Micropropagation of Kratom (Mitragyna speciosa (Korth.) Havil.): Disinfectants, Growth Regulators, and Low-Cost Chemical Disinfection for In Vitro Establishment

Suchonma Sookruksawong

doi:10.69650/ahstr.2024.2887

Authors

Suchonma Sookruksawong Department of Applied Biology, Faculty of Sciences and Liberal Arts, Rajamangala University of Technology Isan, Nakhon Ratchasima 30000, Thailand

DOI:

https://doi.org/10.69650/ahstr.2024.2887

Keywords:

Kratom, low-cost tissue culture, chemical disinfection, plant growth hormones

Abstract

Kratom, Mitragyna speciosa (Korth.) Havil. is a medicinal plant native to Southeast Asia that is renowned for its therapeutic properties and potential in treating various ailments. Despite its significance, the cultivation and propagation of Kratom have been limited. The purpose of the present study was to develop a cost-effective micropropagation protocol for Kratom by investigating the disinfection efficiency and cost-effectiveness of various chemical disinfectants, optimizing plant growth regulator concentrations, and assessing cost-effective media sterilization methods. The results demonstrated that double disinfection with commercial bleach at 20% and 15% for 5 min each was the most cost-effective treatment for surface disinfection of Kratom seeds, achieving a high disinfection rate (96.67±2.89%) and survival rate (73.33±2.89%) at a relatively low cost (1.46 baht per experiment) compared to mercuric chloride (HgCl₂) treatments. The evaluation of benzyladenine (BA) and naphthaleneacetic acid (NAA) effects on in vitro growth revealed that the control treatment (MS (Murashige and Skoog medium) without growth regulators) exhibited the best overall growth performance. Among the low-cost disinfectants tested for the culture medium, while autoclaving offers superior disinfection efficacy, commercial bleach at 2 ml/l emerged as the most cost-effective option, especially for resource-limited operations. The final choice, however, should align with specific operational requirements, including scale, resources, and the need for complete disinfection. The successful establishment of a cost-effective micropropagation protocol using low-cost chemical disinfectants and optimized plant growth regulator concentrations can significantly reduce the production costs associated with tissue culture techniques, making the micropropagation of Kratom more economically viable and accessible for large-scale production. The findings from this research provide insights into cost-effective micropropagation methods for Kratom, which will prove valuable for future studies and applications in the field.

References

Abas, L., Kolb, M., Stadlmann, J., Janacek, D. P., Lukic, K., Schwechheimer, C., Sazanov, L. A., Mach, L., Friml, J., & Hammes, U. Z. (2021). Naphthylphthalamic acid associates with and inhibits PIN auxin transporters. Proceedings of the National Academy of Sciences, 118(1), e2020857118. https://doi.org/10.1073/pnas.2020857118

Alsaedy, N. S., Almilly, R. F., & Al-Shannag, M. (2023). Testing of a potentially used antiseptic consists of povidon iodine, hydrogen peroxide and Aloe vera. Iraqi Journal of Chemical and Petroleum Engineering, 24(1), 105-112. https://doi.org/10.31699/IJCPE.2023.1.12

Amarasinghe, R. M. N. T., Wang, J. H., Xie, W. X., Peng, L. C., Li, S. F., & Li, H. (2018). Seed-sterilization of Rhododendron wardii for micropropagation. Sri Lanka Journal of Food and Agriculture, 4(1), 9-14. https://doi.org/10.4038/sljfa.v4i1.51

Babu, G. A., Mosa Christas, K., Kowsalya, E., Ramesh, M., Sohn, S.-I., & Pandian, S. (2022). Improved sterilization techniques for successful in vitro micropropagation. In S. Gupta & P. Chaturvedi (Eds.), Commercial Scale Tissue Culture for Horticulture and Plantation Crops (pp. 1-21). Springer Nature: Singapore. https://doi.org/10.1007/978-981-19-0055-6_1

Bakhsh, A., Anayol, E., Sancak, C., & Özcan, S. (2016). An efficient and cost effective sterilizing method with least microbial contamination and maximum germination ratio for in vitro cotton (Gossypium hirsutum L.) culture. The Journal of Animal and Plant Sciences, 26(3), 868-873.

Charoenratana, S., Anukul, C., & Aramrattana, A. (2021). Attitudes towards Kratom use, decriminalization and the development of a community-based Kratom control mechanism in Southern Thailand. International Journal of Drug Policy, 95, 103197.

Chotikadachanarong, K. (2014). Influence of commercial bleach on sterilization of persian violet (Exacum affine Balf. F. ex Regel) tissue. Rajabhat Journal of Science, Humanities & Social Science, 14(2), 34-43.

Chotikadachanarong, K. (2018). Effects of media from hydroponics solution and culture temperature on tissue culture of Bacopa caroliniana (Walt.) Rob. The Agricultural Science Journal, 49(1), 88–95-88–95. http://agscij.agr.ku.ac.th/phocadownload/2561-49-1/ASJ-49-1-88-95.pdf

Da Costa Urtiga, C., de Araújo Silva-Cardoso, I. M., & Araujo Figueiredo, S. (2019). Low sodium isocyanurate concentrations as a substitute to medium autoclaving in plant tissue culture. Plant Cell, Tissue and Organ Culture (PCTOC), 139(3), 601-604. https://doi.org/10.1007/s11240-019-01681-9

Duan, Y., Zhang, H., Sun, M., Zhao, F., Xue, T., & Xue, J. (2019). Use of chlorine dioxide to sterilize medium for tissue culture of potato. Scientific reports, 9(1), 10232. https://doi.org/10.1038/s41598-019-46795-4

Fukuzaki, S. (2006). Mechanisms of actions of sodium hypochlorite in cleaning and disinfection processes. Biocontrol science, 11(4), 147-157. https://doi.org/10.4265/bio.11.147

Gaba, V. P. (2005). Plant growth regulators in plant tissue culture and development. Plant development and biotechnology, 87-99. https://doi.org/10.1201/9780203506561.ch8

Hassan, Z., Muzaimi, M., Navaratnam, V., Yusoff, N. H. M., Suhaimi, F. W., Vadivelu, R., Vicknasingam, B. K., Amato, D., von Hörsten, S., Ismail, N. I. W., Jayabalan, N., Hazim, A. I., Mansor, S. M., & Müller, C. P. (2013). From Kratom to mitragynine and its derivatives: Physiological and behavioural effects related to use, abuse, and addiction. Neuroscience & Biobehavioral Reviews, 37(2), 138-151. https://doi.org/10.1016/j.neubiorev.2012.11.012

Jader, H. S., & Obaid, O. H. (2023). Effect different concentrations of growth regulators on callus induction from different explants of Aloe vera L. in vitro. AIP Conference Proceedings, 2776(1), 030006. https://doi.org/10.1063/5.0137255

Katarzyna, W., & Przemysław, B. (2010). The effect of benzyladenine and naphthalene acetic acid on rooting and subsequent growth of Portulaca umbraticola Kunth. Folia Horticulturae, 22(2), 39-44. https://doi.org/10.2478/fhort-2013-0157

Liu, X., Xu, D., Yang, Z., Zhang, N., & Pan, L. (2018). Investigation of exogenous benzyladenine on growth, biochemical composition, photosynthesis and antioxidant activity of Indian sandalwood (Santalum album L.) seedlings. Journal of plant growth regulation, 37(4), 1148-1158. https://doi.org/10.1007/s00344-018-9806-y

Matsumoto, K., Mizowaki, M., Suchitra, T., Murakami, Y., Takayama, H., Sakai, S.-i., Aimi, N., & Watanabe, H. (1996). Central antinociceptive effects of mitragynine in mice: contribution of descending noradrenergic and serotonergic systems. European journal of pharmacology, 317(1), 75-81. https://doi.org/https://doi.org/10.1016/S0014-2999(96)00714-5

Mohamad Zuldin, N. N., Said, I. M., Mohd Noor, N., Zainal, Z., Jin Kiat, C., & Ismail, I. (2013). Induction and analysis of the alkaloid mitragynine content of a Mitragyna speciosa suspension culture system upon elicitation and precursor feeding. The Scientific World Journal, 2013, 209434. https://doi.org/10.1155/2013/209434

Murashige, T., & Skoog, F. (1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia plantarum, 15(3), 473-497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x

Nadhirah, N., Maideen, H., Ab Rahman, Z., & Othman, A. N. (2022). Optimizing in vitro surface sterilization of Cyathea latebrosa Spore. Malaysian Applied Biology, 51(5), 159-163. https://doi.org/10.55230/mabjournal.v51i5.2422

Nair, S., Zhu, A., Jaffry, M., Choudhry, H., & Dastjerdi, M. H. (2023). Povidone-iodine adverse effects and alternatives for ocular procedures. Journal of Ocular Pharmacology and Therapeutics, 39(3), 207-214. https://doi.org/10.1089/jop.2022.0122

Nakaphan, T., Teerachaisakul, M., Puttum, S., Sompimai, K., & Nootim, P. (2016). Traditional uses of Kratom (Mitragyna speciosa Korth) among folk healers in Southern Thailand. http://thailand.digitaljournals.org/index.php/JTTAM/article/download/32871/31831

Nurul, H. D., Shashita, J., & Rozi, M. (2012). An improved surface sterilization technique for introducing leaf, nodal and seed explants of Aquilaria malaccensis from field sources into tissue culture. Asia-Pacific Journal of Molecular Biology and Biotechnology, 20, 55-58.

Pais, A. K., da Silva, A. P., de Souza, J. C., Teixeira, S. L., Ribeiro, J. M., Peixoto, A. R., & da Paz, C. D. (2016). Sodium hypochlorite sterilization of culture medium in micropropagation of Gerbera hybrida cv. Essandre. African Journal of Biotechnology, 15(36), 1995-1998.

Phongprueksapattana, S., Putalun, W., Keawpradub, N., & Wungsintaweekul, J. (2008). Mitragyna speciosa: hairy root culture for triterpenoid production and high yield of mitragynine by regenerated plants. 63(9-10), 691-698. https://doi.org/10.1515/znc-2008-9-1014(Zeitschrift für Naturforschung C)

Phromchan, W. (2023). Changes in Morpho-Physiological Traits on Phytochemical contents in Kratom (Mitragyna speciosa (Korth.) Havil.) Leaves Prince of Songkla University. https://kb.psu.ac.th/psukb/bitstream/2016/19348/1/6310620019.pdf

Prevete, E., Kuypers, K. P. C., Theunissen, E. L., Esposito, G., Ramaekers, J. G., Pasquini, M., & Corazza, O. (2023). Clinical implications of Kratom (Mitragyna speciosa) use: a literature review. Current Addiction Reports, 10(2), 317-334. https://doi.org/10.1007/s40429-023-00478-3

Saingam, D., Assanangkornchai, S., Geater, A. F., & Lerkiatbundit, S. (2016). Factor analytical investigation of Krathom (Mitragyna speciosa Korth.) withdrawal syndrome in Thailand. Journal of Psychoactive Drugs, 48(2), 76-85. https://doi.org/10.1080/02791072.2016.1156791

Sengnon, N., Vonghirundecha, P., Chaichan, W., Juengwatanatrakul, T., Onthong, J., Kitprasong, P., Sriwiriyajan, S., Chittrakarn, S., Limsuwanchote, S., & Wungsintaweekul, J. (2023). Seasonal and geographic variation in alkaloid content of Kratom (Mitragyna speciosa (Korth.) Havil.) from Thailand. Plants, 12(4), 949. https://doi.org/10.3390/plants12040949

Simran Chandrahas, S., & Narasimhan, S. (2021). Sodium dichloroisocyanurate: an eco-friendly chemical alternative for media autoclaving and explant sterilisation in plant tissue culture. International Journal of Research in Pharmaceutical Sciences, 12(1), 107-112. https://ijrps.com/home/article/view/123

Singh, D., Narayanan, S., Vicknasingam, B., Corazza, O., Santacroce, R., & Roman-Urrestarazu, A. (2017). Changing trends in the use of Kratom (Mitragyna speciosa) in Southeast Asia. Human Psychopharmacology: Clinical and Experimental, 32(3), e2582. https://doi.org/10.1002/hup.2582

Sookruksawong, S. (2022). Sterilization and low-cost tissue culture techniques of Dioscorea bulbifera. Science Technology and Social Sciences Procedia, 2022(4), rspg014-rspg014.

Sookruksawong, S. (2023). Explants sterilization techniques of Dioscorea spp. for tissue culture. The 14th Engineering, Science, Technology and Architecture Conference 2023 (ESTACON 2023), (pp. 680-688). Kalasin University.

Sookruksawong, S. (2024). Optimum sterilization method for in vitro cultivation of Mammillaria plumosa Cacti. The 7th National and International Research Conference 2024: NIRC VII 2024 (pp. 789-803). Buriram Rajabhat University.

Suwanlert, S. (1975). A study of Kratom eaters in Thailand. Bull Narc, 27(3), 21-27.

Thepsithar, C., & Thongpukdee, A. (2013). Sterilisation of hyponex medium by chemicals without autoclaving and growth of Phalaenopsis protocorms. World Academy of Science, Engineering and Technology, International Journal of Biological, Biomolecular, Agricultural, Food and Biotechnological Engineering, 7, 403-406.

Thepsithar, C., Thongpukdee, A., & Daorat, A. (2013). Sterilisation of in vitro culture medium of chrysanthemum by plant essential oils without autoclaving. World Academy of Science, Engineering and Technology, International Journal of Biological, Biomolecular, Agricultural, Food and Biotechnological Engineering, 7, 802-805.

Todd, D. A., Kellogg, J. J., Wallace, E. D., Khin, M., Flores-Bocanegra, L., Tanna, R. S., McIntosh, S., Raja, H. A., Graf, T. N., Hemby, S. E., Paine, M. F., Oberlies, N. H., & Cech, N. B. (2020). Chemical composition and biological effects of Kratom (Mitragyna speciosa): in vitro studies with implications for efficacy and drug interactions. Scientific reports, 10(1), 19158. https://doi.org/10.1038/s41598-020-76119-w

Urbano, T., Malavolti, M., Vinceti, M., & Filippini, T. (2024). Mercuric chloride (HgCl2). In P. Wexler (Ed.), Encyclopedia of Toxicology (Fourth Edition) (pp. 117-122). Academic Press. https://doi.org/10.1016/B978-0-12-824315-2.00084-1

Wamaedeesa, R., Ali, B., Chedao, N., & Kanjanawattanawong, S. (2021). Chemical sterilization in MS culture medium for in vitro culture of Philodendron sp.“Ruaysap”. Princess of Naradhiwas University Journal, 13(1), 377-387.

Warner, M. L., Kaufman, N. C., & Grundmann, O. (2016). The pharmacology and toxicology of Kratom: from traditional herb to drug of abuse. International journal of legal medicine, 130(1), 127-138. https://doi.org/10.1007/s00414-015-1279-y

Weber, B. N., Witherell, R. A., & Charkowski, A. O. (2015). Low-cost potato tissue culture with microwave and bleach media preparation and sterilization. American journal of potato research, 92(1), 128-137. https://doi.org/10.1007/s12230-014-9423-7

Webster, S., Mitchell, S., & Ahmad, M. (2003, November). A novel surface sterilization method for reducing microbial contamination of field grown medicinal explants intended for in vitro culture. Biotechnology Centre, UWI, Mona, Kingston.

Wijaya, N. R., Safrina, D., Herera, P. B., & Budiarti, M. (2022). Effect of NAA and BAP application on the growth responses of Mentha piperita L. Journal of Indonesian Medicinal Plant, 15(2), 109-117. https://doi.org/10.22435/jtoi.v15i2.6436

Xiulei, Z., Zhangyou, B., & Ming, R. (2012). Research on explants sterilization technique in Staphylea bumalda DC. tissue culture process. Journal of Anhui Agricultural Sciences, 4, 2110.

Yuniastuti, E., Chintiana, D. G., Sukaya, & Putri, I. I. S. (2022). Effect of NAA and BAP concentrations on rambutan growth (Nephelium lappaceum L.) in Vitro. IOP Conference Series: Earth and Environmental Science, 1107(1), 012029. https://doi.org/10.1088/1755-1315/1107/1/012029

Zulkarnain, Z., & Neliyati, N. (2017). The effect of NAA and BAP on tissue culture of Tangkit pineapple (Ananas comosus (L.) Merr. cv. Tangkit). Biospecies, 10(1), 1-10. https://repository.unja.ac.id/id/eprint/2597