Journal of Applied Phycology

, Volume 21, Issue 2, pp 255–264 | Cite as

Morphogenetic responses from protoplasts and tissue culture of Laminaria digitata (Linnaeus) J. V. Lamouroux (Laminariales, Phaeophyta): callus and thalloid-like structures regeneration

  • Isabelle Mussio
  • Anne-Marie Rusig


The regeneration of meristematic tissues from sporophytes of Laminaria digitata was studied by protoplast and tissue culture. Sequential treatment of explants in sterile seawater with 1% Betadine for 5 min, 1% commercial bleach for 1–2 min and 2% antibiotic treatment supplemented with 1 μM GeO2 overnight enabled viable explants as high as 55%. Different morphogenetic responses were observed from tissue culture on media supplemented with plant growth regulators alone or in combination, mainly filamentous calluses up to 50% according to the media. Dark green compact calluses were observed on two combinations: 4 μM Pi + 2 μM N-(2-chloro-4-pyridyl)-N’-phenylurea (CPPU) and 0.04 μM Pi + 0.44 μM 6-benzylaminopurine. Thalloid-like structures comparable to adventitious buds were regenerated on medium supplemented with 4 μM Pi + 0.45 μM zeatin but at low frequency suggesting a strong genotypic effect. Friable calluses were developed from protoplasts in enriched medium with polyamines and containing 0.40 μM CPPU + 0.45 μM 2,4-dichlorophenoxyacetic acid. In order to produce protoplasts, a one-step enzymatic protocol was developed and yields reached 22 × 106 protoplasts per gram of fresh weight.


Adventitious bud Callus Laminaria digitata Plant growth regulators Protoplast Meristematic explant 



This research was supported by a grant of Conseil Régional de Basse-Normandie. The authors would like to thank Dr Gérard Baux and Dr Philippe Fossier (CNRS, Neurobiologie Cellulaire et Moléculaire) for providing crude extracts of Aplysia vaccaria.


  1. Aguirre-Lipperheide M, Estrada-Rodriguez FJ, Evans LV (1995) Facts, problems and needs in seaweed tissue culture: an appraisal. J Phycol 31:677–688. doi: 10.1111/j.0022-3646.1995.00677.x CrossRefGoogle Scholar
  2. Ar Gall E, Asensi A, Marie D, Kloareg B (1996) Parthenogenesis and apospory in the Laminariales: a flow cytometry analysis. Eur J Phycol 31:369–380. doi: 10.1080/09670269600651601 CrossRefGoogle Scholar
  3. Asensi A, Ar Gall E, Marie D, Billot C, Dion P, Kloareg B (2001) Clonal propagation of Laminaria digitata (Phaeophyceae) sporophytes through a diploid cell-filament suspension. J Phycol 37:411–417. doi: 10.1046/j.1529-8817.2001.037003411.x CrossRefGoogle Scholar
  4. Aziz A, Poinssot B, Daire X, Adrian M, Bezier A, Lambert B, Joubert JM, Pugin A (2003) Laminarin elicits defence responses in grapevine and induces protection against Botrytis cinerea and Plasmopara viticola. Mol Plant Microbe Interact 16:1118–1128. doi: 10.1094/MPMI.2003.16.12.1118 PubMedCrossRefGoogle Scholar
  5. Benet H, Bruss U, Duval JC, Kloareg B (1994) Photosynthesis and photoinhibition in protoplasts of the marine brown alga Laminaria saccharina. J Exp Bot 45:211–220. doi: 10.1093/jxb/45.2.211 CrossRefGoogle Scholar
  6. Benet H, Ar Gall E, Asensi A, Kloareg B (1997) Protoplast regeneration from gametophytes and sporophytes of some species in the order Laminariales (Phaeophyceae). Protoplasma 199:39–48. doi: 10.1007/BF02539804 CrossRefGoogle Scholar
  7. Bradley PM, Cheney DP (1990) Some effects of plant growth regulators on tissue cultures of the marine red alga Agardhiella subulata (Gigartinales, Rhodophyta). Hydrobiologia 204/205:353–360. doi: 10.1007/BF00040256 CrossRefGoogle Scholar
  8. Butler D (1989) Isolation and culture of protoplasts and tissues from Laminaria sp. (Phaeophyta). Dissertation, University of LeedsGoogle Scholar
  9. Butler DM, Ostgaard 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. doi: 10.1093/jxb/40.11.1237 CrossRefGoogle Scholar
  10. Carra A, De Pasquale F, Ricci A, Carimi F (2006) Diphenylurea derivatives induce somatic embryogenesis in Citrus. Plant Cell Tissue Organ Cult 87:41–48. doi: 10.1007/s11240-006-9132-0 CrossRefGoogle Scholar
  11. Cassells AC, Curry RF, Jain SM (2001) Oxidative stress and physiological, epigenetic and genetic variability in plant tissue culture: implications for micropropagators and genetic engineers. Plant Cell Tissue Organ Cult 64:145–157. doi: 10.1023/A:1010692104861 CrossRefGoogle Scholar
  12. Collantes G, Melo C, Candia A (2004) Micropropagation by explants of Gracilaria chilensis Bird, McLachlan and Oliveira. J Appl Phycol 16:203–213. doi: 10.1023/B:JAPH.0000048506.58928.4d CrossRefGoogle Scholar
  13. Davey MR, Anthony P, Power JB, Lowe KC (2005) Plant protoplasts: status and biotechnological perspectives. Biotechnol Adv 23:131–171. doi: 10.1016/j.biotechadv.2004.09.008 PubMedCrossRefGoogle Scholar
  14. Ducreux G, Kloareg B (1988) Plant regeneration from protoplasts of Sphacelaria (Phaeophyceae). Planta 174:25–29. doi: 10.1007/BF00394869 CrossRefGoogle Scholar
  15. Fang ZX, Yan Z, Wang Z (1983) Some preliminary observations on tissue culture in Laminaria japonica and Undaria pinnatifida. Kexue Tongbao 28:247–249Google Scholar
  16. Folefack C, Cosson J (1995) Induction de la callogénèse chez Laminaria digitata (Phéophycées, Laminariales). Acta Bot Gallica 142:145–152Google Scholar
  17. Fries L (1980) Axenic tissue cultures from the sporophytes of Laminaria digitata and Laminaria hyperborea (Phaeophyta). J Phycol 16:475–477. doi: 10.1111/j.1529-8817.1980.tb03062.x CrossRefGoogle Scholar
  18. Garcia-Reina G, Gomez-Pinchetti JL, Robledo DR, Sosa P (1991) Actual, potential and speculative applications of seaweed cellular biotechnology: some specific comments on Gelidium. Hydrobiologia 221:181–194CrossRefGoogle Scholar
  19. Hwang EK, Kim CH, Sohn CH (1994) Callus-like formation and differentiation in Hizikia fusiformis (Harvey) Okamura. Korean J Phycol 9:77–83Google Scholar
  20. Kawashima Y, Tokuda H (1990) Callus formation in Ecklonia cava Kjellman (Laminariales, Phaeophyta). Hydrobiologia 204/205:375–380. doi: 10.1007/BF00040259 CrossRefGoogle Scholar
  21. Kawashima Y, Tokuda H (1993) Regeneration from callus of Undaria pinnatifida (Harvey) Suringar (Laminariales, Phaeophyta). Hydrobiologia 260/261:385–389. doi: 10.1007/BF00049045 CrossRefGoogle Scholar
  22. Kawashima Y, Tokuda H, Kominami H, Kakuno T, Kiyohara M (1992) Effects of plant hormones on the induction of adventitious embryos from calluses of a brown algae, Ecklonia cava (Laminariales). Oceanis 18:1–9Google Scholar
  23. Kimura H, Notoya M (1996) Tissue culture of Ecklonia kurome Okamura (Laminariales, Phaeophyta). Nippon Suisan Gakkai Shi 62:638–641Google Scholar
  24. Kimura H, Notoya M (1997) Tissue culture of Undaria undarioides (Yendo) Okamura (Phaeophyta, Laminariales). J Mar Biotechnol 5:100–102Google Scholar
  25. Kirihara S, Fujikawa Y, Notoya M (1997) Axenic tissue culture of Sargassum confusum C. Agardh (Phaeophyta) as a source of artificial marine forests. J Mar Biotechnol 5:142–146Google Scholar
  26. Kloareg B, Quatrano RS (1987) Isolation of protoplasts from zygotes of Fucus distichus (L.) Powell (Phaeophyta). Plant Sci 50:189–194. doi: 10.1016/0168-9452(87)90073-2 CrossRefGoogle Scholar
  27. 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. doi: 10.1016/0168-9452(89)90194-5 CrossRefGoogle Scholar
  28. Larkin PJ, Scowcroft WR (1981) Somaclonal variation - A novel source of variability from cell cultures for plant improvement. Theor Appl Genet 60:197–214. doi: 10.1007/BF02342540 CrossRefGoogle Scholar
  29. Lawlor HJ, McComb JA, Borowitzka MA (1987) The development of filamentous and callus-like growth in axenic tissue cultures of Ecklonia radiata (Phaeophyta). In: Stadler T, Mollion J, Verdus MC, Karamanos Y, Morvan H, Christiaen D (eds) Algal biotechnology. Elsevier, London, pp 139–150Google Scholar
  30. Lawlor HJ, McComb JA, Borowitzka MA (1989) Tissue culture of Ecklonia radiata (Phaeophyceae, Laminariales): effects on growth of light, organic carbon source and vitamins. J Appl Phycol 1:105–112. doi: 10.1007/BF00003872 CrossRefGoogle Scholar
  31. Lee TF (1985) Aposporous gametophyte formation in stipe explants from Laminaria saccharina (Phaeophyta). Bot Mar 28:179–185CrossRefGoogle Scholar
  32. Lewin J (1966) Silicon metabolism in diatoms. V. Germanium dioxide, a specific inhibitor of diatom growth. Phycologia 6:1–12Google Scholar
  33. Liu X, Kloareg B (1992) Explant axenisation for tissue culture in marine macroalgae. Chin J Oceanology Limnol 10:268–277. doi: 10.1007/BF02843827 CrossRefGoogle Scholar
  34. Loiseaux S, Rozier C (1978) Culture axénique de Pylaiella littoralis (L.) Kjellm. (Phéophycées). Rev Algol N S 13:333–340Google Scholar
  35. Matsumura W (1998) Efficient isolation and culture of viable protoplasts from Laminaria longissima Miyabe (Phaeophyceae). Bull Fac Fish Hokkaido Univ 49:85–90Google Scholar
  36. Matsumura W, Yasui H, Yamamoto H (2000) Mariculture of Laminaria japonica (Laminariales, Phaeophyceae) using protoplast regeneration. Phycol Res 48:169–176. doi: 10.1111/j.1440-1835.2000.tb00213.x CrossRefGoogle Scholar
  37. Matsumura W, Yasui H, Yamamoto H (2001) Successful sporophyte regeneration from protoplasts of Undaria pinnatifida (Laminariales, Phaeophyceae). Phycologia 40:10–20CrossRefGoogle Scholar
  38. Mejjad M, Loiseaux-de-Goër S, Ducreux G (1992) Protoplast isolation, development and regeneration in different strains of Pylaiella littoralis (L.) Kjellm. (Phaeophyceae). Protoplasma 169:42–48. doi: 10.1007/BF01343368 CrossRefGoogle Scholar
  39. Miyanishi N, Iwamoto Y, Watanabe E, Oda T (2003) Induction of TNT-α production from human peripheral blood monocytes with β-1,3-glucanase oligomer prepared from laminarin with b-1,3-glucanase from Bacillus clausii NM-1. J Biosci Bioeng 95:192–195PubMedGoogle Scholar
  40. Mussio I, Rusig AM (2006) Isolation of protoplasts from Fucus serratus and F. vesiculosus (Fucales, Phaeophyceae): factors affecting protoplast yield. J Appl Phycol 18:733–744. doi: 10.1007/s10811-006-9079-x CrossRefGoogle Scholar
  41. Neori A, Ragg NLC, Shpigel M (1998) The integrated culture of seaweed, abalone, fish and clams in modular intensive land-based systems: II. Performance and nitrogen partitioning within an abalone (Haliotis tuberculata) and macroalgae culture system. Aquacult Eng 17:215–239. doi: 10.1016/S0144-8609(98)00017-X CrossRefGoogle Scholar
  42. Notoya M (1988) Tissue culture from the explant of Ecklonia stolonifera Okamura (Phaeophyta, Laminariales). Jpn J Phycol Sorui 36:175–177Google Scholar
  43. Notoya M (1997) Chloroplast changes and differentiation of callus cells in Eckloniopsis radicosa (Kjellman) Okamura (Phaeophyta, Laminariales). J Appl Phycol 9:175–178. doi: 10.1023/A:1007910024753 CrossRefGoogle Scholar
  44. Notoya M, Aruga Y (1989) Tissue culture from the explant of Ecklonia cava Kjellman (Laminariales, Phaeophyta). Jpn J Phycology Sorui 37:302–304Google Scholar
  45. Notoya M, Aruga Y (1990) Tissue culture from the explant of stipe of Eisenia bicyclis (Kjellman) Setchell (Laminariales, Phaeophyta). Jpn J Phycology Sorui 38:387–390Google Scholar
  46. Notoya M, Aruga Y (1992a) Tissue culture of Undaria pinnatifida (Harvey) Suringar (Laminariales, Phaeophyta). Jpn J Phycology Sorui 50:393–395Google Scholar
  47. Notoya M, Aruga Y (1992b) Tissue culture of parthenogenetic and normal young sporophytes of Ecklonia cava Kjellman (Laminariales, Phaeophyta). Jpn J Phycology Sorui 40:53–55Google Scholar
  48. Notoya M, Nagashima M, Aruga Y (1994) Influence of light intensity and temperature on callus development in young sporophytes of three species of Laminariales (Phaeophyta). J Mar Biotechnol 2:15–18Google Scholar
  49. Onsoyen E (1996) Commercial applications of alginates. Carbohydr Eur 14:26–31Google Scholar
  50. Polne-Fuller M, Gibor A (1986) Calluses, cells and protoplasts in studies towards genetic improvement of seaweeds. Aquaculture 57:117–123. doi: 10.1016/0044-8486(86)90188-2 CrossRefGoogle Scholar
  51. Polne-Fuller M, Gibor A (1987) Calluses and callus-like growth in seaweeds: induction and culture. Hydrobiologia 151/152:131–138. doi: 10.1007/BF00046118 CrossRefGoogle Scholar
  52. Qi H, Rorrer KA, Jiang ZD, Rorrer GL (1995) Kinetics of non-photosynthetic callus production from the brown alga Laminaria setchellii (Laminariales). Phycological Res 43:179–182. doi: 10.1111/j.1440-1835.1995.tb00023.x CrossRefGoogle Scholar
  53. Rajakrishna Kumar G, Reddy CRK, Ganesan M, Thiruppathi S, Dipakkore S, Eswaran K, Subba Rao PV, Jha B (2004) Tissue culture and regeneration of thallus from callus of Gelidiella acerosa (Gelidiales, Rhodophyta). Phycologia 43:596–602CrossRefGoogle Scholar
  54. Rajakrishna Kumar G, Reddy CRK, Jha B (2007) Callus induction and thallus regeneration from callus of phycocolloid yielding seaweeds from the Indian coast. J Appl Phycol 19:15–25. doi: 10.1007/s10811-006-9104-0 CrossRefGoogle Scholar
  55. Reddy CRK, Rajakrishna Kumar G, Siddhanta AK, Tewari A, Eswaran K (2003) In vitro somatic embryogenesis and regeneration of somatic embryos from pigmented callus of Kappaphycus alvarezii (Doty) Doty (Rhodophyta, Gigartinales). J Phycol 39:610–616. doi: 10.1046/j.1529-8817.2003.02092.x CrossRefGoogle Scholar
  56. Reddy CRK, Jha B, Fujita Y (2007) Seaweed micropropagation techniques and their potentials: an overview. J Appl Phycol. doi: 10.1007/s1081100792054
  57. Rorrer GL, Cheney DP (2004) Bioprocess engineering of cell and tissue cultures for marine seaweeds. Aquacult Eng 32:11–41. doi: 10.1016/j.aquaeng.2004.03.007 CrossRefGoogle Scholar
  58. Rusig AM, Cosson J (2001) Plant regeneration from protoplasts of Enteromorpha intestinalis (Chlorophyta, Ulvophyceae) as seedstock for macroalgal culture. J Appl Phycol 13:103–108. doi: 10.1023/A:1011136712938 CrossRefGoogle Scholar
  59. Saga N, Sakai Y (1977) Studies on the morphogenesis of Laminariales plants. I. Regeneration of fragments from sporophytes of Laminaria japonica Aresch. Bull Jpn Soc Phycology 25:297–301Google Scholar
  60. Saga N, Sakai Y (1983) Axenic tissue culture and callus formation of the marine brown alga Laminaria angustata. Bull Jpn Soc Sci Fish 49:1561–1563Google Scholar
  61. Saga N, Uchida T, Sakai Y (1978) Clone Laminaria from single isolated cell. Bull Jpn Soc Sci Fish 44:87Google Scholar
  62. Sahoo D, Yarish C (2005) Mariculture of seaweeds. In: Anderson RA (ed) Algal culturing techniques. Academic Press, New York, pp 219–237CrossRefGoogle Scholar
  63. Sasamoto H, Ogita S, Wakita Y, Fukui M (2002) Endogenous levels of abscisic acid and gibberellins in leaf protoplasts competent for plant regeneration in Betula platyphylla and Populus alba. Plant Growth Regul 38:195–201. doi: 10.1023/A:1021515630671 CrossRefGoogle Scholar
  64. Sawabe T, Ezura Y (1996) Regeneration from Laminaria japonica Areschoug (Laminariales, Phaeophyceae) protoplasts isolated with bacterial alginase. Plant Cell Rep 15:892–895. doi: 10.1007/BF00231582 CrossRefGoogle Scholar
  65. Sawabe T, Ezura Y, Yamamoto H (1997) Plant regeneration from protoplasts of Laminaria japonica Areschoug (Laminariales, Phaeophyceae) in a continuous-flow culture system. Plant Cell Rep 17:109–112. doi: 10.1007/s002990050361 CrossRefGoogle Scholar
  66. Titlyanov EA, Titlyanova TV, Kadel P, Lüning K (2006a) New methods of obtaining plantlets and tetraspores from fragments and cell aggregates of meristematic and submeristematic tissue of the red alga Palmaria palmata. J Exp Mar Biol Ecol 339:55–64. doi: 10.1016/j.jembe.2006.07.014 CrossRefGoogle Scholar
  67. Titlyanov EA, Titlyanova TV, Kadel P, Lüning K (2006b) Obtaining plantlets from apical meristem of the red alga Gelidium sp. J Appl Phycol 18:167–174. doi: 10.1007/s10811-006-9091-1 CrossRefGoogle Scholar
  68. Torosantucci A, Bromuro C, Chiani P, De Bernardis F, Berti F, Galli C, Norelli F, Bellucci C, Polonelli L, Costantino P, Rappuoli R, Cassone A (2005) A novel glycol-conjugate vaccine against fungal pathogens. J Exp Med 202:597–606. doi: 10.1084/jem.20050749 PubMedCrossRefGoogle Scholar
  69. Wakita Y, Yokota S, Yoshizawa N, Katsuki T, Nishiyama Y, Yokoyama T, Fukui M, Sasamoto H (2005) Interfamilial cell fusion among leaf protoplasts of Populus alba, Betula platyphylla and Alnus firma: assessment of electric treatment and in vitro culture conditions. Plant Cell Tissue Organ Cult 83:319–326. doi: 10.1007/s11240-005-8428-9 CrossRefGoogle Scholar
  70. Wang XH, Qin S, Li XP, Jiang P, Zeng CK, Qin M (1998) High efficiency induction of callus and regeneration of sporophytes of Laminaria japonica (Phaeophyta). Chin J Oceanol Limnol 16:67–74. doi: 10.1007/BF02849083 CrossRefGoogle Scholar
  71. Yan ZM (1984) Studies on tissue culture of Laminaria japonica and Undaria pinnatifida. Hydrobiologia 116/117:314–316. doi: 10.1007/BF00027692 CrossRefGoogle Scholar
  72. Yokoya NS, Handro W (1996) Effects of auxins and cytokinins on tissue culture of Grateloupia dichotoma (Gigartinales, Rhodophyta). Hydrobiologia 326/327:393–400. doi: 10.1007/BF00047837 CrossRefGoogle Scholar
  73. Yokoya NS, Handro W (2002) Effects of plant growth regulators and culture medium on morphogenesis of Solieria filiformis (Rhodophyta) cultured in vitro. J Appl Phycol 14:97–102. doi: 10.1023/A:1019529306419 CrossRefGoogle Scholar
  74. Zhang Q, Chen JJ, Henny RJ (2006) Regeneration of Syngonium podophyllumVariegatum” through direct somatic embryogenesis. Plant Cell Tissue Organ Cult 84:161–168Google Scholar
  75. Zongxi F, Zuomei Y, Zongcheng W (1983) Some preliminary observations on tissue culture in Laminaria japonica and Undaria pinnatifida. Kexue Tongbao 28:247–249Google Scholar

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© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  1. 1.UMR IFREMER 100 - Physiologie et Ecophysiologie des Mollusques Marins, IFR 146 ICOREUniversité de Caen Basse-NormandieCaen CedexFrance

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