Journal of Applied Phycology

, Volume 30, Issue 6, pp 3299–3310 | Cite as

Effect of plant growth regulators on direct regeneration and callus induction from Sargassum polycystum C. Agardh

  • Siti Nor Safriena Muhamad
  • Anna Pick-Kiong Ling
  • Ching-Lee WongEmail author
8th Asian Pacific Phycological Forum


Seaweed tissue culture is one of the potential technologies that can be used to increase seaweed production to cope with increasing demand. Seaweed tissue culture can be divided into two methods, namely, direct regeneration and callus induction (indirect regeneration). These methods provide several advantages, including generating seaweed cultures that are disease-free and able to mature faster to support mass production. The present study aims to develop a suitable protocol for tissue culture of Sargassum polycystum C. Agardh. We investigated different methods of sterilizing the stipe, stolon and leaf explant surfaces to allow for axenic tissue growth. In addition, we also investigated the effects of different plant growth regulators (PGRs) on seaweeds that are grown via direct regeneration and callus induction method. Our results showed that a combination of physical (brushing using soft brush under microscope) and chemical treatments (detergent, povidone iodine (PI) and antibiotic solution of streptomycin sulphate) was most suitable for growing axenic stipe of S. polycystum. For obtaining axenic leaf and stolon explants, the best treatment option was using germanium oxide (GeO2) as a pre-treatment, followed by treatments with detergent, PI and streptomycin sulphate antibiotic solution. We tested the effects of nine different plant growth regulators on the growth of tissue explants, which include indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), 1-napthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), picloram, forchlorfenuron (CPPU), uniconazole, 6-benzylaminopurine (BAP) and kinetin. Results showed that only bud explant was able to induce callus growth when treated with kinetin (4.65 μM and 13.94 μM) and uniconazole (3.00 μM, 7.00 μM and 10.00 μM). In addition, only stipe explant showed regeneration of adventitious bud when grown in the absence of plant growth regulators.


Sargassum polycystum Phaeophyceae Adventitious bud regeneration Callus induction Plant growth regulator Seaweed tissue culture 


Funding information

The authors would like to thank the funding and technical supports from the Ministry of Education Malaysia (FRGS/1/2013/STWN03/TAYLOR/02/1) and Taylor’s University.


  1. Aguirre- Lipperheide M, Evans LV (1993) A sterilization protocol for the Dictyotales (Phaeophyceae). J Phycol 29:243–251Google Scholar
  2. Aguirre-Lipperheide M, Estrada-Rodriguez FJ, Evans LV (1995) Facts, problems and needs in seaweed tissue culture: an appraisal. J Phycol 31:677–688Google Scholar
  3. Ahemad S, Ismail A, Ariff MRM (2006) The seaweed industry in Sabah, East Malaysia. J Southeast Asian Stud 11:97–107Google Scholar
  4. Ballantine DL (1979) The distribution of algal epiphytes on macrophyte hosts offshore from La Parguera, Puerto Rico. Bot Mar 22:107–110Google Scholar
  5. Baweja P, Sahoo D (2009) Regeneration studies in Grateloupia filicina (JV Lamouroux) C. Agardh: an important carrageenophyte and edible seaweed. Algae 24:163–168Google Scholar
  6. Baweja P, Sahoo D, García-Jiménez P, Robaina RR (2009) Review: seaweed tissue culture as applied to biotechnology: problems, achievements and prospects. Phycol Res 57:45–58Google Scholar
  7. Bodian MY, Lafontaine N, Matard M, Mussio I, Rusig AM (2013) Evaluation of the in vitro methods for micropropagation of Chondracanthus acicularis (Roth) Fredericq (Gigartinales, Rhodophyta): tissue culture and production of protoplasts. J Appl Phycol 25:1835–1845Google Scholar
  8. Bradley PM (1991) Plant hormones do have a role in controlling growth and development of algae. J Phycol 27:317–321Google Scholar
  9. Bradley PM, Cheney DP, Saga N (1988) One step antibiotic disk method for obtaining axenic cultures of multicellular marine algae. Plant Cell Tissue Organ Cult 12:55–60Google Scholar
  10. Chen LM, Taylor ARA (1978) Medullary tissue culture of the red alga Chondrus crispus. Can J Bot 56:883–886Google Scholar
  11. Datu Razali DE, Arsiah B, James A, Roslinah M, Siti RM (2017) On-going assessment of issue in the seaweed farming industry in Sabah, Malaysia. J Asian Acad Appl Business 1:1–11Google Scholar
  12. Fernandes DR, Yokoya NS, Yoneshigue-Valentin Y (2011) Protocol for seaweed decontamination to isolate unialgal cultures. Rev Bras Farmacogn 21:313–316Google Scholar
  13. Fisher DD, Gibor A (1987) Production of protoplasts from the brown alga, Sargassum muticum (Yendo) Fensholt (Phaeophyta). Phycologia 26:488–495Google Scholar
  14. Food and Agriculture Organization of the United Nation (FAO) (2017) FAO aquaculture newsletter. Food and Agriculture Organization of the United Nation, Rome, ItalyGoogle Scholar
  15. Frank M, Guivarc'h A, Krupkova E, Lorenz-Meyer I, Chriqui D, Schmulling T (2002) Tumorous shoot development (TSD) genes are required for coordinated plant shoot development. Plant J 29:73–85PubMedGoogle Scholar
  16. Fritsch FE (1945) The structure and reproduction of the algae. Cambridge University Press, CambridgeGoogle Scholar
  17. 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 221:181–194Google Scholar
  18. George EF (1993) Plant propagation by tissue culture, part 1: the technology, 2nd edn, Exegetics Limited, pp 574–587Google Scholar
  19. Hofmannova J, Schwarzerova K, Havelkova L, Borikova P, Petrasek J, Opatrny Z (2008) A novel, cellulose synthesis inhibitory action of ancymidol impairs plant cell expansion. J Exp Bot 59:3963–3974PubMedPubMedCentralGoogle Scholar
  20. Huang W, Fujita Y (1997) Callus induction and thallus regeneration in some species of red algae. Phycol Res 45:105–111Google Scholar
  21. Ikeuchi M, Sugimoto K, Iwase A (2013) Plant callus: mechanisms of induction and repression. Plant Cell 25:3159–3173PubMedPubMedCentralGoogle Scholar
  22. Iwasaki T, Shibaoka H (1991) Brassinosteroids act as regulators of tracheary element differentiation in isolated Zinnia mesophyll cells. Plant Cell Physiol 32:1007–1014Google Scholar
  23. Izumi K, Kamiya Y, Sakurai A, Oshio H, Takahashi N (1985) Studies of sites of action of a new plant growth retardant (E)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4 triazol-1-yl)-1-penten-3-ol (s-3307) and comparative effects of its stereoisomers in a cell free system from Cucurbita maxima. Plant Cell Physiol 26:821–827Google Scholar
  24. Kantachumpoo A (2013) Studies on phylogeography of Sargassum polycystum C.Agardh in water of Southeast Asia and Japan. Doctorate Thesis, University of Tokyo, JapanGoogle Scholar
  25. Kaur CR, Ang MGH (2009) Report for seminar on developing the seaweed aquaculture sector in Malaysia. Marine Institute of Malaysia (MIMA) Bulletin, pp 32–36Google Scholar
  26. Kirihara S, Fujikawa Y, Notoya M (1997) Axenic tissue culture of Sargassum confusum C. Agardh (Phaeophyta) as a source of seeds for artificial marine forests. J Mar Biotechnol 5:142–146Google Scholar
  27. Krishnaiah D, Sarbatly R, Prasad DMR, Bono A (2008) Mineral content of some seaweeds from Sabah’s South China Sea. Asian J Sci Res 1:166–170Google Scholar
  28. Krupková E, Schmülling T (2009) Developmental consequences of the tumorous shoot development mutation, a novel allele of the cellulose synthesizing KORRIGAN1 gene. Plant Mol Biol 71:641–655PubMedGoogle Scholar
  29. Kumar GR, Reddy CRK, Ganesan M, Thiruppathi S, Dipakkore S, Eswaran K, Subba RPV, Jha B (2004) Tissue culture and regeneration of thallus from callus of Gelidiella acerosa (Gelidiales, Rhodophyta). Phycologia 43:596–602Google Scholar
  30. Kumar GR, Reddy CRK, Jha B (2007) Callus induction and thallus regeneration from callus of phycocolloid yielding seaweeds from the Indian coast. J Appl Phycol 19:15–25Google Scholar
  31. Kumar S, Sahoo D, Levine I (2015) Assessment of nutritional value in a brown seaweed Sargassum wightii and their seasonal variations. Algal Res 9:117–125Google Scholar
  32. Lacey RW, Catto A (1993) Action of povidone-iodine against methicillin-sensitive and-resistant cultures of Staphylococcus aureus. Postgraduate Medical Journal. pp 78Google Scholar
  33. Lewin J (1966) Studies metabolism in diatoms. V. Germanium dioxide, a specific inhibitor of diatom growth. Phycologia 6:1–12Google Scholar
  34. McCracken IR (1988) Purifying algal cultures: a review of chemical methods. Proc Nova Scotian Inst Science pp 145–168Google Scholar
  35. Mooney PA, Staden JV (1985) Effect of seaweed concentrate on the growth of wheat under conditions of water stress. S Afr J Sci 81:632–633Google Scholar
  36. Polne-Fuller M, Gibor A (1987) Calluses and callus-like growth in seaweeds: induction and culture. In: Twelfth International Seaweed Symposium. Springer, Netherlands, pp 131–138Google Scholar
  37. Prince HN, Nonemaker WS, Norgard RC, Prince DL (1978) Drug resistance studies with topical antiseptics. J Pharm Sci 67:1629–1631PubMedGoogle Scholar
  38. Radulovich R, Neori A, Valderrama D, Reddy CRK, Cronin H, Forster J (2015) Farming of seaweed: seaweed sustainability- food and non-food application. Elsevier, AmsterdamGoogle Scholar
  39. Ram M, Vijayaraghavan MR, Babbar SB (2000) Wound response and regeneration in Coelarthrum opuntia. Aquat Bot 68:345–351Google Scholar
  40. Reddy CRK, Kumar GRKK, Siddhanata 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 39:610–616Google Scholar
  41. Reddy CRK, Jha B, Fujita Y, Ohno M (2008) Seaweed micropropagation techniques and their potentials: an overview. J Appl Phycol 20:609–617Google Scholar
  42. Saga N, Motomura T, Sakai Y (1982) Induction of callus from the marine brown alga Dictyosiphon foeniculaceus. Plant Cell Physiol 23:727–730Google Scholar
  43. Saito S, Okamoto M, Kushiro T, Koshiba T, Kamiya Y, Hirai N, Todoroki Y, Sakata K, Nambara E, Mizutani M (2006) A plant growth retardant, uniconazole, is a potent inhibitor of ABA catabolism in Arabidopsis. Biosci Biotechnol Biochem 70:1731–1739PubMedGoogle Scholar
  44. Sasaki E, Ogura T, Takei K, Kojima M, Kitahata N, Sakakibara H, Asami T, Shimada Y (2013) Uniconazole, a cytochrome P450 inhibitor, inhibits trans–zeatin biosynthesis in Arabidopsis. Phytochemistry 87:30–38PubMedGoogle Scholar
  45. Shao K, Wang J, Zhou B (2004) Production and application of filaments of Grateloupia turuturu (Halymeniaceae, Rhodophyta). J Appl Phycol 16:431–437Google Scholar
  46. Shea R, Chopin T (2007) Effects of germanium dioxide , an inhibitor of diatom growth , on the microscopic laboratory cultivation stage of the kelp, Laminaria saccharina. J Appl Phycol 19:27–32Google Scholar
  47. Sriwilaijaroen N, Wilairat P, Hiramatsu H, Takahashi T, Suzuki T, Ito M, Ito Y, Tashiro M, Suzuki Y (2009) Mechanisms of the action of povidone iodine against human and avian influemza a viruses: its effects on hemagglutination and sialidase activities. Virol J 6:124PubMedPubMedCentralGoogle Scholar
  48. Sulistiani E, Soelistyowati DT, Alimuddin, Yani SA (2012) Callus induction of cottoni seaweed (Kappaphycus alvarezii (Doty) Doty) collected from Natuna Islands, Riau Islands Province. Biotropia 19:112–134Google Scholar
  49. Titlyanov EA, Titlyanova TV (2010) Seaweed cultivation: methods and problems. Russ J Mar Biol 36:227–242Google Scholar
  50. Uji T, Nanaumi D, Kawagoe C, Saga N, Miyashita K (2015) Factors influencing the induction of adventitious bud and callus in the brown alga Sargassum horneri (Turner) C. Agardh. J Appl Phycol 28:2435–2443Google Scholar
  51. Yeoman MM (1987) Bypassing the plant. Ann Bot 60:157–174Google Scholar
  52. Yeong HY, Phang SM, Reddy CRK, Khalid N (2014) Production of clonal planting materials from Gracilaria changii and Kappaphycus alvarezii through tissue culture and culture of G. changii explants in airlift photobioreactors. J Appl Phycol 26:729–746Google Scholar
  53. Yokoya NS, Guimaraes MPBS, Handro W (1993) Development of callus like structures and plant regeneration in thallus segments of Grateloupia filiformis Kutzing (Rhodophyta). Hydrobiologia 260:407–413Google Scholar
  54. Yokoya NS, Stirk WA, Van Staden J, Novák O, Turečková V, Strnad M (2010) Endogenous cytokinins, auxins, and abscisic acid in red algae from Brazil. J Phycol 46:1198–1205Google Scholar
  55. Yong WTL, Ting SH, Chin WL, Rodrigues KF, Anton A (2011) In vitro micropropagation of Eucheuma seaweeds. Chem Biol Environ Eng 7:58–60Google Scholar
  56. Yong WTL, Ting SH, Yong YS, Thien VY, Wong SH, Chin WL, Rodrigues KF, Anton A (2014) Optimization of culture conditions for the direct regeneration of Kappaphycus alvarezii (Rhodophyta, Solieriaceae). J Appl Phycol 26:1597–1606Google Scholar
  57. Zamora JL (1986) Chemical and microbiologic characteristics and toxicity of povidone-iodine solutions. Am J Surg 151:400–406PubMedGoogle Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  • Siti Nor Safriena Muhamad
    • 1
  • Anna Pick-Kiong Ling
    • 2
  • Ching-Lee Wong
    • 1
    Email author
  1. 1.School of BiosciencesTaylor’s University, Taylor’s Lakeside CampusSubang JayaMalaysia
  2. 2.Division of Applied Biomedical Sciences and Biotechnology, School of Health SciencesInternational Medical UniversityKuala LumpurMalaysia

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