با همکاری مشترک دانشگاه پیام نور و انجمن بیوتکنولوژی جمهوری اسلامی ایران

نوع مقاله : علمی پژوهشی

نویسندگان

1 استادیار، گروه علوم باغبانی، دانشکده کشاورزی، دانشگاه مراغه، مراغه، ایران.

2 کارشناسی ارشد، گروه علوم باغبانی، دانشکده کشاورزی، دانشگاه مراغه، مراغه، ایران.

3 دانشیار، گروه علوم باغبانی، دانشکده کشاورزی، دانشگاه مراغه، مراغه، ایران و دانشیار، پژوهشی مرکز پژوهش‌های مجلس شورای اسلامی، تهران، ایران.

چکیده

گیاه سنبل‌الطیب (Valeriana officinalis L.) از خانواده Valerianaceae به‌عنوان گیاه دارویی‌ ارزشمند همواره مورد توجه بوده است که از آن برای درمان بیماری‌های عصبی استفاده می‌شود. پژوهش به‌صورت آزمایش فاکتوریل در قالب طرح کاملاً تصادفی شامل سویه‌های باکتری (R1000, LBA9402, C58, A4, GM, ATTCC15834)، سن ریزنمونه جهت تلقیح (10، 20 و 30‌ روز) و ریزنمونه‌های مختلف گیاه سنبل‌الطیب (برگ و ریشه) در سه تکرار انجام گرفت. جهت کشت باکتری‌ها از محیط کشت MYA جامد و جهت تهیه سوسپانسیون باکتری به‌منظور تلقیح از محیط MYA مایع حاوی آنتی‌بیوتیک ریفامپسین با غلظت 50 میلی‌گرم در لیتر استفاده گردید. دو روش تلقیح (روش غوطه‌وری و تزریق) بر درصد ریشه‌زایی ریز‌نمونه‌ها مورد ارزیابی قرار گرفت و روش تزریق در القای ریشه مویین بر ریزنمونه‌ها مؤثر واقع نشد. پس از مشخص شدن بهترین سویه و بهترین ریزنمونه، جهت پرآوری ریشه‌های مویین کلون‌ها در محیط کشت جامد MS ،1/2 MS و MS + 0.2 mg/L NAA کشت گردیدند، مشاهده شد که ریشه‌های قرار داده شده در محیط کشت MS بیشترین زیست‌توده را داشتند. سویه ATCC15834و A4 رشد یافته در محیط MYA روی ریز نمونه برگی 30 روزه مناسب‌ترین سویه‌ها بودند. بیشترین طول ریشه‌ و تعداد ریشه ‌مویین در القای با سویه A4 دیده شد. ریشه‌های مویین القاء شده در محیط کشت MS حاوی 3 درصد ساکارز در دمای 25 درجه سانتی‌گراد بیشترین میزان رشد ریشه‌های مویین را نشان داد. نتایج نشان داد که کاربرد سویه‌های ATTCC15834, R1000, LBA9402 و A4 باعث القای ریشه‌های مویین در گیاه سنبل‌الطیب شد و دو سویه دیگر در القای ریشه‌های مویین ناکارآمد بودند.

کلیدواژه‌ها

موضوعات

عنوان مقاله [English]

Effect of different strains of Agrobacterium rhizogenes, type and age of explant on hairy root production in Valeriana officinalis L.

نویسندگان [English]

  • Mohammad Ali Aazami 1
  • Samieh Ghafury 2
  • Mohammad Bagher Hassanpouraghdam 3

1 Assistant Professor, Department of Horticulture, Faculty of Agriculture, University of Maragheh, Maragheh, Iran.

2 M.Sc. Student, Department of Horticulture, Faculty of Agriculture, University of Maragheh, Maragheh, Iran.

3 Associate Professor, Department of Horticulture, Faculty of Agriculture, University of Maragheh, Maragheh, Iran and Research Associate Professor, Islamic Parliament Research Center, Tehran, Iran.

چکیده [English]

Valeriana officinalis from the valerianaceae is a precious medicinal plant has been employed for the treatment of mental ailments. The study was a factorial experiment in a CRD including bacterial strains (R1000, LBA9402, C58, A4, GM, and ATTCC15834), age of explants for inoculation (10, 20 and 30 days) and different explants of valerian (leaves and roots) in three replications. Solid Manitol Yest exteract Agar (MYA) medium was used for bacterial culture and MYA medium containing rifampicin antibiotic (50 mg/l) was used to prepare the bacterial suspension for inoculation. Two inoculation methods (floating and injection method) were evaluated on the percentage of rooting and the injection method was not effective in hairy root induction on explants. After determining the best strain and the best explants of the clones were cultured in MS, 1/2 MS and MS+0.2 mg/L NAA solid culture medium it was observed that the roots placed in MD medium were the most they had biomass. Strains of ATCC15834 and A4 on the MYA medium on 30 days old leaf explants was the best strain than others. The optimized root length and the top hairy root number were induced by A4 strain. The newly produced hairy root showed the highest growth on MS medium enriched with 3% sucrose at 25°C. Our results showed that the application of ATTCC15834, R1000, LBA9402 and A4 strains induced hairy roots in valerian and the other two strains were ineffective.

کلیدواژه‌ها [English]

  • Agrobacterium rhizogenes
  • Valeriana officinalis L
  • strain
  • Hairy root
Ahlawat S, Saxena P, Ram M., Alam P, Nafis T, Mohd A, Abdin MZ (2012) Influence of Agrobacterium rhizogenes on induction of hairy roots for enhanced production of artemisinin in Artemisia annua L. plants. African J Biotechnol. 11: 8684-91.
Banihashemi O, Khavari-Nejad RA, Yassa N, Najafi F (2015) Induction of hairy root in Atropa komarovii using Agrobacterium rhizogenes ATCC15834. Indian J Fundament. Appl. Life Sci.5(4):22-31.
Bensaddek L, Gillet F, Nava-Saucedo JE, Fliniaux MA (2001) The effect of nitrate and ammonium concentrations on growth and alkaloid accumulation of Atropa belladonna hairy roots. J. Biotech.85: 35-40.
Bensaddek L, Villarreal ML, Fliniaux MA (2008) Induction and growth of hairy roots for the production of medicinal compounds. Int. J. Integr. Biol. 3(1):2-9.
Bonhomme V, Laurain-Mattar D, Lacoux J, Fliniaux MA JacquinDubreuil A (2000) Tropane alkaloid production by hairy roots of Atropa belladonna obtained after transformation with Agrobacterium rhizogenes 15834 and Agrobacterium tumefaciens containing rolA, B, C genes only. J. Biotech. 81(2): 151-158.
Cardarelli M, Mariotti D, Pomponi M, Spano L, Capone I, Costantino P (1987) Agrobacterium rhizogenes T-DNA genes capable of inducing hairy root phenotype. Mol. Gen. Genet.  209(3): 475-480.
Chabaud M, Boisson-Dernier A, Zhang J, Taylor CG, Yu O, Barker DG (2006) Agrobacterium rhizogenes-mediated root transformation. The Medicago truncatula handbook, version November.‏
Dehghan E, Häkkinen ST, Oksman-Caldentey KM, Ahmadi FS (2012) Production of tropane alkaloids in diploid and tetraploid plants and in vitro hairy root cultures of Egyptian henbane (Hyoscyamus muticus L.). Plant Cell, Tissue and Organ Culture 110(1): 35-44.
Dini Torkamani MR, Abas Pour N, Jafari M, Samadi A (2014) Induction and optimization of hairy root growth condition for Valeriana officinalis L. through inoculation by Agrobacterium rhizogenes. J Cell Tissue 5(1): 23-30.
Filizadeh Y, Goodarzi G (2010) Essential Oils from Hairy Root Cultures and Field Cultivated Roots of
Valerian (Valeriana sisymbriifolium). J Med. Plants 9(35): 1-9.
Granicher F, Christen P, Kapetanidis I (1995) Essential oils from normal and hairy roots of Valeriana officinalis var. sambucifolia. Phytochemistry 40(5): 1421-1424.
Guillon S, Guiller JT, Pati PK, Rideau M, Gantet P (2006) Hairy root research: recent scenario and exciting prospects. J. Plant Biol. 9: 341-346.
Gutierrez- valdes N, Hakkinen ST, Lemasson C, Guillet M, Marja K, Caldenty O, Ritala A, Cardon F (2020) Hairy root culture-A versatile tool with multiple applications. Front. Plant Sci. 3: 165-172.
Ho ZB, Du M (2006) Hairy root and its application in plant genetic engineering. J. Integr. Plant Biol. 48(2):121-127.
Ionkova I (2007) Biotechnological approaches for the production of Lignans. Pharmacogn. Rev. 1: 427-443.
Kabirnataj S, Nematzadeh G, Zolala J, Talebi AF (2016) High-efficient transgenic hairy roots induction in chicory: Re-dawn of a traditional herb. Acta Agric. Slov. 107(2): 321-332.
Khan S, Qureshi MI, Alam T, Abdin MZ (2007) Protocol for isolation of genomic DNA from dry and fresh roots of medicinal plants suitable for RAPD and restriction digestion. Afr. J. Biotechnol. 6(3): 175-183.
Mehrotra S, Kukreja AK, Khanuja SPS, Mishra BN (2008) Genetic transformation studies and scale up of hairy root culture of Glycyrrhiza glabra in bioreactor. Electron. J. of Biotechn. 9(1):41-47.
Mi Y, Zhu Z, Qian G, Li Y, Meng X, Xue J, Chen Q, Sun W, Shi Y (2020) Inducing Hairy Roots by Agrobacterium rhizogenes-Mediated Transformation in Tartary Buckwheat (Fagopyrum tataricum). J. Vis. Exp. 157: 67-75.
Morgan J, Barney C, Penn A, Shanks J (2000) Effects of buffered media upon growth and alkaloid production of Catharanthus roseus hairy roots. Appl. Microbiol. Biotechnol. 53: 262-265.
Morton EL, Fuqua C (2012) Unit3D.1 Laboratory maintenance of agrobacterium. Curr. Protoc. Microbiol. 6:1-8.
Murashige T, Skoog F (1962) A revised medium for rapid growth and biossays with tobacco tissue cultures. Physiol. Plant. 15: 473-497.
Murthy HN, Dijkstra C, Anthony P, White DA, Davey MR, Power JB, Hahn EJ, Paek KY (2008) Establishment of Withania somnifera hairy root cultures for the production of Withanolide A. J. Integr. Plant Biol. 50: 975-981.
Nartop P (2018) Engineering of Biomass Accumulation and Secondary Metabolite Production in Plant Cell and Tissue Cultures.Plant metabolites and regulation under environmental stress. Academic Press, pp 169-194.
Nilsson O, Olsson O (1997) Getting to the root: the role of the Agrobacterium rhizogenes rol genes in the formation of hairy roots. Physiol. Plant.100(3): 463-473.
Pakdin-Parizi A, Farsi M, Nematzade GA, Mirshamci A (2015) Impact of different culture media on hairy roots growth of Valeriana officinalis L. Acta Agric.Slov. 103(2): 299-305.
Pandey R, Krishnasamy V, Kumaravadivel N, Rajamani K (2014) Establishment of hairy root culture and production of secondary metabolites in Coleus (Coleus forskohlii). J. Med. Plants Res. 8(1): 58-62.
Petersen SG, Stummann BM, Olesen P, Henningsen KW (1989) Structure and function of root‐inducing (Ri) plasmids and their relation to tumor‐inducing (Ti) plasmids. Physiol. Plant. 77(3): 427-435.
Pitta–Alvarez SI, Spollansky TC, Giulietti AM (2000) The influence of different biotic and abiotic elicitors on the production and profile of tropane alkaloids in hairy root cultures of Brugmansia candida. Enzyme Microb. Tech. 26(2): 252-258.
Ramirez-Estrada K, Vidal-Limon H, Hidalgo D, Moyano E, Golenioswki M, Cusidó R, Palazon J (2016) Elicitation, an Effective strategy for the biotechnological production of bioactive high-added value compounds in plant cell factories. Molecules 21(2): 182-189.
Roychowdhary D, Majumder A, Jha S (2017) Biotechnology for medicinal plants. Agrobacterium rhizogenes-Mediated Transformation in Medicinal Plants: Prospects and Challenges. Springer. pp. 29-68.
Shahzad A, Saeed T (2015). A review on Phytochemistry, Pharmacological properties and Biotechnological studies in Valeriana officinalis L., An important medicinal herb. Hippocratic J. Unani Medicine. 10(1): 53-71.
Sivakumar G, Yu KW, Hahn EJ, Paek KY (2005) Optimization of organic nutrients for ginseng hairy roots production in large-scale bioreactors. Curr. Sci. 26: 641-649.
Sivanandhan G, Selvaraj N, Ganapathi A, Manickavasagam M (2014) An efficient hairy root culture system for Withania somnifera. African J. Biotech. 13: 4141-4147.
Soleimani T, Keyhanfar M, Piri KH, Hasanloo T (2012). Hairy root induction in Burdock (Arctium lappa L.). J. Med Plants 3: 60-69.
Tamakawa T, Sekiguchi S, Kodama T, Smith S, Yeoman MM (1998) Transformation of Chilli Pepper (Capsicum frutescens) with a Phenylalanine Amonia-Lyase gene. J. Plant Biotech. 15: 189-193.
Thwe A, Valan Arasu M, Li X, Ha Park Ch, Kim SJ, Al-Dhabi NA, Un Park S (2016) Effect of different Agrobacterium rhizogenes strains on hairy root induction and phenylpropanoid biosynthesis in tartary buckwheat (Fagopyrum tataricum Gaertn). J. Microb.7:318-327.
Weber RLM, Bodanese-Zanettini MH (2011) Induction of transgenic hairy roots in soybean genotypes by Agrobacterium rhizogenes-mediated transformation. Pesqui. Agropecu. Bras. 46(9): 1070-1075.
Zebarjadi AR, Najafi Sh, Ghasempour HR, Motamedi J (2011) Establishment of a practical tissue culture for producing hairy roots of Valeriana officinalis L. via Agrobacterium rhizogenes. J.Med. Plants Res. 5(20): 345-355.