Journal of Applied Phycology

, Volume 26, Issue 2, pp 729–746 | Cite as

Production of clonal planting materials from Gracilaria changii and Kappaphycus alvarezii through tissue culture and culture of G. changii explants in airlift photobioreactors

  • Hui-Yin Yeong
  • Siew-Moi Phang
  • C. R. K. Reddy
  • Norzulaani Khalid


Global demand for seaweed resources has increased due to their emergent use as sources of biopharmaceuticals, nutraceuticals and biofuels. These high-valued products make possible the use of micropropagation techniques that may be more costly than conventional mariculture. This study reports the successful tissue culture of Kappaphycus alvarezii (Doty) Doty ex P. C. Silva and Gracilaria changii (B. Xia & Abbott) Abbott, Zhang and Xia. Callus induction of K. alvarezii was successfully developed following an explant sterilisation protocol. Callus formation and regeneration of K. alvarezii was observed in solidified Provasoli’s enriched seawater medium. Different culture conditions such as agar concentration, growth hormones, nutrients, irradiance and enrichment media were investigated to determine the suitable conditions for explant culture of G. changii. Proliferations of adventitious shoots were induced under the most suitable culture conditions. G. changii explants were successfully cultured in airlift photo-bioreactors, with no decrease in the carbohydrate content in the G. changii explants. This micropropagation technique can provide a useful alternative system for seedling production of economically important seaweeds.


Tissue culture Callus induction Formation and regeneration Explant culture Plant growth regulators Red seaweeds Kappaphycus Gracilaria Micropropagation Algae biotechnology 



This research was funded by the Ministry of Science, Technology and Innovation, Malaysia (E-Science Fund: 02-01-03-SF0148 and 04-01-03-SF0664; Top-down Project: 06-02-02-003 BTK/ER/016, Grant No: 39-02-03-9002/Oracle 8301902), the Malaysian Toray Science Foundation (Japan) Grant (MTSF 1050-2009A) and the University of Malaya Research Grants (UMRG Grant No: RG100-10SUS, F0233/2004D, P0114/2006A).


  1. Armisen R (1995) World-wide use and importance of Gracilaria. J Appl Phycol 7:231–243CrossRefGoogle Scholar
  2. Atkinson MJ, Bingman C (1997) Elemental composition of commercial seasalts. J Aquaric Aquat Sci 8:39–43Google Scholar
  3. Baweja P, Sahoo D, García-Jiménez P, Robaina RR (2009) Seaweed tissue culture as applied to biotechnology: problems, achievements and prospects. Phycol Res 57:45–58CrossRefGoogle Scholar
  4. Bixler HJ, Porse H (2011) A decade of change in the seaweed hydrocolloids industry. J Appl Phycol 23:321–335CrossRefGoogle Scholar
  5. Chan CX, Teo SS, Ho CL, Rofima Yasmin O, Phang SM (2004) Optimisation of RNA extraction for Gracilaria changii (Gracilariales, Rhodophyta). J Appl Phycol 16:297–301CrossRefGoogle Scholar
  6. Chen LCM, Taylor RA (1978) Medullary tissue culture of the red alga Chondrus crispus. Can J Bot 56:883–886CrossRefGoogle Scholar
  7. Chu WL, Norazmi M, Phang SM (2003) Fatty acid composition of some Malaysian seaweeds. Malays J Sci 22:21–27Google Scholar
  8. Collantes G, Melo C, Candia A (2004) Micropropagation by explants of Gracilaria chilensis Bird, McLachlan and Oliveira Gloria. J Appl Phycol 16:203–213CrossRefGoogle Scholar
  9. Dawes CJ, Koch EW (1991) Branch, micropropagule and tissue culture of the red algae, Eucheuma diculatum and Kappaphycus alvarezii farmed in the Philippines. J Appl Phycol 3:247–257CrossRefGoogle Scholar
  10. Dawes CJ, Trono GC Jr, Lluisma AO (1993) Clonal propagation of Euchuema denticulatum and Kappaphycus alvarezii for Philippine seaweed farms. Hydrobiologia 260/261:379–383Google Scholar
  11. Dawes CJ, Lluisma AO, Trono GC (1994) Laboratory and field growth studies of commercial strains of Eucheuma denticulatum and Kappaphycus alvarezii in the Philippines. J Appl Phycol 6:21–24CrossRefGoogle Scholar
  12. Elhiti M, Stasolla C (2012) In vitro shoot organogenesis and hormone response are affected by the altered level of Brassica napus meristem genes. Plant Sci 190:40–51PubMedCrossRefGoogle Scholar
  13. Food and Agriculture Organization of the United Nations (FAO) (2007) FAO year book, vol. 100/2. FAO, RomeGoogle Scholar
  14. Food and Agriculture Organization of the United Nations (FAO) (2012) World review of fisheries and aquaculture. In: The State of World Fisheries and Aquaculture 2012. FAO, Rome, pp 3–100Google Scholar
  15. Gan SY, Qin S, Rofina Yasmin YD, Phang SM (2003) Transient expression of lacZ in particle bombarded Gracilaria changii (Gracilariales, Rhodophyta). J Appl Phycol 15:315–353CrossRefGoogle Scholar
  16. Gan SY, Rofina Yasmin O, Qin S, Phang SM (2006) Crop improvement in seaweed cultivation. In: Phang SM, Critchley A, Ang P (eds) Advances in seaweed cultivation and utilization in Asia. University of Malaya Maritime Research Center, Kuala Lumpur, pp 81–103Google Scholar
  17. George EF, Hall MA, Klerk GD (2008) Plant propagation by tissue culture, volume 1. The background. Springer, NetherlandsGoogle Scholar
  18. Guillard RR (1975) Culture of phytoplankton for feeding marine invertebrates. In: Smith WL, Chanley MH (eds) Culture of marine invertebrate animals. Plenum, New York, pp 26–60Google Scholar
  19. Gusev MV, Tambiev AH, Kirikova NN, Shelyastina NN, Aslanyan RR (1987) Callus formation in seven species of agarophyte marine algae. Mar Biol 95:593–597CrossRefGoogle Scholar
  20. Hicks G (1994) Shoot induction and organogenesis in vitro: a development perspective. In Vitro Cell Dev 30:10–15CrossRefGoogle Scholar
  21. Ho CL, Teoh S, Teo SS, Rahim RA, Phang SM (2009) Profiling the transcriptome of Gracilaria changii (Rhodophyta) in response to light deprivation. Mar Biotechnol 11:513–519PubMedCrossRefGoogle Scholar
  22. Huang W, Fujita Y (1997) Callus induction and thallus regeneration in some species of red algae. Phycol Res 45:105–111CrossRefGoogle Scholar
  23. Hurtado AQ, Biter AB (2007) Plantlet regeneration of Kappaphycus alvarezii var. adik-adik by tissue culture. J Appl Phycol 19:783–786CrossRefGoogle Scholar
  24. James DE (1978) Culturing algae. Carolina Biological Supply, BurlingtonGoogle Scholar
  25. Kaczyna F, Megnet WH (1993) The effects of glycerol and plant growth regulators on Gracilaria verrucosa (Gigartinales, Rhodophyceae). Hydrobiologia 268:57–64CrossRefGoogle Scholar
  26. Kim JW, Han SK, Kwon SY, Lee HS, Lim YP, Liu JR, Kwak SS (2000) High frequency shoot induction and plant regeneration from cotyledonary hypocotyl explants of cucumber (Cucumis sativus L.) seedlings. J Plant Physiol 157:136–139CrossRefGoogle Scholar
  27. Kochert AG (1978) Carbohydrate determination by the phenol–sulphuric acid method. In: Hellbust JA, Craigie JS (eds) Handbook of phycological methods: physiological and biochemical methods. Cambridge University Press, Cambridge, pp 95–97Google Scholar
  28. Kumar GR, Reddy CRK, Ganesan M, Thiruppathi S, Dipakkore S, Eswaran K, Rao PYS, Jha B (2004) Tissue culture and regeneration of thallus from callus of Gelidiella acerosa (Gelidiaies, Rhodophyta). Phycologia 43:596–602CrossRefGoogle Scholar
  29. 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 19:15–25CrossRefGoogle Scholar
  30. Lim PE, Phang SM (2004) Gracilaria species (Gracilariales, Rhodophyta) of Malaysia including two new records. Malays J Sci 23:71–80Google Scholar
  31. Marinho-Soriano E, Silva TSF, Moreira WSC (2001) Seasonal variation in the biomass and agar yield from Gracilaria cervicornis and Hydropuntia cornea from Brazil. Bioresour Technol 77:115–120PubMedCrossRefGoogle Scholar
  32. McHugh DJ (2003) A guide to the seaweed industry. FAO fisheries technical paper no 441. FAO, Rome, p 105Google Scholar
  33. Phang SM (2006) Seaweed resources in Malaysia: current status and future prospects. Aquat Ecosyst Health Manag 9:185–202CrossRefGoogle Scholar
  34. Phang SM, Shaharuddin S, Noraishah H, Sasekumar A (1996) Studies on Gracilaria changii (Gracilariales, Rhodophyta) from Malaysian mangroves. Hydrobiologia 326/327:347–352Google Scholar
  35. Phang SM, Lim PE, Yeong HY (2010) Malaysian seaweed resources in the South China Sea and their potential economic and ecological applications. J Sci Technol Trop 6:87–109Google Scholar
  36. Polne-Fuller M, Saga N, Gibor A (1986) Algal cell, callus and tissue cultures and selection of algal strains. In: Barclay WR (ed) Algal biomass technologies: an interdisciplinary perspective. Cramer, Berlin, pp 30–36Google Scholar
  37. Reddy CRK, Kumar GRK, 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 Appl Phycol 39:610–616CrossRefGoogle Scholar
  38. Reddy CRK, Jha B, Fujita Y, Ohno M (2008) Seaweed micropropagation techniques and their potentials: an overview. J Appl Phycol 20:609–617CrossRefGoogle Scholar
  39. Schramm W (1991) Cultivation of unattached seaweeds. In: Guiry MD, Blunden G (eds) Seaweed resources in Europe, uses and potential. Wiley, New York, pp 379–408Google Scholar
  40. Shu MH, Appleton D, Zandi K, AbuBakar S (2013) Anti-inflammatory, gastroprotective and antiulcerogenic effects of red algae Gracilaria changii (Gracilariales, Rhodophyta) extract. BMC Compl Alternative Med 13:61CrossRefGoogle Scholar
  41. Sim MC, Lim PE, Gan SY, Phang SM (2007) Identification of random amplified polymorphic DNA (RAPD) markers for differentiating male from female and sporophyte thalli of Gracilaria changii (Rhodophyta). J Appl Phycol 19:763–769CrossRefGoogle Scholar
  42. Siow RS, Teo SS, Ho WY, Mohd. Yunus Abd. S, Phang SM, Ho CL (2012) Molecular cloning and biochemical characterization of galactose-1-phosphate uridylyltransferase from Gracilaria changii (Rhodophyta). J Phycol 48:155–162CrossRefGoogle Scholar
  43. Siow RS, Teoh S, Teo SS, Abd. Shukor MY, Phang SM, Ho CL (2013) Molecular cloning and characterization of GDP-mannose-3′,5′-epimerase from Gracilaria changii. J Appl Phycol. doi: 10.1007/s10811-013-9987-5 Google Scholar
  44. Song SL, Lim PE, Phang SM, Lee WW, Lewmanomont K, Largo DB, Abdul Han N (2013) Microsatellite markers from expressed sequence tags (ESTs) of seaweeds in differentiating various Gracilaria species. J Appl Phycol 25:839–846CrossRefGoogle Scholar
  45. Tan EL (1999) Tissue culture and protoplast isolation of Gracilaria changii (Rhodophyta). Master’s thesis, University of Malaya, Kuala LumpurGoogle Scholar
  46. Teo SS, Ho CL, Teoh S, Lee WW, Tee JM, Abdul Rahim R, Phang SM (2007) Analyses of expressed sequence tags (ESTs) from an agarophyte Gracilaria changii (Gracilariales, Rhodophyta). Eur J Phycol 42:41–46CrossRefGoogle Scholar
  47. Teo SS, Ho CL, Teoh S, Rahim RA, Phang SM (2009) Transcriptomic analysis of Gracilaria changii (Rhodophyta) in response to hyper- and hypo-osmotic stresses. J Phycol 45:1093–1099CrossRefGoogle Scholar
  48. Titlyanov EA, Titlyanova TV, Kadel P, Luning K (2006) 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 339:55–64CrossRefGoogle Scholar
  49. Trewavas AJ (1982) Growth substance sensitivity: the limiting factor in plant development. Physiol Plant 55:60–72CrossRefGoogle Scholar
  50. Wong WH, Goh SH, Phang SM (1994) Antibacterial properties of Malaysian seaweeds. In: Phang SM et al (eds) Algal biotechnology in the Asia-Pacific region. University of Malaya, Kuala Lumpur, pp 75–81Google Scholar
  51. Wong PF, Tan LJ, Nawi H, Abu Bakar S (2006) Proteomics of the red alga, Gracilaria changii (Gracilariales, Rhodophyta). J Phycol 42:113–120CrossRefGoogle Scholar
  52. Yeong HY, Khalid N, Phang SM (2008) Protoplast isolation and regeneration from Gracilaria changii (Gracilariales, Rhodophyta). J Appl Phycol 20:641–651CrossRefGoogle Scholar
  53. Yokoya NS (2000) Apical callus formation and plant regeneration controlled by plant growth regulators on axenic culture of red alga Gracilariopsis tenuifrons (Gracilariales, Rhodophyta). Phycol Res 48:133–142CrossRefGoogle Scholar
  54. 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 14:97–102Google Scholar
  55. Yokoya NS, Kakita H, Obika H, Kitamura T (1999) Effects of environmental factors and plant growth regulators on growth of the red alga Gracilaria vermiculophylla from Shikoku Island, Japan. Hydrobiologia 398/399:339–347Google Scholar
  56. Yokoya NS, West JA, Luchi AE (2004) Effects of plant growth regulators on callus formation, growth and regeneration in axenic tissue cultures of Gracilaria tenuistipitata and Gracilaria perplexa (Gracilariales, Rhodophyta). Phycol Res 52:244–254CrossRefGoogle Scholar
  57. Yow YY, Lim PE, Phang SM (2011) Genetic diversity of Gracilaria changii (Gracilariaceae, Rhodophyta) from west coast, Peninsular Malaysia based on mitochondrial cox1 gene analysis. J Appl Phycol 23:219–226CrossRefGoogle Scholar
  58. Yow YY, Lim PE, Phang SM (2013) Assessing the use of mitochondrial cox1 gene and cox2-3 spacer for genetic diversity study of Malaysian Gracilaria changii (Gracilariaceae, Rhodophyta) from Peninsular Malaysia. J Appl Phycol 25:831–838CrossRefGoogle Scholar
  59. Yunque DAT, Tibubos KR, Hurtado AQ, Critchley AT (2011) Optimization of culture conditions for tissue culture production of young plantlets of carrageenophyte Kappaphycus. J Appl Phycol 23:615–621CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Hui-Yin Yeong
    • 1
  • Siew-Moi Phang
    • 1
    • 2
  • C. R. K. Reddy
    • 3
  • Norzulaani Khalid
    • 2
  1. 1.Institute of Ocean and Earth Sciences (IOES)University of MalayaKuala LumpurMalaysia
  2. 2.Institute of Biological SciencesUniversity of MalayaKuala LumpurMalaysia
  3. 3.Discipline of Marine Biotechnology and EcologyCSIR—Central Salt and Marine Chemicals Research InstituteBhavnagarIndia

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