Exploring Disinfection Methods for Iris pseudacorus L. Rhizome and Seed Under In-Vitro Conditions

Document Type : Full Paper

Authors

1 Department of Horticultural Sciences, Mohaghegh Ardebili University, Ardebil, Iran.

2 Horticultural Department, University of Mohaghegh Ardabili, Ardebil

3 Horticultural Department, University of Mohaghegh Ardabili, Ardebil. Iran.

4 Genetic and Biotechnology Institute of Tabarestan, Sari Agricultural Sciences and Natural Resources University, Sari, Iran

Abstract

Iris pseudacorus L. is one of the native and prominent ornamental species in Iran. Its propagation by tissue culture is prone to sever bacterial and fungal contaminations of the explants, particularly in rhizomes., which causes the failure of conventional sterilization methods. In order to optimize the least dangerous method for sterilization of Iris pseudacorus rhizome and seed under in vitro culture condition, two separate experiments were performed each in a completely randomized design with five replications. 16 and 15 methods were applied for rhizome and seeds steriliztion, respectively. The treated specimens were cultured in a hormone-free MS medium and two weeks later, various indices such as infection percentage, germination percentage, mean leaf length and mean root length were measured. Then intact specimens were transferred to the MS media containing different concentrations of thidiazuron (TDZ) and naphthalen acetic acid (NAA) for proliferation.
 The results showed that A15 treatment (water bath (42 °C) for 60 mins, fluconazole fungicide and antibiotics streptamycin for 60 mins, alcohol 70% for one min. and NaoCl 4% for 10 mins) on rhizome explants with the lowest contamination percentage (11%) and a high germination rate of 88% was the best treatment. Also, A13 treatment with 85% germination and 15% contamination obtained the second place. Seeds-treated with treatment B2 using hot water and 3% sodium hypochlorite and treatment B5 using hydropriming with running water and hypochlorite 2% had the highest germination rate of 71.74% and contamination was almost zero. Meanwhile the highest proliferation was obtained in the culture medium containing MS and a combination of 5 μM TDZ and 6 μM NAA.

Keywords

Main Subjects

Extended Abstract

Introduction

Iris pseudacorus L. is one of the native and prominent ornamental species in Iran. Its propagation by tissue culture is prone to sever bacterial and fungal contaminations of the explants, particularly in rhizomes., which causes the failure of conventional sterilization methods. In addition, low speed propagation by seed and season-dependent propagation by rhizomes, altogether have reduced florists tendency to use this beautiful flower in the ornamental plants industry. The present research aimed to optimize the best and least dangerous disinfection method for the in vitro cultivation of rhizomes and seeds of this species of Iris.

 

Materials and Methods

This research was conducted as two separate trials based on a completely randomized design with 16 treatments on rhizomes and 15 treatments on seeds, in five replications to overcome the microbial infections under in vitro propagation. After disinfecting, the treated explants were cultured in a hormone-free MS medium. Two weeks after cultivation, some parameters, including infection percentage, germination percentage, average leaf length, and average root length, were recorded. After that, healthy samples were cultivated in MS culture medium containing different concentrations of thidiazuron and naphthaleneacetic acid for proliferation.

 

Results and Discussion

Based on the results, compounds such as sodium hydroxide, warm water, and hydro-priming performed the best for seed sterilization. The highest germination rate (71.74 %) and least infection rate (almost zero) were observed in the seed explants of treatments B2 in which warm water and 3% sodium hypochlorite were applied, and also B5 in which hydro-priming with running water and immersion in 2% sodium hypochlorite were used. The sterilization of the rhizomes is much more difficult than that seeds since underground organs are in contact with a wide range of fungal and bacterial infections. Also I. pseudacorus habitats on the margins of marshes, banks of rivers and streams are subjected to wider infections. Based on the findings, sodium hypochlorite does not alone sufficent to control the bacterial and fungal infections of I. pseudacorus L. It was found that the rhizome explants treated by A15 exhibited an infection rate of 11 percent and a germination rate of 88 percent. Also, treatment A13 with a germination rate of 85 percent and an infection rate of 15 percent was the second-best treatment. In general, the use of hot water bath at 42°C and 46°C and the use of fungicides benomyl and fluconazole and antibiotics streptomycin, gentamicin, and clindamycin outperformed antibiotics terbinafine and nystatin. Data analysis of sterilized samples showed that the concentration of 5 μM TDZ and 6 μM NAA hormones resulted in the highest amount of proliferation.

 

Conclusion

The fungicides benomyl and fluconazole along with clotrimazole and antibiotics streptomycin, gentamicin and clindamycin were more effective in controlling intensive fungal and bacterial infections of rhizomes. With the advantage of better effectiveness and greater safety, utilizing hot water bath, along with fungicides and antibiotics can be a more efficient method for providing the required growth conditions for  the rhizomes-derived explants of I. pseudacorus instead of the use of toxic, dangerous, and harmful compounds.

References

ﺑﻬﻠﻮﻟﻲ زﻧﺠﺎﻧﻲ، سحر (1384). ﺑﺮرﺳﻲ ﻛﺸﺖ درون ﺷﻴﺸﻪ‌ای ﮔﻴﺎه آﻟﺴﺘﺮوﻣﺮﻳﺎ. پایان نامه کارشناسی ارﺷﺪ. به راهنمایی یوسف ﺣﻤﻴﺪاوﻏﻠﻲ و عبداله ﺣﺎﺗﻢ زاده.  100ص.
جودی، مهدی و شریف زاده، فرزاد (1383). بررسی اثر هیدروپرایمینگ در ارقام مختلف جو. نشریه بیابان، 15(9)، 101-91.
حاتمی، الناز؛ شکوهیان، علی اکبر؛ قنبری، علیرضا و ناصری، لطف‌علی (1398). تأثیر نانونقره بر آلودگی باکتریایی و شاخص‌های مورفولوژیکی و بیوشیمیایی بادام GN15 کشت بافتی. نشریه علوم باغبانی ایران، 50(1)، 119-128.
دریانی، پریسا؛ زارع، ناصر؛ چمنی، اسماعیل؛ شیخ زاده مصدق، پریسا و جوادی مجدد، داوود (1394). تاثیر نانوذرات نقره بر آلودگی میکروبی و رشد درون شیشه ای جوانه های جانبی و انتهایی ارقام فندق. نشریه فن آوری زیستی در کشاورزی (پژوهش کشاورزی)، 14(1) ، 21-31.
رضوان جو، نوید؛ قنبری، علیرضا؛ محب الدینی، مهدی و ترابی گیگلو، موسی ( 24 بهمن، 1396). تاثیر کابرد آنتی بیوتیک استرپتومایسین در محیط کشت، برای از بین بردن آلودگی باکتریایی ریزنمونه های زغال اخته (Cornus mas L.). کنفرانس بین المللی علوم کشاورزی، گیاهان دارویی و طب سنتی،مشهد، ایران.
شهریاری، امیرغفار؛ باقری، عبدالرضا؛ شریفی، احمد و مشتاقی، نسرین (1390). کنترل آلودگی ریزنمونه های ریزوم گیاه آلسترومریا (Alstroemeria sp.) در شرایط این ویترو. نشریه علوم باغبانی، 25(1)، 115-109.
 فخرفشانی، مسعود؛ باقری، عبدالرضا و شریفی، احمد ( 31 اردیبهشت، 1391). بررسی اثر زمان و غلظت های مختلف نانوذرات نقره بر کنترل آلودگی‌های قارچی و باکتریایی کشت این ویتروی کاپیتول ژربرا (Gerbera Jamesonii)، دوازدهمین کنگره ژنتیک ایران،تهران.
قهرمان، احمد. (1373-1357). فلور رنگی ایران جلد 17. تهران، انشارات موسسه تحقیقات جنگلها و مراتع ایران.
مقیمی، زهرا و صفرنژاد، عباس (1393). بررسی ریزازدیادی و میزان فلاونوئید زالزالک (Crataegus sp.) از طریق کشت بافت. تحقیقات ژنتیک و اصلاح گیاهان مرتعی و جنگلی ایران، 22(2), 181-191.
Arab, M., Yadollahi, M., Hosseini-Mazinani, A., & Bagheri, S. (2014) Effects of antimicrobial activity of silver nanoparticles on in vitro establishment of G × N15 (hybrid of almond × peach) rootstock. Journal of Genetic Engineering and Biotechnology. 12(4), 103–110.
Bohlouli Zanjani, S. (2005). Study of tissue culture in vitro of Alstroemeria. [MSc thesis, University of Guilan]. (In Persian).
Buckseth, T., Singh, R. K., Sharma, A. K., Sharma, S., Modgil, V., & Saraswati, A. (2017). Effect of streptomycin and gentamycin on in vitro growth and cultural contaminants of potato cultivars. International Journal of Current Microbiology and Applied Sciences, 6(12), 4038-4043.
Cao, X., & Hammerschlag, F.A. (2000). Improved shoot organogenesis from leaf explants of highbush blueberry. HortScience, 35(5), 945-947.
Cao, X., Hammerschlag, F. A., & Douglass, L. (2002). A two-step pretreatment significantly enhances shoot organogenesis from leaf explants of highbush blueberry cv. Bluecrop. HortScience, 37(5), 819-821.
Chang, H. S., Chakrabarty, D., Hahn, E. J., & Paek, K. Y.,. (2003). Micropropagation of calla lily (Zantedeschia albomaculata) via in vitro shoot tip proliferation. In Vitro Cellular and Developmental Biology – Plant, 39(12), 129–134.
Daryani, P., Zare, N., Chamani, E., Shiekhzadeh Mosadegh, P., & Javadi, D. (2015). The effect of silver nano-particles on microbial contamination and in vitro growth of apical and auxiliary buds of Biotechnology in Agriculture, 14(1), 21–31. (In Persian).
Doğan, S., & Caglar, G. (2018). In Vitro Shoot Proliferation via Immature Embryos of Iris kirkwoodiae Chaudhary. ANADOLU Journal of Aegean Agricultural Research Institute, 28(2), 48-54 .
Fakhrfshani, M., Bagheri, A., & Sharifi, A. (2012). The effect of time and different concentrations of silver nanoparticles on the control of fungal and bacterial contamination of this vitreous culture of Gerbera Jamesonii Capitol. 12th Iranian Genetic Conference, Tehran, Iran. 20 May. 23–31. (In Persian).
Fisse, J., Batalle, A., & Pera, J. (1987). Endogenous bacteria elimination in ornamental plants. Acta Horticulturae, 212(11), 87–90.
Ghahreman, A. (1978-2003). Flora of Iran in colour,.Research Institute of Forests & Rangelands. Tehran, 1-24. (In Persian).
Gubišová, M., & Gubiš, J. (2019). Growth of potato shoot cultures on media with antibiotics for elimination of bacterial contamination. Agriculture, 65(3), 99–106.
Golle, D. P., Reiniger, L. R. S., Stefanel, C. M., Muniz,M. F. B., & Silva, K. B. (2017). Combination of NAA and TDZ for in vitro multiplication of Eugenia involucrata DC. Revista Árvore, 41(5), 1-7.
Gwon, S. H., Heo, J. Y., & Kim, B. S. (2019). Identification and control of microbial contaminants during in vitro culturing of 'Atlantic' potatos. International Journal of Horticultural Science Technology, 37(4), 528–539.
Han, H., Liao, Q., Marbaha, W., Wang, H., Zhuang, H., & Wang, Q. (2017). Isolation, identification and screening of eEndophytes fungicides for disease control during tissue culture of german irises. Xinjiang. Agricultural Sciences,54(4), 20–25.
Habiba, U., Reza, S., Saha, M. L., khan, M. R., & Hadiuzzaman, S. (2002). Endogenous bacterial contamination during in vitro culture of table banana identification and prevention. Plant Tissue Calture. 12, 117-124.
Hatami, E., Shokoohian, A. A., Ghanbari, A. R., & Naseri, L. A. (2019). Effects of nano silver on bacterial contamination and morphological and biochemical indices of in vitro GN15 almond rootstock. Iranian Journal of Horticultural Science. 50(1), 119–128. (In Persian).
Jena, R. C., & Samal, K. C. (2011). Endogenous microbial contamination during in vitro culture of sweet potato [Ipomoea batatas (L.) Lam]: identification and prevention. Journal of Agricultural Technology, 7(6), 1725–1731.
Jevremović, S., Subotiċ, A., Trifunović, M., Nikolić, M., & Radojević, L. (2008). In vitro plant regeneration of Iris pseudopallida. In J. A. Teixeira da Silva (Ed.) Floriculture. Ornamental and Plant Biotechnology .(pp. 250–252). Global Science Books, Japan.
Judi, M., & Sharifzadeh, F. (2006). Investigation the effect of hydropriming in barley cultivars. Biaban Journal, 15(9), 91–101. (In Persian).
Kromer, K. D. (1985). Regeneration of some monocotyledonous plants from subterranean organs in vitro. Acta Agrobotanica, 38(2), 65–87.
Kulus, D. (2021). Establishment of an efficient in vitro culture system in Dicentra × hybrida. Biology and Life Sciences Fourm, 4(4), 2–5.
Kritzinger, E. M., Jansen. van Vuuren, R., Woodward, B., Rong, I. H., Spreeth, M. H., & Slabbert, M. M. ( 1998). Elimimation of external and internal contaminants in rhizomes of Zantedeschia aethiopica with commercial fungicides and antibiotics. Plant Cell, Tissue and Organ. Culture, 52(2), 61–65.
Leifert, C., Camotta, H., & Waites, W. M. (1992). Effect of combinations of antibiotics on micropropagated Clematis, Delphinium, Hosta, Iris and Photinia. Plant Cell, Tissue and Organ Culture, 29(2), 153–160.
Lin, H. S., De Jeu, M. J., & Jacobsen, E. (1997). Direct shoot regeneration from excised leaf explants of in vitro grown seedling of Alestromeria. Plant cell reports, 16(11), 770–774.
Lu, M. Y., Du, Y., & Bi, X. Y. (2017). Research on seed dormancy and germination characteristics of five wild Iris species. Acta Botanica Boreali-Occidentalia Sinica, 37(9), 1823–1830.
Marcotrigiano, M., McGlew, S. P., Hackett, G., & Chawla, B. (1996). Shoot regeneration from tissuecultured leaves of the American cranberry (Vaccinium macrocarpon). Plant cell, tissue and organ culture, 44(3), 195-199.
Marinescu M., Teodorescu A., & Sutan N. (2013). Preliminary results on the in vitro propagation by leaf explants and axillary buds of Iris aphylla L. J. Horticult. For. Biotechnol. 17, 279–282.
Mbah, E. I., & Wakil, S. M. (2012). Elimination of bacteria from in vitro yam tissue cultures using antibiotics. Journal of Plant Pathology, 94(1), 53–58.
 
Mol, J., Vivas Salim, M. J., Chemparathy, Sh. M., Karim, R., & Balakrishnan T. U. (2016). An efficient protocol for raising contamination free micropropagation of Zingiber Officinale (Ginger). Journal of Pharmaceutical and Biological Sciences, 4(5), 145–148.
Moghimi, Z., & Safarnejad, A. (2014). Assessment of micropropagation and flavonoid content of hawthorn  through tissue culture. Iranian Journal of Rangelands and Forests Plant Breeding and Genetic Research, 22(2), 181-191. (In Persian).
Oo, K. T., Oo, K. S., & Mon, Y. Y. (2018). Establishment of efficient surface sterilization protocol on different types of field grown strawberry explants (Fragaria × ananassa Dutch.). Journal of Scientific and Innovative Research, 7(3), 70–74.
Park, H. Y., Kim, K. S., Ak, G., Zengin, G., Cziáky, Z., Jekö, J., Adaikalam, K., Song, K., Kim, D. H., & Sivanesan, I. (2021). Establishment of a rapid micropropagation system for Kaempferia parviflora Wall. Ex Baker: Phytochemical analysis of leaf extracts and evaluation of biological activities, Plants. 10(4), 683–698.
Patel, A., Patil, G., Mankad, M., & Subash, N. (2018). Optimization of surface sterilization and manipulation of in vitro conditions for reduced browning in pomegranate (Punica granatum L.) variety Bhagava. International Journal of Chemical Studies, 6(3), 8–23.
Pedersen, C., & Brandt, K. (1992). A method for disinfection of underground rhizome of Alestromeria and Heliconia. Acta Horticulturae, 325(69), 499–504.
Reed, B. M., & Abdelnour-Esquivel, A.(1991). The use of zeatin to initiate in vitro cultures of Vaccinium species and cultivars. Horticultural Science, 26(11), 1320-1322.
Reuther, G. (1977). Embryoide differenzierungsmuster im kallusder Gattungen Iris und Asparagus. Berichte der Deutschen Botanischen Gesellschaft, 90(2), 417–437.
Rezvanjoo, N., Ghanbari, A., Mohebulddini, M., & Torabi Giglo, M. (2018, Februery, 13). The effect of streptomycin antibiotic application in culture medium to eliminate bacterial contamination of (Cornus mas L.) explants. International Conference of Agricultural Sciences, Medicinal Plants and Traditional Medicine, Mashhad, Iran. (In Persian).
Shahriari, A. G., Bagheri, A., Sharifi, A., & Mushtaqi, N. (2011). Infection control of rhizome samples of Alstroemeria (Alstroemeria sp.) under in vitro conditions. Journal of Horticultural Sciences, 25(1), 109–115. (In Persian).
Sinha Ray, S., & Ali, N. (2016). Biotic Contamination and Possible Ways of Sterilization: A Review with Reference to Bamboo Micropropagation. Agriculture, Agrobusiness and Biotechnology. 59(2), 1–12.
Sivanesan, I., Muthu, M., Gopal, J., Tasneem, S., Kim, D.-H., & Oh, J.-W. (2021). A fumigation-based uurface sterilization approach for plant tissue culture. International Journal of Environmental Research and Public Health, 18(3), 2282.
Srivastava, P., Kasoju, N., Bora, U., & Chaturvedi, R.(2010). Accumulation of betulinic, oleanolic, and ursolic acids in in vitro cell cultures of Lantana camara L. and their significant cytotoxic effects on HeLa cell lines. Biotechnol Bioproc E. .15:1038–1046.
Sun, Y. J., Zhang, K., Wang L., & Qiu, X. J. (2006). NaOH scarification and stratification improve germination of Iris lactea var. chinensis seed. HortScience, 41(3), 773–774.
Teixeira da Silva, J. A., Winarto, B., Dobránszki, J., & Zeng, S. (2015). Disinfection procedures for in vitro propagation of Anthurium. Folia Horticulturae, 27(1), 3–14.
Tikhomirova, L. (2017). Morphogenesis and histology of cultures of Iris ensata Thunb. Generative Organs. In Vitro Cellular and Developmental Biology - Plant. 53, 270-3.
Tikhomirova, L. (2020). Scientific framework for selecting explants of Iris L. genus for direct shoot regeneration. IOP Conf. Series: Earth and Environmental Science. 421 052014.
Torres, G. R. C., Coutinho, F. P., Araújo, B. G. P., Abreu, G. P., & Júnior, R. S. (2019). Thermotherapy as a microbial contaminant-reducing agent in micropropagation of bamboo. Agricultural and Biological Sciences, 32(3), 690–697.
Wang, L., Du, Y., Rahman, M. M., Tang, B., Fan, L. J., & Kilaru, A. (2018). Establishment of an efficient in vitro propagation system for Iris sanguinea. Scientific reports, 8(1), 17100. doi: 10.1038/s41598-018-35281-y.
Young, P. M., Hutchins, A. S., & Canfield, M. L. (1984). Use of antibiotics to control the bacteria in shoot cultures of woody plants. Plant Science Letters, 34(1–2), 203–209.
Iranian Journal of Horticultural Science
Volume 55, Issue 1
June 2024
Pages 153-175

Files

History

  • Receive Date: 16 February 2023
  • Revise Date: 29 August 2023
  • Accept Date: 10 September 2023

Share

How to cite

Statistics