Protoplast is an importanttooi for parasexual modification of genetic content of plant cells (Vasil and Vasil, 1980). Production of algal protoplasts and their fusion are relatively new fields and lag far behind that of terrestrial plants (Berliner 1981, 1983; Cheney et af., 1986). To date, protoplasts have been isolated from several algae, most of which are blue green algae and green algae (Adamich and Hemmingsen, 1980). Protoplasts have been obtained from some marine brown algae (Kloareg and Quantrano, 1987)

Kaladharan, P

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Proc. First Natl. Semi. Mar. Biotech., 1998 .. " , 'II PROTOPLASTS - A POWERFUL TOOL IN GENETIC MANIPULATION OF COMMERCIAL SEAWEEDS P. KALADHARAN Central Marine Fisheries Research Institute, Cochin - 682 014, India INTRODUCTION Protoplast is an importanttooi for parasexual modification of genetic content of plant cells (Vasil and Vasil, 1980). Production of algal protoplasts and their fusion are relatively new fields and lag far behind that of terrestrial plants (Berliner 1981, 1983; Cheney et af., 1986). To date, protoplasts have been isolated from several algae, most of which are blue green algae and green algae (Adamich and Hemmingsen, 1980). Protoplasts have been obtained from some marine brown algae (Kloareg and Quantrano, 1987). In the Rhodophyta, viable protoplasts have been isolated from only two marine genera Porphyra and Graci/aria. Protoplasts have been isolated and subsequently regenerated from four Porphyra species - P. yezoenzis (Saga and Sakei 1984; Fujita and Migita 1985), P. suborbiculata (Tang 1982; Mizukami et al., 1995), P. perforata (Polne-Fuller and Gibor 1984; Saga et al., 1986) and P. nereocystis (Waaland et al., 1990). Cheney et al., (1986) reported the successful isolation of viable protopiasts from Graci/aria tikvahiae and Gracilariopsis leman~iformis, hut their efforts to regenerate whole plants were unsuccessful. NEED FOR GENETICALLY MAMPlJLATED SEAWEEDS In India seaweed resources are commercially exploited for the production of phycocolloids such as agar and sodium alginate. Seaweed industries in India depend entirely on Graci/aria edulis, G. verucosa and Gelidiella acerosa for agar and species of Sargassum and Turbinaria for sodium alginate from the natural beds along the coasts of Tamil Nadu and the Gulf of Kutch. The Indian species of agar yielding Gracilaria are inherently poor yielders of agar (10-12% dry weight) with poor quality (gel strength of 100-150 gtcm2), though they are foliose type and can grow luxuriantly. However, species of Gelidium and Gelidiella are high yielding resources with better gel strength, hut they are crustose type and slow growing seaweeds making their exploitation very difficult. Carrageenan is another important phycocolloid obtained from Bypnea valentiae and H. musciformis in India and the Eucheuma species from the Philippines. The Indian species of Carrageenophytes yield 20% carrageenan, whereas Eucheuma species can yield 40%. The phycocolloid industry in India would become commercially attractive' if a' genetica'lIy 83 manipulated seaweed with fast growth, high biomass production coupled with better yield and quality of product could be achieved through protoplast isolation and somatic hybridization techniques. Isolation of protoplasts Plant cells differ from animal cells in two respects: they have a cellulose cell wall and are held together by their middle lamellae. Isolated protoplasts are living cells from which the walls have been removed. Protoplasts are isolated either mechanically through plasmolysis or enzymatically treating them with cell wall degrading enzymes. Techniques for the removal of the cell wall using cell wall degrading enzymes were developed in the 1960s (Cocking, 1960) and these made possible the production oflarge number of healthy protoplasts from many plant tissues. Since then, protoplasts have become recognized as powerful research tool in physiology, biochemistry and 1nore recently in molecular biology and genetic engineering. Cells are separated fromtissue samples by a sonicator and then treated with a mixture of pectinase, cellulase and macerozyme with reciprocal shaking (40-50 strokes/min). Protoplasts are isolated at 22° C - 28° C. Commercial preparations of cell waUlysing enzymes extracted from fungi as well as sea snail gut enzymes are available in the Market (Table 1). Agar solublising enzymes extracted from marine species of Osci/aloria, an epiphyte on Gracilaria edulis is tried and success is being evaluated in isolation of viable protoplasts from G. edulis (Kaladharan and Seetha, Pers. Commun.) Protoplast preparation almost always contain cellular debris in the form of undigested cuticle, vascular elements, cell walls, tissue 84 fragments, etc. which must be removed rapidly and adequately. Much of the debris can be removed by filtration through miracloth or stainless or nylon filters followed by washing on millipore filters or by centrifugation at low speed «500 rpm). Table 1. Composition of em,yme solution for eell wall degradation (Cltell, 1987) Composition Concentration Sea snail enzyme mixture 2.0g Onozuka R-IO cellulase l.5g R-IO pectinase 0.5 g Sorbitol 9.2g CaClz ·2H2O 80fUg NaHl04. Hp 10mg Ca(Hl04\ . HzO 10mg Distilled water 100ml pH 7 Techniques for the isolation and culture of protoplasts from seaweeds have been developed only recently and the amount of background research in this field is limited. The first report of protoplast isolation was from the green alga Enteromorpha inteslinalis (Millner et al., 1979). Since then protoplasts have been isolated from two genera of red seaweeds viz. Porphyra and Graci/aria, two other genera of green seaweeds viz. Viva and Monostroma and six other genera of brown seaweeds viz. Dictyota, Laminaria, Macrocystis, Vndaria, Sargassum and Sphacelaria. Characteristics of protoplasts The isolated protoplasts assume a spherical shape and acquire some properties common to animal cells. They show budding and can take up even virus particles and chloroplasts by endocytosis. A unique feature of the seaweed protoplast is its ability to synthesis a new cell wall around it in vitro. This reformed cell undergoes divisions to produce a clump of cells (Nagata and Takebe 1970; Takcbe and Nagata, 1973). It is als~ possible to induce differentiation of organs in this tissue and obtain entire plants by suitable experimental manipulation (Vasil and Vasil 1980). Plants derived from protoplasts ar~ useful for the study of the phenomenon of somaclonal variation. The removal of cell wall permits modification of the genetic content through protoplast fusion or by gene transfer. As protoplasts can be regenerated into whole plants, one of their most powerful application is as vehicles for the transfer offoreign genes. One basic prerequisite for potential use of ~rotoplasts in such studies, is the ability to Isolate them readily in large numbers and to culture them in vitro to form cell colonies as welI as whole plants. The period for which the protoplasts remain naked is most important, because it is during this time that they can be specially SUbjected to experimentation. Culture of protoplasts Protoplasts are cultured in vitro as suspension and drop cultures. Protoplasts are suspended in a liquid medium at a density of about 10s/ml and cultured 'in very shallow layers (soft agarose two-layer medium Mizukami et ai., 1995) in 25 ml Erlenmeye; flasks, with or without shaking. Protoplasts suspension at a density of 104 or 10s/ml are placed in 50111 drops in plastic petri dishes sealed with parafilm and incubated. Inorde; to provide adequate humidity in the cultures, drops of water are placed in the middle of the petri dish and monitored regularly with the aid ~f an. ~nverted microscope. Low light mtellSltJes are favourable (50 IlE/m2/S) from the source of white fluorescent tubes 'at desired photoperiod. The medium commonly used for culture of seaweed protoplasts is given in Table 2. Sorbitol or mannitol are used to maintain the osmoticum of pro top lasts. Table 2. Composition ofre-II eulturemediumfor seaweed protoplasts (Cit en, 1982). Composition Concentration (,uM) NaN03 NH4NO l NazSi03 • 9Hp Naz EDTA FcEDTA FeCI) . 6Hp MgS04·7HP NazMn04 • 2Hp H380J NaHl04. Hp CaClz · 2H20 KCL Phenylacetic acid Pyridoxine-HCI p-Hydroxyphenylacetic acid V-33 PI-5X* Kinetin a-NAA JAA Seawater 0.5 0.25 0.2 10 lO 2 5 S 20 0.5 2.0 0.1 00.1 0.2 2.0ml 2.0ml 1.0 1.0 2.0 1000 ml ;) V-3: Thiamine - HCI 0.5 mg Nicotinic acid 0 1 C h , . . mg. a-pantot enate 0/ mg. Biotin 1 p.g. Folic acid 2 mg. Thymine 5 mg. Cobalamme 1 p.g. Inositol 5 mg. Cyanocobalamin Ip.g. *PI-%;,,:MnCI2 7 p.M. ZnCl1 8 x ]0" p.M. CoCI 2 x lO·l p.M. CuCI/fP 2 x 10 -J p.M. 1 PI 5~ and V-3, 2 ml of stock solutio~ into 1000 ml-given reqUIred amounts. The pH ofTC-I 1 medium adjusted to 7.5.- I , •• tIl,,' 85 \ \, Fusion of protoplasts (Somatic hybridization) Genetic manipulation of seaweeds can be achieved by fusion of protoplasts either by chemically using polyethylene glycol (PEG), poly-L-ornithine (PLO), etc. or induced electrically through cell fusion system (Fig. 1). Some cations (Ca++) are also found to increase fusion frequencies. It is therefore possible to produce new intraspecific, interspecific and even intergenei"ic hybrid cells and to regenerate these into whole thallus (K a o and Michayluk, 1974). This technique offers the possibility of transferring genes respons ible for disease resistance , improving th e yield and quality of colloids or stress tolerance Fig. 1. Cell/usioll system btx ecm 2001 model ·86 to the new hybrid strains when this can not be accomplished by s:exual crossing. Protoplast fusion also results in new chloroplastic and mitochondrial gene combinations (Cybridization), so that the cytoplasmic characters can be manipulated and modified. New and unique cytoplasmic e ntities can therefore be created for experimental investigation and possible eventual exploitation in varietal improvement in seaweeds. Although isolation and culture of protoplasts have b e en taken up in many seaweeds, r egeneration has been achieved In El1teromorpha, Ulva, Monostroma and Sphacelaria, but not in any of the complex red or brown seaweeds except Porphyra (Table 3). CONCLUSION Seaweeds are the prime source of phycocolloids such as agar, carrageenan and algin which have important commerical, food and medicinal uses. Although research on seaweed protoplasts lags very far behind that of land crops, steady and intensive works hy a handful of workers during the current decade have rendered spectacular advances in regeneration of protoplasts and even interspecific somatic hybrids. Besides strain im provement through protoplast fusion and somatic hybridization, the expertise on culture of seaweed protoplasts can enable to understand and eventually manipulate the biosynthesis of commercially important cell wall polysaccharides in vitro so as to achieve better yield and superior quality. Table 3. Isolation and culture o/protoplasts/romseaweeds Species Isolation condition Enteromorpha intestinalis 4%driseiase, l.2M sorbitol O.4%pectinase, pH 6.0 Porphyra suborbiculata enzyme extract from Turbo coronatus VIva linza & Monostroma angicava E. linza, Mzostericola & Upertusa Laminariajaponica & P. yezoensis P. peiforata Graci/aria tikvahiae & G. lameneiformis Macrocystis pyrifera & Sargassum muticum E. intestinalis & U angustata P. leucosticta Sphacelaria sp. L. saccharina & L. digitata and cellulase RIO 2% Cellulase RIO 4% & pectolyase 2% in 2M gluscose Cell ulase RIO 1M Mannitol pH 6 crude extract of Strongylocentrotus intermedius in 1.2M Mannitol, O.IM Tris, pH 7 crude extract of Haliotis sp., 0.6M Sorbitol in seawater, pH 6.0 Cellulase RIO 3% macerozyme RIO 3% agarase 1 % & spectolyase 5% extract of Haliotis sp. macerozyme 1 %, pectinase I % and cellulase RIO 2% in l.OM sorbitol, pH 6.0 Cellulase 3% & acerozyme 1% extract of Littorina . lit/orina. cellulase RIO 1.5% & pectinase 0.5% in 0.5M sorbitol cellulysin 2%, pectolyase Lyase Y23 0.5% Alginate Iyases 0.25VA/ml 0.8 mannitol, pH 5.8 cellulase 2%, mannuronate lyase 0.5-5 VArnI, Guluronate lyase 1-2 VA/ml BSAO 0.4%, pH 6.5 Results 85-90% viable cell wall regeneration and cell division . Plant re~eneration Plant regeneration Plant regeneration cell wall regeneration and cell division good yield of protoplasts, no regeneration Plant regeneration from callus fonned Reference Milner eta/., 1979 Tang, 1982 Saga & Sakai, 1984 Polne-fuller & Gibor, 1984 Cheney eta/.,1986 Saga et al.. 1986 cell division, Chen, 1987 plant regeneration in TC 11 medium Plant regeneration from apical cellprotoplasts viability> 80% wall regeneration 87 .. Adamich, M. and B.B. Hemmingsen 1980. Protoplast and spheroplast production. In : E. Grantt (ed .) Handbook of Rhycological Methods - Developmental and Cytological Methods. Cambridge University Press, Cambridge. pp. 153-170. Berliner, M. 1981. Protoplasts of eukaryotic algae. Int. Rev. Cytol., 16: 1-19. Berliner, M. 1983. Protoplasts of nonvascular plants. Int. Rev. Cytoi., 73 : 5-19. Carlson, P. S. 1973. Somatic cell genetics of higher plants. In: F .H. Ruddle, (ed.) Genetic Mechanisms of Development, Acad. Press, New York. pp. 329-353. Chaleff, R.S. and P.S. Carlson 1974. Somatic cell genetics of higher plants. Int. rev. Cytol., 8 : 267-278. Cocking, E.C. 1960. A method for the isolation of plant protoplasts and vacuoles. Nature, 187 : 927-929. Chen, L.c. 1982. Callus-like formation from Irish Moss. Bioi. Bull. Nat'l Taiwan Normal Univ. Taiwan, 17: 63-67. Chen, L.c. 1987. Protoplast morphogenesis of POIphyra leucosticta in culture. Bot. Mar., 30 : 399-403. Cheney, D., E. Mar, N. Saga and 1. van del' Meer 1986. Protoplast isolation and cell division in the agar producing seaweed Gracilaria (Rhodophyta). J. Phycoi., 22: 238-243. Fujita, Y. and S. Migita 1985. Isolation and culture of protoplasts from some seaweeds. Bull. Fac. Fish. Nagasaki Univ. 57 : 39-45. Kao, K.N. and M.R. Michayluk 1974. A method for high frequency intergeneric fusion of plant protoplasts. Planta, 115: 355-367. Kloareg, B. and R. Quatrano 1987. Isolation of pro top lasts from zygotes of Fuclls distichus (L.) Powell (Phaeophyta). Plant Science, 50 : 189-194. Kloareg, B . and R. Quatrano 1989. Isolation of protoplasts of viable protoplasts from Macrocystis pyrifera (L.) C. Ag. (Phaeophyta). Plant Science, 62: 105-112. 88 Millner, P.A., M.E. Callow and L.V. Evans 1979. Preparation of protoplasts from the green alga Enteromorpha intestinalis (L.) Link. Planta, 147: 174-177. Mizukami, Y., M. Okauchi, H. Kito, S. Ishimoto, T. Ishida and M. Fuseya 1995. Culture and development of electrically fused protoplasts from red marine alga Porphyra yezoensis and P. sllborbiculata. Aquaculture, 132: 361-367. Nagata, T. and I. Takebe 1970. Cell wall regeneration and cell division in isolated tobacco mesophyll protoplasts. Planta, 92: 301-308. Polne-Fuller, M. and A. Gibor 1984. Developmental studies in POIphyra. I. Blade differentiation in POIphyra pel/orata as expressed by morphology, enzymatic digestion and protoplast regeneration. 1. Phycoi. , 20 : 609-616. Saga, N. and Y. Sakai 1984. Isolation of protoplasts from Laminaria and POlphyra. Bulletin of the Japanese Society of Scientific Fisheries, 50 : 1085. Saga, N., M. Polne-Fuller and A. Gibor 1986. Protoplasts from seaweeds: Production and fusion. Nova Hedwegia, 83: 37-43. Tang, Y. 1982. Isolation and cultivation of the vegetative cells and pro top lasts of Porphyra suborbiclllata Kjellm. Journal of Shan dong College of Oceanology 12 : 37-50. Takebe, I. and T. Nagata 1973. Culture of isolated tobacco meophyll protoplasts. In : Protoplasts etfusion de cel/lies somatiques vegetales. Coloq. Int. C.N.R.S. France, 212 : 175-187. Vasil, I.K. and V. Vasil 1980. Isolation and culture of protoplasts In: I.K.Vasil (ed.) Perspectives in Plant cell and tissue clliture. Int. rev. Cytol. (Supp!.) lIB: 10-19. Waaland, J.R., L.G. Dickson and B.A. Watson 1990. Protoplast isolation and regeneration in the marine red alga Porphyra nereocystis. Planta, 181 : 522-528. SEAWEED BIOTECHNOLOGY N. KALIAPERUMAL Regional Centre of Central Marine Fisheries Research Institute, Ramnad District - 623 520, Tamil Nadu Proc. First Natl. Semi. Mar. Biotech., 1998: 89-98 Contents Introduction Need and Advantages of Seaweed Culture Methods of Seaweed Cultivation • Vegetative propagation method • Fixed off bottom culture • Floating raft/cage culture • Bottom culture • Green house culture • Spray culture • Raceways culture • Tissue culture • Reproductive propagation method Prospects in Application of Biotechnology in Seaweed Culture for Commercial Scale Production in India Acknowledgement References 

Protoplasts - a powerful tool in genetic manipulation of commercial seaweeds

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Protoplasts - a powerful tool in genetic manipulation of commercial seaweeds

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