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Sterilisation of in vitro Culture Medium of Chrysanthemum by Plant Essential Oils without Autoclaving

Authors: Chockpisit Thepsithar, Aree Thongpukdee, Apichya Daorat


The alternative technique for sterilization of culture medium to replace autoclaving was carried out. For sterilization of culture medium without autoclaving, some commercial pure essential oils, bergamot oil, betel oil, cinnamon oil, lavender oil and turmeric oil, were tested alone or in combinations with some disinfectants, 10% povidone-iodine and 2% iodine + 2.4% potassium iodide. Each essential oil or combination was added to 25-mL Murashige and Skoog (MS) medium before medium was solidified in a 120-mL container, kept for 2 weeks before evaluating sterile conditions. Treated media, supplemented with essential oils, were compared to control medium, autoclaved at 121 degree Celsius for 15 min. In vitro sterile conditions were found 20 – 100% from these treated media compared to 100% sterile condition from autoclaved medium. Treated media obtained 100% sterile conditions were chosen for culturing chrysanthemum shoots. It was found that 10% povidoneiodine in combination with cinnamon oil (3:1) and 2% iodine + 2.4% potassium iodide in combination with lavender oil (1:3) at the concentration of 36 3L/25 mL medium provided the promising growth of shoot explants.

Keywords: Sterilizing agents, essential oils, disinfectants, MS medium, in vitro culture, chrysanthemum, sterilization of medium without autoclaving

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[1] A. D. Russell, “Similarities and differences in the responses of microorganisms to biocides.” Journal of Antimicrobial Chemotherapy, vol. 52, no. 5, pp. 750–763, 2003.
[2] United State Patent 5750402. “Compositions and methods to prevent microbial contamination of plant tissue culture media.” (Online). Available; http://www. 5750402.html.
[3] U. Habiba, S. Reza, M. L. Saha, M. R. Khan, and S. Hadiuzzaman, “Endogenous bacterial contamination during in vitro culture of table banana: Identification and prevention.” Plant Tissue Culture, vol. 12, no. 2, pp. 117–124, 2002.
[4] S. L. Teixeira, “The development of a new protocol that uses sodium hypochlorite to replace the autoclaving procedure for establishing axenic in vitro banan (Musa sp.) culture.” Agricell Report, vol. 47, no. 3, pp. 17–18, 2006.
[5] T. Yanagawa, M. Nagai, T. Ogino, and M. Maeguchi, “Application of disinfection to orchid seeds, plantlet and media as a means to prevent in vitro contamination.” Lindleyana, vol. 10, pp. 33–36, 2006.
[6] M. Chansean, and I. Syoichi, “Conservation of Wild Orchids in Cambodia by Simple Aseptic Culture Method.” Proceedings of NIOC, pp. 13–19, April, 2007.
[7] W. Deein, C. Thepsithar, and P. Karawak, “Effect of disinfectants on sterile conditions of VW medium and growth of Dendrobium seeds. The 12th National Horticulture Congress, Horticulture Toward AEC Under Climate Changes, Bangkok, Thailand, p. 77, 9-12 May 2013.
[8] C. Thepsithar, and A. Thongpukdee. “Sterilisation of Hyponex medium by chemicals without autoclaving and growth of Phalaenopsis protocorms.” World Academy of Science, Engineering and Technology, vol. 78, pp. 786–789, 2013.
[9] R. A. Sawant, and P. N. Tawar, “Use of sodium hypochlorite as medium sterilant in sugarcane micropropagation at commercial scale.” Sugar Tech, vol. 13, no. 1, pp. 27-35, 2011.
[10] C. Thepsithar, A, Thongpukdee, S. Bunmee, and V. Chanasak, “Effects of sterilizing agents to microbial contamination and plant growth in vitro. Agricultural. Sci. J., vol. 40, no. 3 (suppl.), pp. 107-110, 2009.
[11] W. Deein, C. Thepsithar, and A. Thongpukdee, “In vitro culture medium sterilization by chemicals and essential oils without autoclaving and growth of chrysanthemum nodes.” World Academy of Science, Engineering and Technology, vol. 78, pp. 1014–1017, 2013.
[12] M. M. Hoque, S. Rattila, M. A. Shishir, M. L. Bari, Y. Inatsu, and S. Kawamoto, “Antibacterial activity of ethanol extract of betel leaf (Piper betle L.) against some food borne pathogens.” Bangladesh Journal of Microbiology, vol. 28, no. 1, 58–63, 2011.
[13] M. Seema, S. S. Sreenivas, N. D. Rekha, and N. S. Devaki, “In vitro studies of some plant extracts against Rhizoctonia solani Kuhn infecting FCV tobacco in Karnataka Light Soil, Karnataka, India.” Journal of Agricultural technology, vol. 7, no. 5, pp. 1321–1329, 2011.
[14] D. Srichana, A. Phumruang, A. and Chongkid, B. “Inhibition effect of betel leaf extract on the growth of Aspergillus flavus and Fusarium verticillioides.” Thammasat International Journal of Science and Technology, vol. 14, no. 3, pp. 74–77, 2009.
[15] T. S. A. T. Kamazeri, O. A. Samah, M. Taher, D. Susanti, and H. Qaralleh, “Antimicrobial activity and essential oils of Curcuma aerugonosa, Curcuma mangga and Zingiber cassumunar from Malaysia.” Asian Pacific Journal of Tropical Medicine, vol. 5, no. 3, 202–209, March, 2012.
[16] B. Joshi, G. P. Sah, B. B. Basnet, M. R. Bhatt, D. Sharma, K. Subedi, J. Pandey, and R. Malla, “Phytochemical extraction and antimicrobial properties of different medicinal plants: Ocimum sanctum (tulsi), Eugenia caryophyllata (clove), Achyranthes bidentata (datiwan) and Azadirachta indica (neem).” Journal of Microbiology and Antimicrobials, vol. 3, no. 1, pp. 1–7, January, 2011.
[17] I. N. Fit, G. Rapuntean, S. Rapuntean, F. Chirila, and G. C. Nadas, “Antibacterial effect of essential vegetal extracts on Staphylococcus aureus compared to antibiotics.” Notulae Botanicae Horti Agrobotanici Cluj-Napoca, vol. 37, 117–223, 2009.
[18] L. Hui, L. He, L. Huan, L. Xiaolan, and Z. Aiguo, “Chemical composition of lavender essential oil and its antioxidant activity and inhibition against rhinitis related bacteria.” African Journal of Microbiology Research, vol. 4, pp. 309–313, 2010.
[19] F. G. Kirbaslar, A. Tavman, B. Dulger, and G. Turker, “Antimicrobial activity of Turkish Citrus peel oils.” Pakistan Journal of Botany, vol. 41, pp. 3207–3212, 2009.
[20] S. S. Allawi, J. M. Auda, H. Q. Hameed, and T. I. Ali, “The effect of Curcuma longa (turmeric) rhizomes extracts on pathogenic bacteria in comparison with standard antibiotics.” Journal of Biotechnology Research Center 3, pp. 15–20, 2009.
[21] T. Murashige, and F, Skoog, “A revised medium for rapid growth and bioassays with tobacco tissue culture.” Physiologia Plantarum, vol. 15, no. 3, pp. 473–497, 1962.
[22] D. B. Duncan, “Multiple range and multiple F test. Biometrics, vol. 11, pp. 1–42, 1995.
[23] S. Ranjan, N. Dasgupta, P. Saha, M. Rakshit, and C. Ramalingam, “Comparative study of antibacterial activity of garlic and cinnamon at different temperature and its application on preservation of fish. Advances in Applied Science Research, vol. 3, no. 1, pp. 495-501, 2012.