نوع مقاله : مقالات پژوهشی
نویسندگان
گروه باغبانی، دانشکده کشاورزی، دانشگاه زنجان، زنجان، ایران
چکیده
رفع آلودگی بهعنوان یک چالش اساسی در تکنیک کشت سلول، بافت و اندام گیاهی به‌شمار می‌رود. با اینکه روش‌های مختلفی برای این منظور وجود دارد، امّا توسعه روش‌های ضدعفونی مختص هر گونه یک عامل مهم در استقرار و موفقیت سیستم کشت بافت به‌شمار می‌رود. لذا، پژوهش حاضر بهمنظور بررسی تأثیر زمان و غلظت‌های مختلف هیپوکلریت سدیم، قارچکش کاربندازیم و کلرید جیوه در مهار آلودگی پیازهای لاله واژگون (Fritillaria spp.) در محیط کشت بافت بهصورت طرح کاملاً تصادفی در آزمایشگاه بیوتکنولوژی دانشگاه زنجان اجرا شد. تیمارهای آزمایش شامل قارچکش 0/1 درصد در زمان‌های مختلف (صفر، 30،25، 35 و 40 دقیقه)، پنج سطح هیپوکلریت سدیم (صفر ، 1، 1/5، 2، 2/5 و 3 درصد) در زمان‌های مختلف (صفر ، 7، 9، 10، 12 و 15 دقیقه)، الکل 70 درصد با زمان (صفر ، 60 و 90 ثانیه) و کلرید جیوه (صفر ، 0/1 و 0/2 درصد) بود. نتایج پژوهش حاضر نشان داد که با وجود اینکه کمترین درصد آلودگی در زمان 40 دقیقه قارچکش کاربندازیم، الکل 70 درصد در زمان 90 ثانیه، غلظت 3 درصد و زمان 15 دقیقه هیپوکلریت سدیم و غلظت 0/2 درصد کلرید جیوه حاصل شد، امّا میزان زندهمانی ریزنمونه‌ها به کمترین میزان کاهش یافت و باعث قهوه‌ای شدن ریزنمونه‌ها شد. طبق نتایج حاصل، بهترین تیمار با رفع آلودگی 80 درصد و درصد زندهمانی بالا، زمان 30 دقیقه قارچکش، زمان 60 ثانیه الکل 70 درصد، هیپوکلریت سدیم 2 و 2/5 درصد بهترتیب به‌مدت 12 و 10 دقیقه و کلرید جیوه 0/1 درصد به‌مدت هفت دقیقه بود. لذا استفاده از تیمارهای ذکر شده در شرایط کشت درون شیشه‌ای، جهت کاهش آلودگی و ریزازدیادی پیازهای گیاه لاله واژگون پیشنهاد می‌گردد.
کلیدواژه‌ها
موضوعات
عنوان مقاله [English]
The Effect of Time and Different Concentrations of Sodium Hypochlorite, Carbendazim Fungicide and Mercuric Chloride in Inhibiting the Contamination of Fritillaria spp. Bulbs in Tissue Culture Environment
نویسندگان [English]
- Letafat Moradi
- Seyyed Najmodin Mortazavi
- Mitra Alaei
Department of Horticulture, Faculty of Agriculture, University of Zanjan, Zanjan, Iran
چکیده [English]
Introduction
Success in tissue culture technique, especially in bulbous plants, depends on the microbial contamination control during in vitro culture. Decontamination is considered as a fundamental challenge in the technique of cell, tissue and plant organ culture. Although there are various methods for this purpose, the development of disinfection methods specific to each species is considered an important factor in the establishment and success of the tissue culture system. Applying different treatments can control the microbial contamination and consequently increase the percentage of explant survival. This plant is among the poisonous plants of pastures and the chemical compounds present in it have important medicinal effects, so that the existence of alkaloid and glycosidic compounds has been reported for it. These chemical compounds are used to treat rheumatic pains, lymphatic system diseases and also as liver cleansers. This ornamental plant also has the ability to produce pots and its export is possible in pots . Currently, in order to overcome the issues of the usual methods of propagation of this ornamental plant and to produce pollution-free plants, the use of in vitro cultivation methods in this plant has become very important. bulbes is one of the most common explants of inverted tulip tissue culture, but it often faces very high internal and external fungal and bacterial contamination. In the in vitro culture of onion plants, the existence of fungal and bacterial contaminations are one of the main problems that can affect the efficiency of this type of propagation methods. Fungal and bacterial contaminations are one of the most important problematic factors in different in vitro culture methods. Internal contamination usually appears several weeks after planting. These contaminations may not be seen externally, but they affect the growth, division and greening of small samples. Mercuric chloride is mainly used as a surface disinfectant along with sodium hypochlorite and it controls the spectrum of bacteria and fungi and increases the disinfection efficiency. The results of the study showed that mercuric chloride relatively controls the bacterial and fungal contamination of the samples, but it reduces the survival percentage of the samples in high concentration. It seems that some plants are sensitive to mercuric chloride and determining the appropriate amount of this substance plays a very important role in preparing a healthy and living specimen. Determining the duration of disinfectant treatment was also considered in various researches, so that sometimes carbendazim 1% fungicide for 20 minutes, 70% alcohol for 60 seconds, sodium hypochlorite 2.5% for 10 minutes. And 0.1% mercury chloride was used for 7 minutes to sterilize aloe vera sample. Considering the importance of plant tissue culture techniques and the need to create new protocols to reduce contamination and increase the number of healthy cultures, the main goal of this study is to create an efficient protocol for the disinfection of Fritillaria spp. onion explants in order to increase the efficiency of in vitro cultures.
Materials and Methods
This study aimed to investigate the effect of time and different concentrations of sodium hypochlorite, carbendazim fungicide and mercuric chloride in inhibiting the contamination of inverted tulip bulbs (Fritillaria spp.) in tissue culture environment. So, an experiment was done as a completely randomized design at four replications in the biotechnology laboratory of Zanjan University during 2023. The experimental treatments consisted of 0.1% fungicide at different times (30, 25, 35 and 40 minutes), 5 levels of sodium hypochlorite (0,1, 1.5, 2, 2.5 and 3%) at different times (7, 9, 10, 12 and 15 minutes), 70% Ethanol at two different times (0,60 and 90 seconds) and mercury chloride (0,0.1 and 0.2%). Bulbs that collected from nature were transferred to the Biotechnology Laboratory of the Faculty of Agriculture, Department of Horticulture, Zanjan University and kept them in the refrigerator at 4°C for two weeks. After this period of time, the bulbs were washed with detergent and then remained in water for 30 minutes. Then they were disinfected by using the above-mentioned treatments: It should be noted that at the end of each step, Fritillaria bulbs were washed with sterile distilled water. Also, after the end of the disinfection treatments, the desired explants were cultured in MS basic culture medium. The statistical analysis of this experiment was carried out using SAS software, version 9.1. The differences between mean values were compared using Duncan’s multiple range test method at the 5% significant level (p < 0.05).
Results and Discussion
The control of fungal and bacterial contamination, especially in relation to bulbous and bulbous plants that are in direct contact with the culture medium, is one of the basic steps in in vitro culture conditions. Applying different treatments including the use of fungicides, alcohol, sodium hypochlorite and mercuric chloride in the cultivation environment can help control fungal and bacterial contamination in the Fritillaria spp. plant. However, the application of such treatments can have a negative effect on the regeneration potential of the explants, so it is important to choose the appropriate treatment in a way that controls the fungal and bacterial contamination and on the other hand preserves the regeneration potential of the explants. This study investigated the effectiveness of various disinfection protocols on Laleh-Avgagun onion explants. While the lowest contamination (0%) was achieved using 0.1% fungicide for 40 minutes, 70% alcohol for 90 seconds, 3% sodium hypochlorite for 15 minutes, and 0.1% mercury chloride, this treatment significantly reduced explant survival and regeneration, causing browning. Conversely, applying 0.1% fungicide for 30 minutes, 70% alcohol for 60 seconds, 2% or 2.5% sodium hypochlorite for 12 or 10 minutes respectively, and 0.1% mercury chloride effectively reduced contamination while maintaining explant survival. This optimized protocol improved the growth and regeneration ability of the explants.Therefore, in the conditions of in vitro cultivation, the use of the mentioned treatments is recommended to control contamination and optimal reproduction of the bulbs of Fritillaria spp. plant.
کلیدواژه‌ها [English]
- Disinfection
- Extinction
- In vitro
- Mercuric chloride
- Micropropagation
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- Abdi, G., Salehi, H., & Khosh-Khui, M. (2008). Nano silver: A novel nanomaterial for removal of bacterial contaminants in valerian (Valeriana officinalis) tissue culture. Acta Physiologiae Plantarum, 30, 709-714. https://doi.org/10.1007/s11738-008-0169-z
- Afifian, M. (2010). Identification and determination of some active ingredients of Fritillaria imperialis and relation of it to the habitat conditions, western area of Isfahan province. M.Sc. Thesis Natural Resources Faculty, Isfahan University of Technology, Iran.
- Ahmadi, E., Nasr, S.M.H., Jalilvand, H., & Savadkoohi, S.K. (2012). Contamination control of microbe Ziziphus spina [christti] seed in vitro Trees, 26, 1299-1304. https://doi.org/10.1007/s00468-012-0705-8
- Alam, J., Alam, I., Sharmin, S.A., Rahman, M., Anisuzzaman, M., & Alam, M.F. (2010). Micropropagation and antimicrobial activity of Operculina turpethum' (Syn.'Ipomoea turpethum'), an endangered medicinal plant. Plant Omics, 3(2), 40-46.
- Allen, T.W., Enebak, S.A., & Carey, W.A. (2004). Evaluation of fungicides for control of species of Fusarium on longleaf pine seed. Crop Protection, 23(10), 979-982. https://doi.org/10.1016/j.cropro.2004.02.010
- Altan, F., Bürün, B., & Sahin, N. (2010). Fungal contaminants observed during micropropagation of Lilium candidum and the effect of chemotherapeutic substances applied after sterilization. African Journal of Biotechnology, 9(7), 991-995. https://doi.org/10.5897/AJB08.090
- Barnett, J.P., & McGilvray, J.M. (2002). Reducing Seed and Seedlings Pathogens Improves Longleaf Pine Seedlings Production. In: Gen. Tech. Rep. SRS-56. Asheville, NC: US Department of Agriculture, Forest Service, Southern Research Station. p. 19-20.
- Cowan, M.M. (1999). Plant products as antimicrobial agents. Clinical Aicrobiology Reviews, 12(4), 564-582. https://doi.org/10.1128/cmr.12.4.564
- Eed, A.M., Reddy, S.A., Reddy, K.M., Silva, J.A.T., Reddy, P.V., Beghum, H., & Venkatsubbaiah, P.Y. (2010). Effect of antibiotics and fungicides on the in vitro production of Citrus limonia Osbeck nodal segment and shoot tip explants. The Asian and Australasian Journal of Plant Science and Biotechnology, 4(1), 66-70.
- Eslamzadeh, N., Hoseini, H., Moradi, H., & Dehkordi, F. (2009). Introduction of new habitat for the Fritillaria, a GIS. Production and Processing of Agricultural and Horticultural Products Journal, 8(16), 556-582.
- Gholami, M. (2007). Micropropagation of of inverted tulip (Fritillaria imperialis ). Doctoral Dissertation, M.Sc. Thesis, University of Avicenna. Hamedan, Iran.
- Hamidoghli, S., Chamani, E., Hamidoghli, Y., & Talei, N. (2015). Effect of different plant growth regulators on direct bulblet regeneration from scale explants of Fritillaria imperialis. Isfahan University of Technology Journal of Crop Production and Processing, 5(16), 211-218. https://doi.org/10.18869/acadpub.jcpp.5.16.211
- Harum, N. (2013). Surface sterilization procedures for leaves explants of rhododendron (Rhododendron javanicum (Blume) Benn) cultured in vitro. The third Basic Science International Conference, 21, 1-4.
- Hosseini, B., & Alizadeh, M. (2013). Effect of biomass and BAP hormone on in vitro regeneration of medicinal herb (Hyssopus officinalis L.). Journal of Horticulture, 27, 207-201. (In persian)
- Karaoğlu, C., Çöcü, S., Ipek, A., Parmaksız, I., Uranbey, S., Sarıhan, E., & Khawar, K. M. (2006, October). In vitro micropropagation of saffron. In II International Symposium on Saffron Biology and Technology, 739(223-227). https://doi.org/10.17660/ActaHortic.2007.739.28
- Kashif, M. (2005). Effect of duration of mercuric chloride treatment on culture viability, contamination and mortality of various accessions of grapes. Sarhad Journal of Agriculture (Pakistan), 21(1).
- Kharrazi, M., Tehranifar, A., Nemati, H., & Bagheri, A. (2016). Investigation of different methods of Amaryllis (Hippeastrum× johnsonii) culturing and propagation for increasing the proliferation rate during in vitro and greenhouse conditions (Doctoral dissertation, Ph.D. Dissertation, Ferdowsi University of Mashhad, Mashhad, Iran. (In Persian).
- Khatibzadeh, R., Azizi, M., Arouiee, H., & Farsi, M. )2013(. Effects of sterilization protocol and prechilling treatment on in vitro seed germination of Levisticum officinale Koch. Journal of Horticultural Science, 27(2), 130-138. https://doi.org/10.22067/JHORTS4.V0I0.24810
- Mahmoud, S.N., & Al-Ani, N.K. (2016). Effect of different sterilization methods on contamination and viability of nodal segments of Cestrum nocturnum International Journal of Research Studies in Biosciences, 4(1), 4-9. https://doi.org/10.20431/2349-0365.0401002
- Manole, C.G., Balan, V., Mencinicopschi, I.C., Golea, D., Rodino, S., & Butu, A. (2012). The influence of growth regulators concentrations on in vitro micropropagation of Ribes rubrum species. Scientific Bulletin, Series F, Biotechnologies, XVI, 2012. ISSN-L 2285-1364.
- Mihaljević, I., Dugalić, K., Tomaš, V., Viljevac, M., Pranjić, A., Cmelik, Z., Puskar, B., & Jurkovic, Z. (2013). In vitro sterilization procedures for micropropagation of' oblacinska' sour cherry. Journal of Agricultural Sciences, 58, 117-126. https://doi.org/10.2298/JAS1302117M
- Mng'omba, S.A., du Toit, E.S., Akinnifesi, F.K., & Sileshi, G. (2012). Efficacy and utilization of fungicides and other antibiotics for aseptic plant cultures. Faculty of Natural and Agricultural Sciences, University of Pretoria, Pretoria.
- Mohammadi Deh-cheshmeh, M., Khalighi, A., & Naderi, R. (2007). Indirect somatic embryogenesis from petal explant of endangered wild population of Fritillaria imperialis. Pakistan Journal of Biological Sciences, 10(11), 1875-1879. https://doi.org/10.3923/pjbs.2007.1875.1879
- Mohammadi Deh-cheshmeh, M., Khalighi, A., Naderi, R., Sardari, M., & Ebrahimie, E. (2008). Petal: A reliable explant for direct bulblet regeneration of endangered wild populations of Fritillaria imperialis L. Acta Physiologiae Plantarum, 30(3), 395-399. https://doi.org/10.1007/s11738-007-0126-2
- Pernezny, K., Nagata, R., Raid, R.N., Collins, J., & Carroll, A. (2002). Investigation of seed treatments of management of bacterial leaf spot of lettuce. Journal of Plant Disease, 151-155. https://doi.org/10.1094/PDIS.2002.86.2.151
- Reed, B.M., Buckley, P.M., & DeWilde, T.N. (1995). Detection and eradication of endophytic bacteria from micropropagated mint plants. In Vitro Cellular and Developmental Biology-Plant, 31, 53-57. https://doi.org/10.1007/BF02632228
- Sadr, S.S., Zakizadeh, H, Naghavi, M.R., Olfati, J., & Hazrati, K. (2019). Introducing a tissue culture protocol for fritillaria (Fritillaria raddeana) via seed and bulb scales. Iranian Journal of Horticultural Science, 49(4), https://doi.org/10.22059/ijhs.2017.230135.1219
- Saggoo, M., & Kaur, R. (2010). Studies in North Indian Aloe vera: Callus induction and regeneration of plantlets. Archives of Applied Science Research, 2(2), 241-245.
- Sain, M., & Sharma, V. (2013). International journal of pure and applied bioscience Catharanthus roseus (an anti-cancerous drug yielding plant)—A review of potential therapeutic properties. International Journal of Pure and Applied Bioscience, 1(6), 139-142.
- Shamsian, S., Omidi, M., & Torabi, C. (2016). effect of growth regulators and active charcoal on the proliferation of Aloe vera in vitro. Iranian Journal of Medicinal and Aromatic Plants Research, 32, 281-289.35.
- Silva, J.A., & Dobránszki, J. (2014). Sonication (Ultrasound) affects in vitro growth of hybrid cymbidium. Botanica Lithuanica (1392-1665), 20(2). https://doi.org/10.2478/botlit-2014-0014
- Smith, R.H. (2013), Amsterdam Academic Press, Plant Tissue Culture. Techniques and Experiments. pp. 188, £54.99. ISBN 978-012-415920-4.
- Teixeira da Silva, J.A., & Dobranszki, J. (2014). Sonication (ultrasound) affects In vitro growth of hybrid Cymbidium. Bot Lith, 20, 121-130
- Telem, R.S., Sadhukhan, R., Mandal, N., Wani, S.H., Sarkar, H.K., & Naorem, B.S. (2016). Estimating the efficiency of different explants for direct in vitro multiple shoots development in chrysanthemum. International Journal of Agriculture, Environment and Biotechnology, 9(3), 345-352. https://doi.org/10.5958/2230-732X.2016.00045.0
- Yildiz, M., & Er, C. (2002). The effect of sodium hypochlorite solutions on in vitro seedling growth and shoot regeneration of flax (Linum usitatissimum). Naturwissenschaften, 89, 259-261. https://doi.org/10.1007/s00114-002-0310-6
- Zamanzadeh Darban, M., & Joulii Tehrani, M. (2020) Adventurer in Zanjan book. Travel and tourism books, Shakib Publications, p. 40.
- Zhang, L.Z., Wei, N., Wu, Q.X., & Ping, M.L. (2007). Anti-oxidant response of Cucumis sativus to fungicide carbendazim. Pesticide Biochemistry and Physiology, 89(1), 54-59. https://doi.org/10.1016/j.pestbp.2007.02.007
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