بررسی عامل‌های مؤثر در القای ریشۀ موئین و افزایش میزان رزمارینیک‌اسید زرین‌گیاه (Dracocephalum kotschyi Boiss)

نوع مقاله : مقاله پژوهشی

نویسندگان

1 دانشیار، دانشکدۀ کشاورزی، دانشگاه ارومیه، ارومیه، ایران

2 دانشجوی سابق کارشناسی ارشد، دانشکدۀ کشاورزی، دانشگاه ارومیه، ارومیه، ایران

3 استادیار دانشکدۀ کشاورزی، دانشگاه ارومیه، ارومیه، ایران

چکیده

زرین­گیاه یا همان بادرنجبویۀ دنایی با نام علمی Dracocephalum kotschyi متعلق به تیرۀ نعناعیان و جنس بادرشبو (Dracocephalum) است که به علت پراکنش گستردۀ آن در ایران با نام­های چندی از آن یاد می­شود. ترکیب‌های فلاونوئید موجود در زرین­ گیاه، خاصیت ضدسرطانی دارد و استفاده از روش­های مختلف مانند تولید ریشه­های موئین می‌تواند به تولید ترکیب‌هایی با ارزش دارویی بالا کمک کند. در این بررسی تأثیر چهار نوع ریزنمونه (لپه یا کوتیلدون، محور ریزلپه یا هیپوکوتیل، برگ و میانگره( و سن ریزنمونه (برگ یک‌هفته‌ای، دوهفته‌ای و چهارهفته‌ای) بر میزان القای ریشۀ موئین گیاه زرین­گیاه بررسی شد. تأیید تراریختی با استفاده از آغازگر اختصاصی ژن rolB به روش PCR انجام شد. به‌منظور شناسایی محیط کشت مناسب برای رشد و افزونش ریشه­های موئین، تأثیر چهار نوع محیط کشت پایۀ مایع MS، 1/2MS، 1/4 MS وB5  و محرک UV-B در سه سطح: 3 دقیقه، 6 دقیقه و 9 دقیقه بر تغییر ویژگی‌های بیوشیمیایی و رزمارینیک اسید ریشه­های موئین بررسی شد. بیشترین درصد القاء ریشه در ریزنمونۀ برگ یک‌هفته‌ای (66/56 درصد) و لپه (66/36 درصد) مشاهده شد. بیشترین وزن­تر (34/4 گرم) و وزن خشک (31/0 گرم) در محیط کشت 1/4MS و کمترین وزن­تر (04/1 گرم) و وزن خشک (10/0 گرم) در محیط کشتB5  ثبت شد. بیشترین فعالیت پاداکسندگی (آنتی­اکسیدانی) در تیمار شش دقیقه (33/92 درصد) و کمترین میزان فعالیت پاداکسندگی در ریشه­های غیرتراریخت (63 درصد) به دست آمد.نتایج تجزیۀ HPLCنشان داد که تولید رزمارینیک اسید در ریشه­های تراریخت تیمارشده با UV-B با زمان شش دقیقه، 5/2 برابر بیشتر از ریشه­های غیر تراریخت (g /mg DW 9/34 در مقایسه با،µg /mg DW 8/13) است.

کلیدواژه‌ها

موضوعات


عنوان مقاله [English]

Study of factors affecting hairy roots induction and Rosmarinic Acid production in Dracocephalum kotschyi Boiss

نویسندگان [English]

  • Bahman Hosseini 1
  • Nasrin Ayyobi 2
  • Mohammad Fattahi 3
1 Associate Professor, Faculty of Agriculture, Urmia University, Iran
2 Former M.Sc. Student, Faculty of Agriculture, Urmia University, Iran
3 Assistant Professor, Faculty of Agriculture, Urmia University, Iran
چکیده [English]

Abstract: The flavonoid compounds in the Dracocephalum kotschyi has anti-cancer properties and using various methods such as hairy roots production can used to produce valuable secondary metabolites. In this study, the effect of explant type (cotyledon, hypocotyl, leaf and internode) and explants age (1 week leaf, 2 weeks leaf and 4 weeks old leaf) on hairy roots induction percent were surveyed. The transgenic status of hairy roots was confirmed by PCR using rolB genes-specific primers. In order to find optimum culture media for hairy roots growth and proliferation, four types of liquid basal media including, MS, 1/2MS, 1/4 MS and B5 and UV-B elicitor effects in three levels (3 min, 6 min and 9 min) on biochemical characteristics and Rosmarinic acid (RA) contents were investigated. The maximum hairy roots induction was obtained in 1 week leaves old (56.66 %); cotyledon explants (36.66 %), respectively. The highest fresh (4.34 gr) and dry weight (0.31 gr) and lowest fresh (1.04 gr) and dry weight (0.10 gr) was recorded in 1/4 MS and B5 media, respectively. The maximum and minimum antioxidant activities (92.33% and 63 %) were obtained in UV-B exposure time to 6 min radiation and non-transformed hairy roots. High performance liquid chromatography analysis revealed that the production of RA in UV-B (6 min) elicited hairy roots of D. kotschyi was almost 2.5-fold higher than that of the non-transformed roots (34.9 µg /mg DW compared to 13.8 µg /mg DW).

کلیدواژه‌ها [English]

  • Agrobacterium rhizogenes
  • antioxidant activitiy
  • Catalase
  • Total Flavonoid
  • Zarrin-giah
Agrawal, S. B. (1992). Effects of supplemental UV-B radiation on photosynthetic pigment, protein and glutathione contents in green algae. Environmental and Experimental Botany, 32 (2), 137-143.
Allen, D. J., Nogues, S. & Baker, R. N. (1998). Ozonedepletion and increased UV-B radiation: is there a real threat to photosynthesis. Journal of Experimental Botany, 328, 1775-1788.
Bensaddek, L., Gillet, F., Nava-Saucedo, J. E. & Fliniaux, M. A. (2001). The effect of nitrate and ammonium concentrations on growth and alkaloid accumulation of Atropa belladonna hairy roots. Journal of Biotechnology, 85, 35-40.
Bischof, K., Peralta, G., Krabs, G., Van de Poll, W. H., PerezLIore´ns, J. L. & Anneke, M. (2002). Effects of solar UV-B radiation on canopy structure of Ulva communities from southern Spain. Journal of Experimental Botany, 53(379), 2411-2421.
Chawla, H. S. (2000). Introduction to plant biotechnology. (3rd ed.). CRC Press Book.
Chiou, A., Karathanos, V. T., Mylona, A., Salta, F. N., Preventi, F. & Andrikopoulos, N. K.) 2007(. Currants (Vitis vinifera L.) content of simple phenolics and antioxidant activity. Food Chemistry, 102, 516-522.
Dhakulkar, S. & Bhargava, S. (2004). Induction of hairy roots in Gemelina arborea, using Agrobacterum rhizogens. Plant Cell Reports, 49, 105-107.
Dai, Q., Yan, B., Huang, S., Liu, X. & Peng, S. (1997). Response of oxidative stress defense system in rice (Oryza sativa) leaves with supplemental UVB radiation. Physiologia Plantarum, 101, 301-308.
Fattahi, M. (2012). Assessment of morphological diversity, phytochemical and production of capillary roots in daenensis lemon balm. Ph. D. thesis. Faculty of Agriculture Tehran University, Iran. (in Farsi)
Fattahi, M. & Fattahi, B. (2011). Medicinal plant. Jahad Daneshgahi, Tehran. Iran.
Furness, N., Upadhyaya, M. K. & Ormrod, D. P. (1999). Seedling growth and leaf surface morphological responses of three rangeland weeds to ultraviolet-B radiation. Weed Science, 47, 427-434.
Ghahreman, A. (2000). Botanical basic anatomy and morphology of vegetative organs and operates the world's largest plant them in groups. (5th ed.). Tehran University Press.
Gandi, S. & Giri, A. (2012). Genetic transformation of Centella asiatica by Agrobacterium rhizogenes. Journal of Pharmacognosy, 3(2), 82-84.
Gould, K. S., Markham, K. R., Smith, R. H. & Goris, J. J. (2000). Functional role of anthocyanine in the leaves of Quintinia serrate. Journal of Experimental Botany, 51, 1107-1115.
Gitz, D. C., Gitz, L. L., Mcclure, J. W. & Huerta, A. J. (2004). Effects of a PAL inhibitor on phenolic accumolation and UV-B tolerance in Spirodela intermedia. Journal of Experimental Botany, 55, 919-927.
Giordano, C. V., Mori, T., Sala, O. E., Scope, A. L., Caldwell, M. M. & Ballare, C. L. (2003). Functional acclimation to solar UV-B radiation in Gunnera magellanica, a native plant species of Southernmost Patagonia. Plant, Cell and Environment, 26, 2027-2036.
Hoekema, A., Hirsch, P. R. & Hooykaas, P. J. J. (1983). A binary plant vector strategy based on separation of vir-region and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature, 303, 179-180.
Hopkins, L., Bond, M. A. & Tobin, A. K. (2002).Ultraviolete-B radiation reduces the rates of cell division and elongation in the primary leaf of wheat (Triticum aestivum L.). Plant, Cell and Environment, 25, 617-625.
Hofmann, W., Swinny, E. E., Bloor, S. J., Markham, K. R., Ryan, K. G., Campbell, B. D., Jordan, B. R. & Fountain, D. W. (2000). Responses of nine Trifolium repens L. populations to ultraviolet-B radiation: differential flavonol glycoside accumulation and biomass production. Annals of Botany, 86:527-573.
Hoque, E. & Remus, G. (1999). Natural UVscreening mechanisms of Norway spruce (Picea abies L. karts) needles. Photochemistry and Photobiology, 69, 177-192.
Inze, D. & Montagu, M. V. (2002). Oxidative stress in plants. (1st ed.) Taylor and Froncis press.
Kabirnotaj, S., Zolali, J., Nematzade, G. H. & Shokri, A. (2012). Optimization of hairy root culture establishment in Chicory plants (Cichorium intybus) through inoculation by Agrobacterium rhizogenes. Journal of Agricultural Biotechnology, 4(2), 75-61. (in Farsi)
Karmarkar, S. H. & Keshavachndran, R. (2001). Genetic transformation and hairy root induction in Holostemma ada-kodien K. Shum-A vulnerable medicinal plant. Indian Journal of Experimental Bioloy, 39, 1263-1267.
Khan, S., Irfan, Q. M., Kamaluddin, A. T. & Abdin, M. Z. (2007). Protocol for isolation of genomic DNA from dry and fresh roots of medicinal plants suitable for RAPD and restriction digestion. African Journal of Biotechnology, 6, 175-178.
Khezri, M. (2013). The impact of some chemical triggers production of hairy root induction of β Caryophyllene Teucrium (Teucrium polium L). M.Sc. thesis. Agriculture facultuy of Orumiyeh University, Iran. (in Farsi)
Liu, C. Z., Guo, C., Wang, Y. C. & Ouyang, F. (2002). Effect of light irradiation on hairy root growth and artemisinin biosynthesis of Artemisia annua L. Process Biochemistry, 38(4), 581-585.
Mapelli, S., Shorina, M., Brambilla, I. & kuznetsov, V. (2006). Biochemical and physiological events following exposure to UV-B radaiation in ice plants. Plant Physiology, 6, 33-44.
Mashayekhi, K. (2007). Somatic embryogenesis Plant. Makhtomgholi Fraghi (Sarli) press.
Nourozi, A. (2013). Effect of different strains of Agrobacterium Rayzvzhnz to induce hairy root in the Mexican goal (Agastache foeniculum). Msc. Thesis. Agriculture facultuy of Orumiyeh University, Iran. (in Farsi)
Park, S. U. & Facchini, P. J. (2000). Agrobacterium rhizogenes mediated transformation of Opium poppy, Papaver somniferum L. and California poppy, Eschscholzia californica Cham., root cultures. Journal of Experimental Botany, 347, 1005-1016.
Petersen, M., Häusler, E., Karwatzki, B. & Meinhard, J. (1993). Proposed biosynthetic pathway for rosmarinic acid in cell cultures of Coleus blumei Benth. Planta, 189, 10-14.
Petersen, M. (1997). Cytochrome P450-dependent hydroxylation in the biosynthesis of rosmarinic acid in Coleus. Phytochemistry, 45, 1165-1172.
Pirian, K., Piri, K. & Ghiyasvand, T. (2012). Hairy roots induction from Portulaca oleracea using Agrobacterium rhizogenes to Noradrenaline, sproduction. International Research Journal of Applied and Basic Sciences, 3(3), 642-649.
Qing, L., Callaghan, T. V. & Yuanyuan, Z. (2004). Effects of Elevated Solar UV-B Radiation from Ozone Depletion on Terrestrial Ecosystems. Journal of Mountain Science, 1(3), 276-288.
Rahnama, H., Hasanloo, T., Shams, M. K. & Sepehrifar, R. (2008). Silymarrin production in hairy root culture of Silybum marianum Gearth. Iranian Journal of Biotechnology, 6, 113-118. (in Farsi)
Saravankumar, A., Aslam, A. & Shajahan, A. (2012). Developmant and optimaization of hairy root cultur systems in Withania somnifera (L.) Dunal for withaferin-A production. African Journal of Biotechnology, 11(89), 16412-16420.
Sevon, N. & Oksman-Caldentey, K. M. (2002). Agrobacterium rhizogenes mediated transformation: root cultures as a source of alkaloids. Planta Medica, 68, 859-868.
Shin, Y., Liu, R. H., Nock, J. F., Holliday, D. & Watkins, C. B. (2007). Temperature and relative humidity effects on quality, total ascorbic acid, phenolics and flavonoid concentrations, and antioxidant activity of strawberry. Postharvest Biology and Technology, 45, 349-357.
Shirazi, Z., Piri, K., Mirzaie, A. & Hasanloo, T. (2012). Glycyrrhizin and isoliquiritigenin production by hairy root culture of Glycyrrhiza glabra. Journal of Medicinal Plants Research, 6(31), 4640-4646. (in Farsi)
Sidhu, Y. (2010). In vitro micropropagation of medicinal plants by tissue culture. The Plymouth Student Scientist, 4(1), 432-449.
Slinkard, K. & Singleton, V. L. (1977). Total phenol analysis: automation and comparison with manual methods. American Journal of Enology and Viticulture, 28(1), 49-55.
Solemani, T., Keyanfar, M., Piri, H. & Hasanloo, T. (2012). Morphological Evaluation of hairy roots induced in Artemisia annu L and investigating elictation effects on the hairy roots biomass production. International Journal of Agriculture: Research and Review, 2, 1005-1013. (in Farsi)
Wellman, E. (1971). Phytochrome-mediated flavon glycoside synthesis in cell suspension culture of Petroselinum hortense after pre irradiation with ultraviolet light. Planta, 101, 283-286.
Wen Wang, J. & Yong Wu, J. (2010). Tanshinone biosynthesis in Salvia miltiorrhiza and production in plant tissue cultures. Application Microbiology Biotechnology, 88, 437-449.
Xiao, Y., Di, P., Chen, J., Liu, Y., Chen, W. & Zhang, L. (2009). Characterization and expression profiling of 4-hydroxyphenylpyruvate dioxygenase gene (Smhppd) from Salvia miltiorrhiza hairy root cultures. Molecular Biology Reports, 36, 2019-2029.
Yang, S. H., Liu, X. F., Guo, D. A. & Zhen, J. H. (2006). Induction of hairy roots and anthraquinone production in Rheum palmatum. Zhongguo Zhong Yao Za Zhi, 18, 1496-1499.