Abstract
Protoplasts were isolated enzymatically from the carrageenophyte red alga Grateloupia turuturu (Halymeniales, Rhodophyta) that occurs along the coast of the French Channel in Normandy. Effects of the main factors on the protoplast yield were identified to improve the isolation protocol. The optimal enzyme composition for cell wall digestion and protoplast viability consisted of 2% cellulase Onozuka R-10, 0.5% macerozyme R-10, 2% crude extract from viscera of Haliotis tuberculata, 0.8 M mannitol, 20 mM sodium citrate, 0.3% bovine serum albumin at 25°C, and 4-h incubation period. The protoplasts were approximately 5–15 μm in diameter, liberated mainly from the surface cell layers. Maximum yield was 1.5 × 107 protoplasts g-1 fresh tissue. The protoplasts underwent initial division after 14 days with a high density level of 1 × 106 cells mL-1 in culture medium and developed into microthalli of a line of two to six cells.
This is a preview of subscription content,
to check access.




References
Araki T, Lu Z, Morishita T (1998) Optimization of parameters for isolation of protoplasts from Gracilaria verrucosa (Rhodophyta). J Mar Biotechnol 6:193–197
Athukorala Y, Jung WK, Park PJ, Lee YJ, Kim SK, Vasanthan T, No HK, Jeon YJ (2008) Evaluation of biomolecular interactions of sulfated polysaccharide isolated from Grateloupia filicina on blood coagulation factors. J Microbiol Biotechnol 18:503–511
Barreiro R, Quintela M, Barbara I, Cremades J (2006) RAPD differentiation of Grateloupia lanceola and the invasive Grateloupia turuturu (Gigartinales, Rhodophyta) in the Iberian Peninsula. Phycologia 45:213–217
Björk M, Ekman P, Wallin A, Pedersén M (1990) Effects of growth rate and other factors on protoplast yield from four species of Gracilaria (Rhodophyta). Bot Mar 33:433–439
Butler DM, Østgaard K, Boyen C, Evans LV, Jensen A, Kloareg B (1989) Isolation conditions for high yields of protoplasts from Laminaria saccharina and L. digitata (Phaeophyceae). J Exp Bot 40:1237–1246
Cabioch J, Castric-Fey A, L’Hardy-Halos MT, Rio A (1997) Grateloupia doryphora et Grateloupia filicina var. luxurians (Rhodophyta, Halymeniaceae) sur les côtes de Bretagne (France). Cryptogam, Algol 18:117–137
Chattopadhyay K, Mateu CG, Mandal P, Pujol CA, Damonte EB, Ray B (2007) Galactan sulfate of Grateloupia indica: isolation, structural features and antiviral activity. Phytochemistry 68:1428–1435
Chen LCM (1987) Protoplast morphogenesis of Porphyra leucosticta in culture. Bot Mar 30:399–403
Chen YC, Chiang YM (1994) Development of protoplasts from Grateloupia sparsa and G. filicina (Halymeniaceae, Rhodophyta). Bot Mar 37:361–366
Chen YC, Chiang YM (1995) Ultrastructure of cell wall regeneration from isolated protoplasts of Grateloupia sparsa (Halymeniaceae, Rhodophyta). Bot Mar 38:393–399
Cheney DP, Mar E, Saga N, van der Meer J (1986) Protoplast isolation and cell division in the agar-producing seaweed Gracilaria (Rhodophyta). J Phycol 22:238–243
Chiang YM (1993) The developmental sequence of the marine red alga Grateloupia filicina in culture. The Korean Journal of Phycology 8:231–237
D’Archino R, Nelson WA, Zuccarello GC (2007) Invasive marine red alga introduced to New Zealand waters: first record of Grateloupia turuturu (Halymeniaceae, Rhodophyta). NZ J Mar Freshw Res 41:35–42
Denis C, Le Jeune H, Gaudin P, Fleurence J (2009) An evaluation of methods for quantifying the enzymatic degradation of red seaweed Grateloupia turuturu. J Appl Phycol 21:153–159
Farnham WF (1980) Studies on aliens in the marine flora of southern England. In: Price JH, Irvine DEG, Farnham WF (eds) The shore environment 2: Ecosystems. Academic Press, London, pp 875–914
Fujiwara-Arasaki T, Mino N, Kuroda M (1984) The protein value in human nutrition of edible marine algae in Japan. Hydrobiologia 116(117):513–516
García-Jiménez P, Robaina RR, Luque A, Tsekos I (1996) Glycerol-activated cellular division and biosynthetic activity during growth and morphogenesis of carpospore seedlings of Grateloupia doryphora (Cryptonemiales, Rhodophyta). Phycologia 35:261–269
García-Jiménez P, Rodrigo M, Robaina RR (1998) Influence of plant growth regulators, polyamines and glycerol interaction on growth and morphogenesis of carposporelings of Grateloupia cultured in vitro. J Appl Phycol 10:95–100
Gavio B, Fredericq S (2002) Grateloupia turuturu (Halymeniaceae, Rhodophyta) is the correct name of the non-native species in the Atlantic known as Grateloupia doryphora. Eur J Phycol 37:349–359
Gómez-Pinchetti JLG, Björk M, Pedersén M, Reina GG (1993) Factors affecting protoplast yield of the carrageenophyte Solieria filiformis (Gigartinales, Rhodophyta). Plant Cell Rep 12:541–545
Harlin MM, Villalard-Bohnsack M (2001) Seasonal dynamics and recruitment strategies of the invasive seaweed Grateloupia doryphora (Halymeniaceae, Rhodophyta) in Narragansett Bay and Rhode Island Sound, Rhode Island, USA. Phycologia 40:468–474
Huang W, Fujita Y (1997) Callus induction and thallus regeneration in some species of red algae. Phycol Res 45:105–111
Huang W, Fujita Y, Ninomiya M, Ohno M (1999) Seed production and cultivation of Grateloupia turuturu (Cryptonemiales, Rhodophyta) by callus culture. Bulletin of Marine Sciences and Fisheries 19:1–7
Hudson JB, Kim JH, Lee MK, DeWreede RE, Hong YK (1999) Antiviral compounds in extracts of Korean seaweeds: evidence for multiple activities. J Appl Phycol 10:427–434
Iima M, Kinoshita T, Kawaguchi S, Migita S (1995) Cultivation of Grateloupia acuminata (Halymeniaceae, Rhodophyta) by regeneration from cut fragments of basal crusts and upright thalli. J Appl Phycol 7:583–588
Ishikawa Y (1984) New seaweed resources 2: cultivation of Grateloupia filicina. Suisan no Kenkyu 3:57–60
Kevekordes K, Beardall J, Clayton MN (1993) A novel method for extracting protoplasts from large brown algae. J Exp Bot 44:1587–1593
Kim KY, Nam KA, Kurihara H, Kim SM (2008) Potent α-glucosidase inhibitors purified from the red alga Grateloupia elliptica. Phytochemistry 69:2820–2825
Kloareg B, Quatrano RS (1987) Isolation of protoplasts from zygotes of Fucus distichus (L.) Powell (Phaeophyta). Plant Sci 50:189–194
Kloareg B, Quatrano RS (1988) Structure of the cell walls of marine algae and ecophysiological functions of the matrix polysaccharides. Oceanogr Mar Biol: Annu Rev 26:259–315
Kloareg B, Polne-Fuller M, Gibor A (1989) Mass production of viable protoplasts from Macrocystis pyrifera (L.) C. Ag. (Phaeophyta). Plant Sci 62:105–112
Kuhlenkamp R, Müller DG (1994) Isolation and regeneration of protoplasts from healthy and virus-infected gametophytes of Ectocarpus siliculosus (Phaeophyceae). Bot Mar 37:525–530
Le Gall Y, Braud JP, Kloareg B (1990) Protoplast production in Chondrus crispus gametophytes (Gigartinales, Rhodophyta). Plant Cell Rep 8:582–585
Liu QY, Chen LCM, Taylor ARA (1992) Ultrastructure of cell wall regeneration by isolated protoplasts of Palmaria palmata (Rhodophya). Bot Mar 35:21–33
Liu H, Yu W, Dai J, Gong Q, Yang K, Lu X (2004) Cryopreservation of protoplasts of the alga Porphyra yezoensis by vitrification. Plant Sci 166:97–102
Marston M, Villalard-Bohnsack M (2002) Genetic variability and potential sources of Grateloupia doryphora (Halymeniaceae, Rhodophyta), an invasives species in Rhode Island waters (USA). J Phycol 38:649–658
Mejjad M, Loiseaux-de-Goër S, Ducreux G (1992) Protoplast isolation, development, and regeneration in different strains of Pilayella littoralis (L.) Kjellm. (Phaeophyceae). Protoplasma 169:42–48
Migita S (1988) Cultivation of Grateloupia filicina (Rhodophyta, Cryptonemiales) by regeneration of crusts. Nippon Suisan Gakkaishi 54:1923–1927
Mollet JC, Verdus MC, Kling R, Morvan H (1995) Improved protoplast yield and cell wall regeneration in Gracilaria verrucosa (Huds.) Papenfuss (Gracilariales, Rhodophyta). J Exp Bot 46:239–247
Mussio I, Rusig AM (2006) Isolation of protoplasts from Fucus serratus and F. vesiculosus (Fucales, Phaeophyceae): factors affecting protoplast yield. J Appl Phycol 18:733–740
Mussio I, Rusig AM (2009) Morphogenetic responses from protoplasts and tissue culture of Laminaria digitata (Linnaeus) J.V. Lamouroux (Laminariales, Phaeophyta): callus and thalloid-like structures regeneration. J Appl Phycol 21:255–264
Pang SJ, Xiao T, Shan TF, Wang ZF, Gao SQ (2006) Evidences of the intertidal red alga Grateloupia turuturu in turning Vibrio parahaemolyticus into non-culturable state in the presence of light. Aquaculture 260:369–374
Plouguerné E, Hellio C, Deslandes E, Véron B, Stiger-Pouvreau V (2008) Anti-microfouling activities in extracts of two invasive algae: Grateloupia turuturu and Sargassum muticum. Bot Mar 51:202–208
Polne-Fuller M, Gibor A (1984) Development studies in Porphyra. I. Blade differentiation in Porphyra perforata as expressed by morphology, enzymatic digestion and protoplast regeneration. J Phycol 20:609–616
Provasoli L (1968) Media and prospects for the cultivation of marine algae. In: Watanabe A, Hattori A (eds) Culture and collection of algae. Japanese Society Plant Physiology, Hakone, pp 63–75
Qin S, Jiang P, Tseng CK (2004) Molecular biotechnology of marine algae in China. Hydrobiologia 512:21–26
Reddy CRK, Dipakkore S, Kumar GR, Jha B, Cheney DP, Fujita Y (2006) An improved enzyme preparation for rapid mass production of protoplasts as seed stock for aquaculture of macrophytic marine green algae. Aquaculture 260:290–297
Ribera MA, Boudouresque CF (1995) Introduced marine plants, with special reference to macroalgae: mechanisms and impact. Prog Phycol Res 11:187–268
Robaina RR, Garcia P, Garcia-Reina G, Luque A (1990a) Morphogenetic effect of glycerol on tissue cultures of the red seaweed Grateloupia doryphora. J Appl Phycol 2:137–143
Robaina RR, Garcia-Reina G, Luque A (1990b) The effects of the physical characteristics of the culture medium on the development of red seaweeds in tissue culture. Hydrobiologia 204/205:137–142
Robaina RR, García-Jiménez P, Luque A (1996) Light and glycerol driven development of Grateloupia doryphora (Rhodophyta) in vitro. Sci Mar 60:283–289
Saga N, Polne-Fuller M, Gibor A (1986) Protoplasts from seaweeds: production and fusion. Beih Nova Hedwigia 83:37–43
Salvador RC, Serrano AE (2005) Isolation of protoplasts from tissue fragments of Philippine cultivars of Kappaphycus alvarezii (Solieriaceae, Rhodophyta). J Appl Phycol 17:15–22
Saunders GW, Withall RD (2006) Collections of the invasive species Grateloupia turuturu (Halymeniales, Rhodophyta) from Tasmania, Australia. Phycologia 45:711–714
Shao K, Wang J, Zhou B (2004) Production and application of filaments of Grateloupia turuturu (Halymeniaceae, Rhodophyta). J Appl Phycol 16:431–437
Simon C, Ar Gall E, Deslandes E (2001) Expansion of the red alga Grateloupia doryphora along the coasts of Brittany (France). Hydrobiologia 443:23–29
Verlaque M, Brannock PM, Komatsu T, Villalard-Bohnsack M, Marston M (2005) The genus Grateloupia C. Agardh (Halymeniaceae, Rhodophyta) in the Thau Lagoon (France, Mediterranean): a case study of marine plurispecific introductions. Phycologia 44:477–496
Villalard-Bohnsack M, Harlin MM (1997) The appearance of Grateloupia doryphora (Halymeniaceae, Rhodophyta) on the northeast coast of North America. Phycologia 36:324–328
Villalard-Bohnsack M, Harlin MM (2001) Grateloupia doryphora (Halymeniaceae, Rhodophyta) in Rhode Island waters (USA): geographical expansion, morphological variations and associated algae. Phycologia 40:372–380
Wang SC, Bligh SWA, Shi SS, Wang ZT, Hu ZB, Crowder J, Branford-White C, Vella C (2007) Structural features and anti-HIV-1 activity of novel polysaccharides from red algae Grateloupia longifolia and Grateloupia filicina. Int J Biol Macromol 41:369–375
Wong SL, Chang J (2000) Salinity and light effects on growth, photosynthesis, and respiration of Grateloupia filicina (Rhodophyta). Aquaculture 182:387–395
Yamaguchi K, Araki T, Aoki T, Tseng CH, Kitamikado M (1989) Algal cell wall-degrading enzymes from viscera of marine animals. Nippon Suisan Gakkaishi 55:105–110
Yeong HY, Khalid N, Phang SM (2008) Protoplast isolation and regeneration from Gracilaria changii (Gracilariales, Rhodophyta). J Appl Phycol 20:641–651
Yokoya NS, Handro W (1996) Effects of auxins and cytokinins on tissue culture of Grateloupia dichotoma (Gigartinales, Rhodophyta). Hydrobiologia 326(327):393–400
Yokoya NS, Handro W (1997) Thallus regeneration and growth induced by plant growth regulators and light intensity in Grateloupia dichotoma (Rhodophyta). In: Kitamura T (ed) Proceedings of I.T.I.T. International Symposium on New Technologies from Marine-Sphere. Agency of Industrial Science and Technology (AIST/MITI) and New Energy and Industrial Technology Development Organization (NEDO), Takamatsu, pp 83–86
Yokoya NS, Guimarães SMPB, Handro W (1993) Development of callus-like structures and plant regeneration in thallus segments of Grateloupia filiformis Kützing (Rhodophyta). Hydrobiologia 260/261:407–413
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Lafontaine, N., Mussio, I. & Rusig, AM. Production and regeneration of protoplasts from Grateloupia turuturu Yamada (Rhodophyta). J Appl Phycol 23, 17–24 (2011). https://doi.org/10.1007/s10811-010-9527-5
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10811-010-9527-5