some Ulvaria indicated nuclear fusion following plasmogamy of fused zoospores. The chromosome counts in field collected plants and also 10 week-old control plants showed haploid nuclei with 10 chromosomes, while diploid nuclei with 20 chromosomes were found in germlings of fusion products. Nevertheless, some germlings and fusion products of Ulvaria contained multinucleate cells, presumably resulting from zoospore fusion without nuclear fusion. Recently, Kito et al. (1998) for the first time demonstrated successful trans-divisional protoplast fusion and regeneration of somatic hybrids between Monostroma and Porphyra. The resultant hybrid progeny have also been found to have characteristic fatty acid components as well as DNA of either parent indicating possible genetic recombination in hybrid progeny. In most of the studies, the heterokaryons (heteroplasmic fusion products) have been identified from homokaryons and un-fused parental protoplasts based on visual observation, since the seaweeds and their cells have fascinating colors. Nevertheless, this method of screening is less reliable particularly when identifying the heterokaryons of similar color cells. Therefore, it is essential to develop reliable screening methods, such as differential fluorescence labeling, genetic complementation, physiological complementation or restoration of growth capacity in hybrids upon fusion of inactivated cells with chemical poisons (ethidium bromide, sodium azide or irradiation with X- and gamma rays), for selection of heterokaryons as in higher plants. Genetic transformation The development of genetic transformation procedures for macroalgae is in its inception and in formative stages as compared to crop plants. As expected, the red and brown seaweeds with commercial importance have become the first preferred choice of target species for carrying out genetic transformation studies. The seaweed species for 626 J Appl Phycol (2008) 20:619–632 Table 3 Status of protoplasts fusion and regeneration of somatic hybrids in seaweeds Fusion species Status Reference Ulva linza×Monostroma angicava U. pertusa×U. conglobata U. pertusa×Enteromorpha prolifera Enteromorpha×Porphra yezoensis E. linza×E. linza Gracilaria chilensis×G. tikvahiae Ulvaria oxysperma×U. oxysperma Porphyra yezoensis (red)×P. yezoensis (green) P. yezoensis×P. pseudolinearis P. yezoensis×P. haitanensis P. yezoensis×P. tenera (green) P. yezoensis (green)×P. suborbiculata P. yezoensis×P. vietnamensis P. tenera×P. suborbiculata P. linearis×P. miniata P. suborbiculata×P. tenuipedalis P. yezoensis×Bangia atropurpurea P. pseudolinearis×B. atropurpurea P. yezoensis×Monostroma nitidum Callus formation Plant development Plant development Protoplast fusion Plant development Plant development Plant development Plant development Plant development Callus formation Callus formation Callus formation Callus formation Callus formation Callus formation Callus formation Callus formation Callus formation Plant development Zhang 1983 Reddy and Fujita 1989 Reddy et al. 1992 Saga et al. 1986 Kapraun 1989a Cheney 1990 Kapraun 1990a Fujita and Migita 1987 Fujita and Saito 1990 Dai et al. 1993 Araki and Morishita 1990 Mizukami et al. 1995 Matsumoto et al. 1995 Matsumoto et al. 1995 Chen et al. 1995 Achiha and Nakajima, 1995 Fujita 1993 Fujita 1993 Kito et al. 1998 a Zoospore fusion which genetic transformation has been undertaken are shown in Table 4. The first report of accomplishment of genetic engineering in a macro alga was that of Kurtzman and Cheney (1991), who successfully demonstrated the transient expression of the bacterial β-glucoronidase gene (gus) in the carrageenan-producing red alga Kappaphycus alvarezii. The gus gene was fused to the CaMV 35S promoter and introduced into the algal tissues by microparticle bombardment. Similar results of transient expression of 35S-gus gene construct in the electroporated protoplasts of Porphyra (Kubler et al. 1994) and Ulva (Huang et al. 1996) have also been reported. Subsequently, a number of such studies have been accomplished in different species of economically important seaweeds, such as Porphyra, Gracilaria, Grateloupia, Laminaria and Undaria, by utilizing various gene transfer methods reported in the literature (Kuang et al. 1998; Qin et al. 1994, 1998a, 2003). Liu et al. (2003) for the first time attempted to use homologous promoters for foreign gene expression in Porphyra protoplasts using a portion of 18SrDNA in a vector pQD-GUS, and compared the expression of GUS protein with that of the parent pBS-GUS vector. Table 4 Status of genetic engineering in marine macroalgae Species Method of gene transfer Status of expression Reference Acetabularia mediterranea Chondrus crispus Gracilaria changii Kappaphycus alvarezii Laminaria japonica L. japonica L. japonica L. digitata Porphyra leucosticta P. miniata P. tenuipedalis P. yezoensis P. yezoensis P. yezoensis P. yezoensis P. yezoensis Ulva lactuca Undaria pinnatifida U. pinnatifida Micro-injection Vector mediated Biolistic particle Biolistic particle Biolistic particle Biolistic particle Biolistic particle Vector mediated Electroporation Electroporation Electroporation Electroporation Electroporation Vector mediated Vector mediated Electroporation Electroporation Vector mediated Biolistic particle Stable stable Transient Transient Transient Transient Stable Stable Transient Transient Transient Transient Transient Stable Transient Transient Transient Transient Stable Neuhaus et al. 1986 Collen et al. 2006 Gan et al. 2003 Kurtzman and Cheney 1991 Qin et al. 1998a Qin et al. 1998b Jiang et al. 2003 Roeder et al. 2005 Lin et al. 2001 Kubler et al. 1994 Achiha 1995 Kuang et al. 1998 He et al. 2001 Cheney et al. 2001 Liu et al. 2003 Mizukami et al. 2004 Huang et al. 1996 Yu et al. 2002 Qin et al. 2003 J Appl Phycol (2008) 20:619–632 627 The resultant transformants showed increased GUS activity with pQD-GUS compared with those of pBS-GUS. Bernasconi et al. (2004) described Agrobacterium-mediated transformation of Porphyra yezoensis expressing the bacterial nitroreductase gene (nfsI) and capable of detoxifying trinitrotoluene (TNT). Of the methods used for gene transfer to macroalgae, biolistic bombardment method has been reported to be more effective when thallus explants were used. The Chinese scientists have achieved success in stable transformation of Laminaria japonica using biolistic method. The transformants showed stable expression of LacZ gene (lacZ) and CAT gene (cat) in parthenogenetically regenerated sporophytes (Qin et al. 2005). However, the SV40 promoter reported to be more efficient than CaMV35S in expression of above mentioned genes in Laminaria. Endogenous tubulin promoter has also been used to determine its transcription promoting abilities for foreign gene gusA in protoplasts of Porphyra yezoensis using vector pATubGUS (Gong et al. 2007). For the first time, the Expressed sequence tags (ESTs) from protoplasts and thalli have been used to identify the genes involved in cell wall regeneration and stress responses in Chondrus crispus and Laminaria digitata (Collen et al. 2006; Roeder et al. 2005). The fraction of stress-related ESTs were five times higher in the protoplast than in the thallus library of C. crispus (Collen et al. 2006). The ESTs that were statistically over-represented in protoplasts included: glutathione S-transferases, heat shock proteins, and vanadium bromoperoxidase along with several genes of unknown function, whereas in thallus NADH dehydrogenase, peroxidase ESTs were over-represented together with several genes of unknown function (Collen et al. 2006; Roeder et al. 2005). Since, the protoplast regeneration systems are constantly being developed for all the important seaweeds, it would become much easier and more reliable to select transformants from untransformed ones if protoplasts are used rather than seaweed explants whose regeneration protocols are under-developed. Promoter selection Efficient expression of foreign genes in transformants depends on the promoter used and is crucial for genetic engineering. Promoters from seaweeds or seaweed associated infective virus or bacteria have rarely been isolated and studied. The major drawback in developing reliable methods for transformation of macroalgae is the lack of availability of native algal promoters. The two promoters that have been used most often for constitutive expression are the NOS (nopaline synthase) promoter from A. tumefaciencs T-DNA and 35S promoter from cauliflower mosaic virus (CaMV). The studies carried out to find better promoters for kelp transformation revealed that CaMV35S and fcp (from diatom fucoxanthin, chlorophyll a/c binding protein gene) promoter were found to be more efficient in stable expression of foreign genes than Ubiquitin (from maize) and amt (adenine methyltransferase) promoter (Table 5). Another study using SV40 promoter (from simian virus) resulted in uniform expression of lacZ reporter gene in regenerated Laminaria sporophytes indicating its high transcription recognition efficiency irrespective of tissue specificity (Jiang et al. 2003). The same promoter has also been confirmed to work well with Undaria for both transient and stable expression. There is considerable work going on to understand different gene regulatory domains in algal system for using such regulatory domains as possible algal promoters for efficient stable expression of foreign genes. Table 5 Promoters and reporters genes used for genetic transformation of macroalgae Species Promoter Reporter gene Marker gene Reference Acetabularia mediterranea Kappaphycus alvarezii Porphyra miniata Ulva lactuca Laminaria japonica L. japonica P. yezoensis P. tenera P. leucosticta P. yezoensis Gracilaria changii P. yezoensis L. japonica Undaria pinnatifida P. yezoensis P. yezoensis P. yezoensis SV40 CaMV35S CaMV35S CaMV35S CaMV35S/SV40 CaMV35S/SV40 CaMV35S CaMV35S CaMV35S CaMV35S SV40 Rubisco SV40 SV40 18S rDNA pYez-Rub Tubulin Nil gus gus gus lacZ/cat lacZ/cat gus gus lac Z gus/gfp lacZ gus lacZ/gal lacZ/gus gus gus/gfp/luc gus G418 (pSVneo) — npt-II (pB1121) npt-II (pB1121) — — npt-II — — — — — — — — — amp-R Neuhaus et al. 1986 Kurtzman and Cheney 1991 Kubler et al. 1994 Huang et al. 1996 Qin et al. 1998a Qin et al. 1998b Kuang et al. 1998 Okauchi and Mizukami, 1999 Lin et al. 2001 Cheney et al. 2001 Gan et al. 2003 Hado et al. 2003 Jiang et al. 2003 Qin et al. 2003 Liu et al. 2003 Mizukami et al. 2004 Gong et al. 2007 628 Screening for transformants Another important factor in genetic transformation studies is the selection system for recovery of transgenic cells which depends on the gene present in the expression cassette and selective agent employed. Kanamycin and the hygromycin are two of the commonly used selective agents for transgenic plant cells. The selection is based upon the expression of bacterial neomycin phosphotransferase gene (nptII) and the hygromycin phosphotransferase gene (hptII). Herbicides and other phytotoxic chemicals (bialaphos, chlorsulfuron, sulfonamide, etc.) have also been employed in a few cases. For example, bialaphos resistance gene (bar) from fungus encodes a phosphinothricin acetyltransferase that is able to detoxify the herbicide. Susceptibility of Laminaria and Undaria to nine antibiotics (lincomycin, ampicillin, streptomycin, kanamycin, neomycin, chloramphenical, hygromycin, zeocin, G-418) and one herbicide (basta) has been studied (Qin et al. 1998b). The results indicated that both the species were sensitive only to chloramphenical, hygromycin and basta, and subsequently either one has been used as selectable markers for screening kelp transformants. The recent work in our laboratory with protoplasts of green seaweeds such as Ulva and Monostroma have also shown considerable resistance to antibiotics (hygromycin and kanamycin). This finding has further expanded the scope of the studies to screen for more broad-spectrum antibiotics and herbicides for developing reliable selection systems for recovery of transformants following transformation. Conclusion Reliable procedures are now available to isolate and culture protoplasts-to-plant regeneration from a wide range of seaweeds including anatomically most complex taxa like Laminaria, Undaria and Gracilaria that are among the commercially important genera. Further, the success in protoplast isolation and regeneration of protoplasts provided a tool and impetus to study of somatic cell hybridisation. The studies on interspecific and intergeneric protoplast fusion clearly demonstrated the possibilities of developing new and in certain cases improved strains in seaweeds. The natural colors of seaweeds, to some extent, facilitate direct visual identification of heterokaryons, leading to misidentification of protoplast products of similar color. Hence, it is essential to develop reliable methods, such as differential fluorescence labeling, genetic complementation or physiological complementation, for isolating bi-parental fusion products or heterokaryons. Although protoplasts have been successfully used for transient expression of foreign genes in some seaweeds, the evidence for efficient stable expression is lacking due to poor understanding of different J Appl Phycol (2008) 20:619–632 gene regulatory domains in algal systems. Nevertheless, the following aspects have to be critically studied for effective realization of benefits associated with exploitation of protoplasts for genetic improvement and metabolic engineering of seaweeds for sustainable yields and products: & & & & & Methods for producing viable axenic material with greater consistency are required. Most of the seaweed tissue and protoplast culture studies have so far been developmental studies and have not been applied to select genotypes. It is imperative that homozygous lines for fast growth and high yielding strains of quality gel forming polysaccharides are established. Protoplast fusion studies require development of new methods for recovery of hetero-karyons from those of homo-karyons. It is also necessary to generate data on susceptibility of seaweeds to different broad-spectrum antibiotics and herbicides. This information is useful for selection and recovery of transformants following the genetic engineering techniques. 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