CALLUS INDUCTION IN Kappaphycus alvarezii USING INDOLE-3-ACETIC ACID (IAA) and 6-BENZYLAMINOPURINE (BAP) FOR SEEDSTOCK DEVELOPMENT
Abstract
The commercially important red seaweed Kappaphycus alvarezii is extensively cultivated for carrageenan production. Despite its economic value, large-scale reproduction and genetic enhancement remain limited due to its low regeneration potential. This study aimed to optimize plant growth regulator (PGR) concentrations for efficient callus induction in K. alvarezii. A completely randomized design was employed, comprising five treatments with varying concentrations of indole-3-acetic acid (IAA) and 6-benzylaminopurine (BAP), along with a control lacking PGRs. A total of 180 explants from meristematic tissues of acclimatized thalli were cultured (30 explants per treatment). The highest callus induction rate (88%) was achieved with 1.50 mg/L IAA and 5 mg/L BAP (Treatment F), with visible callus formation beginning around day 38. A progressive color change from brown to white was observed, indicating active cellular proliferation. Other treatments exhibited lower induction rates, ranging from 0% (control) to 61% (Treatment D). These findings underscore the critical influence of auxin–cytokinin interactions on callogenesis and offer an optimized hormonal regime for improving in vitro culture efficiency. The established protocol provides a valuable platform for future large-scale propagation and genetic improvement strategies in K. alvarezii, contributing to the advancement of seaweed biotechnology.
Keywords
Full Text:
PDFReferences
Avila-Victor, C. M., Arjona-Suárez, E. J., Iracheta-Donjuan, L., Valdez-Carrasco, J. M., Gómez-Merino, F. C., & Robledo-Paz, A. (2023). Callus type, growth regulators, and phytagel on indirect somatic embryogenesis of coffee (Coffea arabica L. var. Colombia). Plants (Basel), 12(20), 3570. https://doi.org/10.3390/plants12203570
Bano, A. S., Khattak, A. M., Basit, A., Alam, M., Shah, S. T., Ahmad, N., Gilani, S. A. Q., ... Mohamed, H. I. (2022). Callus induction, proliferation, enhanced secondary metabolites production and antioxidants activity of Salvia moorcroftiana L. as influenced by combinations of auxin, cytokinin, and melatonin. Brazilian Archives of Biology and Technology, 65, e22210200. https://doi.org/10.1590/1678-4324-2022210200
Basyuni, M., Puspita, M., Rahmania, R., Albasri, H., Pratama, I., Purbani, D., Aznawi, A. A., ... Kajita, T. (2024). Current biodiversity status, distribution, and prospects of seaweed in Indonesia: A systematic review. Heliyon, 10(10), e31073. https://doi.org/10.1016/j.heliyon.2024.e31073
Bennur, P. L., O’Brien, M., Fernando, S. C., & Doblin, M. S. (2025). Improving transformation and regeneration efficiency in medicinal plants: Insights from other recalcitrant species. Journal of Experimental Botany, 76(1), 52-75. https://doi.org/10.1093/jxb/erae189
Bielach, A., Hrtyan, M., & Tognetti, V. (2017). Plants under stress: Involvement of auxin and cytokinin. International Journal of Molecular Sciences, 18(7), 1427. https://doi.org/10.3390/ijms18071427
Chandimali, N., Park, E. H., Bak, S.-G., Lim, H.-J., Won, Y.-S., & Lee, S.-J. (2024). Seaweed callus culture: A comprehensive review of current circumstances and future perspectives. Algal Research, 77, 103376. https://doi.org/10.1016/j.algal.2023.103376
Chandler, J., & Werr, W. (2015). Cytokinin–auxin crosstalk in cell type specification. Trends in Plant Science, 20(5), 291-300. https://doi.org/10.1016/j.tplants.2015.02.003
Chen, Y. M., Huang, J. Z., Hou, T. W., & Pan, I. C. (2019). Effects of light intensity and plant growth regulators on callus proliferation and shoot regeneration in the ornamental succulent Haworthia. Botanical Studies, 60(1), 10. https://doi.org/10.1186/s40529-019-0257-y
Das, A. K., & Prasad, K. (2015). Extraction of plant growth regulators present in Kappaphycus alvarezii sap using imidazolium-based ionic liquids: Detection and quantification by using HPLC-DAD technique. Analytical Methods, 7, 9064-9067. https://doi.org/10.1039/C5AY02210J
Fadilah, S., Pong-Masak, P. R., Farman, A., & Ratnawati, P. (2024). Genetic diversity, growth, and carrageenan quality of Kotoni (red seaweed) across three cultivation sites in Eastern Indonesia. Indonesian Aquaculture Journal, 19(2), 167-178. https://doi.org/10.15578/iaj.19.2.2024.167-178
FAO. (2024). The State of World Fisheries and Aquaculture 2024 – Blue Transformation in Action. Rome. https://doi.org/10.4060/cd0683en
Fehér, A. (2023). A common molecular signature indicates the pre-meristematic state of plant calli. International Journal of Molecular Sciences, 24(17), 13122. https://doi.org/10.3390/ijms241713122
Ge, F., Luo, X., Huang, X., Zhang, Y., He, X., Liu, M., Lin, H., ... Shen, Y. (2016). Genome-wide analysis of transcription factors involved in maize embryonic callus formation. Physiologia Plantarum, 158(4), 452-462. https://doi.org/10.1111/ppl.12470
Hayashi, L., Yokoya, N. S., Kikuchi, D. M., & Oliveira, E. C. (2008). Callus induction and micropropagation improved by colchicine and phytoregulators in Kappaphycus alvarezii (Rhodophyta, Solieriaceae). Journal of Applied Phycology, 20, 653-659. https://doi.org/10.1007/s10811-007-9234-z
Hlaing, W., & Jarukamjor, K. (2024). Plantlet regeneration from callus cultures of Kappaphycus alvarezii for cultivation in coastal waters at Myeik Archipelago, Myanmar. Pakistan Journal of Biological Sciences, 27(9), 479-486. https://doi.org/10.3923/pjbs.2024.479.486
Hong, C., Lee, H., Shim, S., Park, O., Kim, J., Lee, K., Oh, E., ... Seo, P. J. (2024). Histone modification-dependent production of peptide hormones facilitates acquisition of pluripotency during leaf-to-callus transition in Arabidopsis. New Phytologist, 242(3), 1068-1083. https://doi.org/10.1111/nph.19637
Ikeuchi, M., Sugimoto, K., & Iwase, A. (2013). Plant callus: Mechanisms of induction and repression. Plant Cell, 25(9), 3159-3173. https://doi.org/10.1105/tpc.113.116053
Jiksing, C., Ongkudon, M. M., Thien, V. Y., Rodrigues, K. F., & Yong, W. T. L. (2022). Recent advances in seaweed seedling production: A review of eucheumatoids and other valuable seaweeds. Algae, 37(2), 105-121. https://doi.org/10.4490/algae.2022.37.5.11
Kester, D. R., Duedall, I. W., Connors, D. N., & Pytkowicz, R. M. (1967). Preparation of artificial seawater. Limnology and Oceanography, 12(1), 176-179. https://doi.org/10.4319/lo.1967.12.1.0176
Khan, N., Sudhakar, K., & Mamat, R. (2023). Seaweed farming: A perspectives of genetic engineering and nano-technology application. Heliyon, 9(4), e15168. https://doi.org/10.1016/j.heliyon.2023.e15168
Klimek-Chodacka, M., Kad³uczka, D., £ukasiewicz, A., Malec-Pala, A., Barañski, R., & Grzebelus, E. (2020). Effective callus induction and plant regeneration in callus and protoplast cultures of Nigella damascena L. Plant Cell, Tissue, and Organ Culture (PCTOC), 143(3), 693-707. https://doi.org/10.1007/s11240-020-01953-9
Kruglova, N. (2022). Callus formation and callusogenesis in vitro in cereals: The role of hormonal balance (review). Izvestia Ufimskogo Nauchnogo Tsentra Ran, 0(1), 52-59. https://doi.org/10.31040/2222-8349-2022-0-1-52-59
Kumar, Y., Poong, S., Gachon, C., Brodie, J., Sade, A., & Lim, P. (2020). Impact of elevated temperature on the physiological and biochemical responses of Kappaphycus alvarezii (Rhodophyta). Plos One, 15(9), e0239097. https://doi.org/10.1371/journal.pone.0239097
Lee, J.-H., Bashir, K. M. I., Tirtawijaya, G., Negara, B. F. S. P., & Choi, J.-S. (2024). Establishment of effective callus induction in the economically important brown seaweed Ecklonia cava. Applied Sciences, 14(8), 3480. https://doi.org/10.3390/app14083480
Lim, P., Tan, J., Phang, S., Nikmatullah, A., HÓng, Ð., Sunarpi, H., & Hurtado, A. Q. (2013). Genetic diversity of Kappaphycus doty and Eucheuma J. Agardh (Solieriaceae, Rhodophyta) in Southeast Asia. Journal of Applied Phycology, 26(2), 1253-1272. https://doi.org/10.1007/s10811-013-0197-y
Long, Y., Yang, Y., Pan, G., & Shen, Y. (2022). New insights into tissue culture plant-regeneration mechanisms. Frontiers in Plant Science, 13, 926752. https://doi.org/10.3389/fpls.2022.926752
Ma, J., Li, Q., Zhang, L., Cai, S., Liu, Y., Lin, J., Huang, R., ... Xu, T. (2022). High auxin stimulates callus through SDG8-mediated histone H3K36 methylation in Arabidopsis. Journal of Integrative Plant Biology, 64(12), 2425-2437. https://doi.org/10.1111/jipb.13387
Maradhy, E., Nazriel, R., Sutjahjo, S., Rusli, M., Widiatmaka, W., & Sondita, M. (2022). Evaluation of water suitability for sustainable seaweed (Kappaphycus alvarezii) cultivation to support science technopark in North Kalimantan. Jurnal Pengelolaan Sumberdaya Alam dan Lingkungan (Journal of Natural Resources and Environmental Management), 11(3), 490-503. https://doi.org/10.29244/jpsl.11.3.490-503
Martinez, M., Jorquera, L., Poirrier, P., Díaz, K., & Chamy, R. (2021). Effect of the carbon source and plant growth regulators (PGRs) in the induction and maintenance of an in vitro callus culture of Taraxacum officinale (L) Weber ex F.H. Wigg. Agronomy, 11(6), 1181. https://doi.org/10.3390/agronomy11061181
Mazzoni, S., Brumós, J., Zhao, C., Alonso, J., & Stepanova, A. (2021). Auxin interactions with other hormones in plant development. Cold Spring Harbor Perspectives in Biology, 13(10), a039990. https://doi.org/10.1101/cshperspect.a039990
Meiyana, M., Dhoe, S., Minjoyo, H., & Rivaie, A. (2023). Growth of Eucheuma cottonii seaweed from tissue culture with different time acclimatization in Hurun Bay. E3s Web of Conferences, 442, 02026. https://doi.org/10.1051/e3sconf/202344202026
Mo, V., & Reddy, C. (2016). Investigation of explants sterilization process, effect of light intensity, and concentration of agar for callus induction of Kappaphycus alvarezii (Doty) doty (Rhodophyta) in vitro. Vietnam Journal of Biotechnology, 14(3), 515-522. https://doi.org/10.15625/1811-4989/14/3/9868
Mo, V. T., Cuong, L. K., Tung, H. T., Huynh, T. V., Nghia, L. T., Khanh, C. M., Lam, N. N., & Nhut, D. T. (2020). Somatic embryogenesis and plantlet regeneration from the seaweed Kappaphycus striatus. Acta Physiologiae Plantarum, 42(104). https://doi.org/10.1007/s11738-020-03102-3
Muyong, J. S., & Tahiluddin, A. B. (2024). Interaction of nutrient enrichment and farming method on performance of the red seaweed Kappaphycus alvarezii. Aquatic Botany, 191, 103743. https://doi.org/10.1016/j.aquabot.2023.103743
Neves, F. A. S., Simioni, C., Bouzon, Z. L., & Hayashi, L. (2015). Effects of spindle inhibitors and phytoregulators on the micropropagation of Kappaphycus alvarezii (Rhodophyta, Gigartinales). Journal of Applied Phycology, 27, 437-445. https://doi.org/10.1007/s10811-014-0309-3
Nie, S., Wang, Y., Yan, Y., Liu, S., Guo, W., Yang, L., & Shen, H. (2023). Effects of a new plant growth regulator on callus induction from immature embryo explants of Korean pine (Pinus koraiensis). Forests, 14(12), 2413. https://doi.org/10.3390/f14122413
Noor, W., Lone, R., Kamili, A. N., & Husaini, A. M. (2022). Callus induction and regeneration in high-altitude Himalayan rice genotype SR4 via seed explant. Biotechnology Reports (36). https://doi.org/10.1016/j.btre.2022.e00762
Ozden, M., & Karaaslan, M. (2011). Effects of cytokinin on callus proliferation associated with physiological and biochemical changes in Vitis vinifera L. Acta Physiologiae Plantarum, 33, 1451-1459. https://doi.org/10.1007/s11738-010-0681-9
Pereira, L. (2021). Macroalgae. Encyclopedia, 1(1), 177-188. https://doi.org/10.3390/encyclopedia1010017
Perianez-Rodriguez, J., Manzano, C., & Moreno-Risueno, M. A. (2014). Post-embryonic organogenesis and plant regeneration from tissues: Two sides of the same coin? Frontiers in Plant Science, 5, 219. https://doi.org/10.3389/fpls.2014.00219
Pradhan, B., & Ki, J.-S. (2023). Biological activity of algal derived carrageenan: A comprehensive review in light of human health and disease. International Journal of Biological Macromolecules, 238, 124085. https://doi.org/10.1016/j.ijbiomac.2023.124085
Provasoli, L. (1968). Media and prospects for the cultivation of marine algae. Proceedings of the US-Japan Conference, Hakone, 12-15 September 1966, 63-75.
Rademacher, W. (2015). Plant growth regulators: Backgrounds and uses in plant production. Journal of Plant Growth Regulation, 34(4), 845-872. https://doi.org/10.1007/s00344-015-9541-6
Radkhah, A. R., Eagderi, S., Poorbagher, H., & Atash Afrazeh, S. (2024). Investigating the potential of toxins from cyanobacterial growth in bioterrorism: The need for protective-management strategies for aquaculture center employees. Journal of Biosafety, 16(4), 55-80. http://journalofbiosafety.ir/article-1-550-fa.html
Rupert, R., Rodrigues, K., Thien, V., & Yong, W. (2022). Carrageenan from Kappaphycus alvarezii (Rhodophyta, Solieriaceae): Metabolism, structure, production, and application. Frontiers in Plant Science, 13. https://doi.org/10.3389/fpls.2022.859635
Segaran, T. C., Azra, M. N., Mohd Noor, M. I., Danish-Daniel, M., Burlakovs, J., Lananan, F., Xu, J., Kari, Z. A., & Wei, L. S. (2024). Knowledge mapping analysis of the global seaweed research using CiteSpace. Heliyon, 10(7), e28418. https://doi.org/10.1016/j.heliyon.2024.e28418
Siddiqui, S. A., Agrawal, S., Brahmbhatt, H., & Rathore, M. S. (2022). Metabolite expression changes in Kappaphycus alvarezii (a red alga) under hypo- and hyper-saline conditions. Algal Research, 63, 102650. https://doi.org/10.1016/j.algal.2022.102650
Sosnowski, J., Truba, M., & Vasileva, V. (2023). The impact of auxin and cytokinin on the growth and development of selected crops. Agriculture, 13(3), 724. https://doi.org/10.3390/agriculture13030724
Statistics Indonesia (Badan Pusat Statistik / BPS). (2023). Exports of seaweed and other algae by major countries of destination, 2012–2022. Retrieved from https://www.bps.go.id/
Stirk, W., & Staden, J. (2014). Plant growth regulators in seaweeds. 125-159. https://doi.org/10.1016/b978-0-12-408062-1.00005-6
Sulistiani, E., Soelistyowati, D. T., Alimuddin, A., & Yani, S. A. (2012). Callus induction and filaments regeneration from callus of cottonii seaweed (Kappaphycus alvarezii (Doty) collected from Natuna Islands, Riau Islands Province. Biotropia, 19(2), 103-114. https://doi.org/10.11598/btb.2012.19.2.254
Suryati, E., Rosmiati, R., Parenrengi, A., & Tenriulo, A. (2015). In vitro growth rate of Kappaphycus alvarezii micropropagule and embryo by enrichment medium with seaweed extract. Indonesian Aquaculture Journal, 10(1), 13. https://doi.org/10.15578/iaj.10.1.2015.13-17
Tanaka, Y., Ashaari, A., Mohamad, F., & Lamit, N. (2020). Bioremediation potential of tropical seaweeds in aquaculture: Low-salinity tolerance, phosphorus content, and production of UV-absorbing compounds. Aquaculture, 518, 734853. https://doi.org/10.1016/j.aquaculture.2019.734853
Tirtawijaya, G., Negara, B. F. S. P., Lee, J.-H., Cho, M.-G., Kim, H. K., Choi, Y.-S., Lee, S.-H., & Choi, J.-S. (2022). The influence of abiotic factors on the induction of seaweed callus. Journal of Marine Science and Engineering, 10(4), 513. https://doi.org/10.3390/jmse10040513
Tuskan, G. A., Mewalal, R., Gunter, L. E., Palla, K. J., Carter, K., Jacobson, D. A., Jones, P. C., Garcia, B. J., Weighill, D. A., Hyatt, P. D., Yang, Y., Zhang, J., Reis, N., Chen, J. G., & Muchero, W. (2018). Defining the genetic components of callus formation: A GWAS approach. Plos One, 13(8), e0202519. https://doi.org/10.1371/journal.pone.0202519
Wang, W., Li, H., Lin, X., Yang, S., Wang, Z., & Fang, B. (2015). Transcriptome analysis identifies genes involved in adventitious branches formation of Gracilaria lichenoides in vitro. Scientific Reports, 5, 17099. https://doi.org/10.1038/srep17099
Wang, W., Li, H., Lin, X., Zhang, F., Fang, B., & Wang, Z. (2016). The effect of polar auxin transport on adventitious branches formation in Gracilaria lichenoides in vitro. Physiologia Plantarum, 158(3), 356-365. https://doi.org/10.1111/ppl.12464
Yegappan, R., Selvaprithiviraj, V., Amirthalingam, S., & Jayakumar, R. (2018). Carrageenan based hydrogels for drug delivery, tissue engineering, and wound healing. Carbohydrate Polymers, 198, 385-400. https://doi.org/10.1016/j.carbpol.2018.06.086
Yin, R., Chen, R., Xia, K., & Xu, X. (2024). A single-cell transcriptome atlas reveals the trajectory of early cell fate transition during callus induction in Arabidopsis. Plant Communications, 5(8), 100941. https://doi.org/10.1016/j.xplc.2024.100941
DOI: http://dx.doi.org/10.15578/iaj.20.1.2025.1-10

This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License.

Indonesian Aquaculture Journal is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License.