Contamination control in Iranian seedless barberry micropropagation

Document Type : Research Paper


1 Department of Horticultural Science and Landscape, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran.

2 Khorasan Science and Technology Park (KSTP), Mashhad, Iran.

3 Department of Ornamental Plants, Research Center for Plant Sciences, Ferdowsi University of Mashhad, Mashhad, Iran.


Iranian seedless barberry is a species with high degree of contamination in in vitro culture. The current study was aimed to investigate the effects of mercuric chloride (HgCl2), sodium hypochlorite (NaOCl) + HgCl2, HgCl2 + 8-hydroxyquinoline, cefotaxime + streptomycin, HgCl2 + shoot type, HgCl2 + explant position and HgCl2 + shoot color on the reduction of contamination. The results showed that the combined use of NaOCl and HgCl2 was more effective than their single use, but the contamination rate was relatively high. Interestingly, the results showed that explants sterilized with 100 mg/L HgCl2 and cultured in the medium containing 150 mg/L 8-hydroxyquinoline exhibited the lowest fungal contamination (15%), while those treated with 50 mg/L HgCl2 and cultured in the 8-hydroxyquinoline-free medium showed the highest fungal contamination (96%). Furthermore, treatment of 75 mg/Lcefotaxime + 70 mg/L streptomycin exhibited no bacterial contamination compared to the control with 58% bacterial contamination. Explants prepared from suckers and treated with 150 mg/L HgCl2 showed the lowest rates of fungal (16%) and bacterial (13%) contamination. The results also showed that explants collected from upper position had the lowest rates of fungal (20.87%) and bacterial (18.4%) infection. Explants taken from pinkish-colored shoots showed surprisingly the lowest rates of fungal contamination (13.33%), bacterial contamination (10%) and bud mortality (36.47%). In general, choice of explants from upper position of pinkish-colored suckers, sterilization of them with 100 mg/L HgCl2 for 5 min and their culture in medium containing 150 mg/L 8-hydroxyquinoline can be recommended for reducing the contamination.


Article Title [فارسی]

کنترل آلودگی در ریزازدیادی زرشک بیدانه ایرانی

Abstract [فارسی]

زرشک بیدانه ایرانی گونه‌ای با درجه آلودگی بالا در کشت بافت می‌ باشد. مطالعه حاضر با هدف بررسی تأثیر کلرید جیوه، هیپوکلرید سدیم + کلرید جیوه، کلرید جیوه + 8 هیدروکسی کینولین، سفوتاکسیم + استرپتومایسین، کلرید جیوه + نوع شاخه، کلرید جیوه + موقعیت ریزنمونه و کلرید جیوه + رنگ شاخه بر کاهش آلودگی اجرا گردید. بر اساس نتایج حاصل، استفاده ترکیبی از هیپوکلرید سدیم و کلرید جیوه مؤثرتر از استفاده یگانه کلرید جیوه بود، اما میزان آلودگی نسبتاً بالا بود. نتایج به‌طور شگفت‌انگیزی نشان داد که ریزنمونه­ های ضدعفونی­شده با 100 میلی‌گرم بر لیتر کلرید جیوه و کشت آن‌ ها در محیط حاوی 150 میلی­گرم بر لیتر 8 هیدروکسی کینولین کمترین میزان آلودگی  قارچی (%15) را داشتند، درحالی‌که ریزنمونه ­های تیمار شده با 50 میلی‌گرم کلرید جیوه و کشت آن‌ ها در محیط فاقد 8-هیدروکسی کینولین بیشترین میزان آلودگی قارچی (%96) را دارا بودند. همچنین، در مقایسه با تیمار کنترل با 58% آلودگی باکتریایی، تیمار 75 میلی‌گرم بر لیتر سفوتاکسیم + 70 میلی‌گرم بر لیتر استرپتومایسین فاقد هر ‌گونه آلودگی باکتریایی بود. ریزنمونه ­های تهیه‌شده از پاجوش و تیمار شده با 150 میلی‌گرم بر لیتر کلرید جیوه کمترین میزان آلودگی قارچی (%16) و باکتریایی (%13) را نشان دادند. همچنین نتایج حاکی از آن بود که ریزنمونه­ های جمع‌آوری‌شده از موقعیت بالایی، از کمترین میزان آلودگی قارچی (87/%20) و باکتریایی (4/%18) برخوردار بودند. ریزنمونه ­های تهیه شده از شاخه‌های صورتی‌رنگ به‌طور شگفت‌انگیزی کمترین میزان آلودگی قارچی (33/%13)، آلودگی باکتریایی (%10) و مرگ جوانه (47/%36) را نشان دادند. به‌طورکلی، انتخاب ریزنمونه­ ها از موقعیت بالایی پاجوش با رنگ صورتی، ضدعفونی آن‌ها با 100 میلی‌گرم بر لیتر کلرید جیوه به مدت 5 دقیقه و کشت آن‌ها در محیط حاوی 150 میلی‌گرم بر لیتر 8-هیدروکسی کینولین برای کاهش آلودگی پیشنهاد می‌ گردد.

Keywords [فارسی]

  • 8-هیدروکسی کینولین
  • آلودگی
  • جوانه
  • ریزازدیادی
  • زرشک بیدانه ایرانی
  • کلرید جیوه
  • نوع شاخه
Alemardan A, Asadi W, Rezaei M, Tabrizi L and Mohammadi S, 2013. Cultivation of Iranian seedless barberry (Berberis integerrima ‘Bidaneh’): a medicinal shrub. Industrial Crops and Products 50: 276-287.
Azizi M, Aghabozorgi M, Farsi M, Tehranifar A, ZolAli J and Ghabooli M, 2007. Study on the adventitious root formation in some horticultural crops using Agrobacterium rhizogenes. Journal of Agricultural Sciences and Technology 21(2) Special Issue in Horticlture Sciences): 79-90 (In Persian with English abstract).
Bohra P, Waman AA, Sathyanarayana BN, Umesha K, Anu SR, Swetha HG and Gourish RK, 2014. Aseptic culture establishment using antibiotics with reference to their efficiency and phytotoxicity in difficult-to-establish native Ney Poovan banana (Musa, AB). Proceedings of the National Academy of Sciences, India Section B: Biological Sciences 84: 257-263.
Bonga JM, Klimaszewska KK and von Aderkas P, 2010. Recalcitrance in clonal propagation, in particular of conifers. Plant Cell, Tissue and Organ Culture 100: 241-254.
Cardoso JC and da Silva JAT, 2013. Gerbera micropropagation. Biotechnology Advances 31(8): 1344-1357.
Chang SH, Ho CK, Chen ZZ and Tsay JY, 2001. Micropropagation of Taxus mairei from mature trees. Plant Cell Reports 20: 496-502.
Chen B, 2009. In vitro propagation of a medicinal plant: Tripterygium wilfordii Hook f. Forestry  Studies in China 11: 174-178.
Chowdhury P, Das M, Sikdar SR and Pal A, 2004. Influence of the physiological age and position of the nodal explants on micropropagation of field-grown Dendrocalamus strictus Nees. Plant Cell Biotechnology and Molecular Biology 5(1-2): 45-50.
Dangi M, Gill MIS and Saini HK, 2009. Factors affecting in vitro shoot regeneration in banana cv. Grand Naine. Indian Journal of Plant Physiology 14: 39-43.
Desai P, Patil G, Dholiya B, Desai S, Patel F and Narayanan S, 2018. Development of an efficient micropropagation protocol through axillary shoot proliferation for pomegranate variety ‘Bhagwa.’ Annals of Agrarian Science 16(4): 444-450.
Dhaka N and Kothari SL, 2005. Micropropagation of Eclipta alba (L.) Hassk-an important medicinal plant. In Vitro Cellular & Developmental Biology- Plant 41(5): 658-661.
Hossain HMMT, Kim Y-H and Lee Y-S, 2010. The apical bud as a novel explant for high-frequency in vitro plantlet regeneration of Perilla frutescens L. Britton. Plant Biotechnology Reports 4: 229-235.
Kafi M, Balandary A, Rashed-Mohasel MH, Koochaki A and Molafilabi A, 2002. Berberis: Production and Processing. Zaban va Adab Press (In Persian).
Kafi M, Balandary A, Rashed-Mohasel MH, Koochaki A and Molafilabi A, 2002. Berberis: Production and Processing. Zaban va Adab Press  (In Persian).
Krishna H and Singh SK, 2007. Biotechnological advances in mango (Mangifera indica L.) and their future implication in crop improvement-a review. Biotechnology Advances 25(3): 223-243.
Leifert C, Camotta H and Waites WM, 1992. Effect of combinations of antibiotics on micropropagated Clematis, Delphinium, Hosta, Iris and Photinia. Plant Cell, Tissue and Organ Culture 29: 153-160.
Meier K and Reuther G, 1994. Factors controlling micropropagation of mature Fagus sylvatica. Plant Cell, Tissue and Organ Culture 39: 231-238.
Misra P, Gupta N, Toppo DD, Pandey V, Mishra MK and Tuli R, 2010. Establishment of long-term proliferating shoot cultures of elite Jatropha curcas L. by controlling endophytic bacterial contamination. Plant Cell, Tissue and Organ Culture 100: 189-197.
Mohammadi M, Bagheri A, Marashi H, Moshtaghi N and Balandary A, 2011. Investigation into seasonal effect and browning inhibitor on callus regeneration of seedless barberry (Berberis vulgaris var. asperma). Plant Tissue Culture and Biotechnology 21(2): 161‐168.
Mukherjee P, Husain N, Misra SC and Rao VS, 2010. In vitro propagation of a grape rootstock, deGrasset (Vitis champinii Planch.): effects of medium compositions and plant growth regulators. Scientia Horticulturae 126: 13-19.
Nadel BL, Altman A, Pleban S and Hüttermann A, 1991. In vitro development of mature Fagus sylvatica L. buds. I. The effect of medium and plant growth regulators on bud growth and protein profiles. Journal of Plant Physiology 138(5): 596-601.
Nadha HK, Salwan R, Kasana RC, Anand M and Sood A, 2012. Identification and elimination of bacterial  8(30): 93-97.
Pardos JA, 1981. In vitro plant formation from stem pieces of Quercus suber L. In: Colloque International Sur la Culture in Vitro Des Essences Forestières, IUFRO Section, 31 August - 4 September, Fontainebleau, AFOCEL, France. Pp. 186-190.
Pattnaik S and Chand PK, 1996. In vitro propagation of the medicinal herbs Ocimum americanum L. syn. O. canum Sims. (hoary basil) and Ocimum sanctum L. (holy basil). Plant Cell Reports 15: 846–850.
Pérez-Tornero O, Burgos L, 2007. Apricot micropropagation. In: Jain SM and Häggman H (eds.). Protocols for Micropropagation of Woody Trees and Fruits. Pp. 267-278. Springer, Dordrecht, The Netherlands.
Poonawala IS, Jana MM and Nadgauda RS, 1999. Factors influencing bud break and rooting and mass-scale micropropagation of three Phragmites species: P. karka, P. communis and P. australis. Plant Cell Reports 18: 696-700.
Rai MK, Asthana P, Jaiswal VS and Jaiswal U, 2010. Biotechnological advances in guava (Psidium guajava L.): recent developments and prospects for further research. Trees 24: 1-12.
Rao AM, Sree KP and Kishor PBK, 1995. Enhanced plant regeneration in grain and sweet sorghum by asparagine, proline and cefotaxime. Plant Cell Reports 15: 72-75.
Ríoz Leal D, Sánchez-Olate M, Avilés F, Materan ME, Uribe M, Hasbún R and Rodríguez R, 2007. Micropropagation of Juglans regia L. In: Jain SM and Häggman H (eds.). Protocols for Micropropagation of Woody Trees and Fruits. Pp. 381-390. Springer, , Dordrecht, The Netherlands.
Shekhawat NS, Rai MK, Phulwaria M, Rathore JS, Gupta AK, Purohit M, Patel AK, Kataria V and Shekhawat S, 2014. Tree biotechnology with special reference to species of fragile ecosystems and arid environments. In: Ramawat KG, Mérillon JM and Ahuja MR (eds.). Tree Biotechnology. Pp. 187-222. CRC Press, USA.
Shi NO and Cheng ZM, 1994. 580 FB 085 Stimulation of embryogenesis by antibiotics from American elm cotyledons. HortScience 29: 515. 10.21273/HORTSCI.29.5.515a
Singh P, Patel RM and Kadam S, 2013. In vitro mass multiplication of pomegranate from cotyledonary nodal explants cv. Ganesh. African Journal of Biotechnology 12(2): 2863-2868.
Sivanesan I, Song JY, Hwang SJ and Jeong BR, 2011. Micropropagation of Cotoneaster wilsonii Nakai-a rare endemic ornamental plant. Plant Cell, Tissue and Organ Culture, 105(1): 55-63.
Swart PA, Kulkarni MG, Bairu MW, Finnie JF and Van Staden J, 2012. Micropropagation of Romulea sabulosa Schltr. ex Beg.- a potential ornamental plant. Scientia Horticulturae 135: 151-156.
Tao F, Zhang Z, Zhou J, Yao N and Wang D, 2007. Contamination and browning in tissue culture of Platanus occidentalis L. Forestry  Studies in China 9: 279-282.
Tiwari KN, Sharma NC, Tiwari V and Singh BD, 2000. Micropropagation of Centella asiatica (L.), a  valuable medicinal herb. Plant Cell, Tissue and Organ Culture 63(3): 179-185.
van Doorn WG, Schurer K and de  Witte Y, 1989. Role of endogenous bacteria in vascular blockage of cut rose flowers. Journal of Plant Physiology 134(3): 375-381.
Vieitez AM, Carmen San-Jose M and Vieitez E, 1985. In vitro plantlet regeneration from juvenile and mature Quercus robur L. Journal of Hhorticultural Science 60: 99-106.
Wojtania A, Pulawska J and Gabryszewska E, 2005. Identification and elimination of bacterial contaminants from Pelargonium tissue cultures. Journal of Fruit and Ornamental Plant Research 13: 101-108.
Wu JH, Miller SA, Hall HK and Mooney PA, 2009. Factors affecting the efficiency of micropropagation from lateral buds and shoot tips of Rubus. Plant Cell, Tissue and Organ Culture 99: 17-25.
Yang G and Read PE, 1992. Pre-forcing treatments influence bud break and shoot elongation in forced woody species. Journal of Environmental Horticulture 10(2): 101-103.
Yepes LM and Aldwinckle HS, 1994. Factors that affect leaf regeneration efficiency in apple, and effect of antibiotics in morphogenesis. Plant Cell, Tissue and Organ Culture 37: 257-269.
Zacchini M and De Agazio M, 2004. Micropropagation of a local olive cultivar for germplasm preservation. Biologia Plantarum 48: 589-592.