توليد ريشه‌هاي مويين با استفاده از اگروباكتريوم رايزوژنز در گياه دارويي تاتوره تماشايي (Datura innoxia L.) و بررسي اثر محرك‌هاي زيستي ساليسيليك اسيد و متيل جاسمونات بر محتواي آتروپين و اسكوپولامين

The Production of Hairy Roots from (Datura innoxia L.) by Agrobacterium rhizogenes and Effects of Salicylic Acid and Methyl Jasmonate Biological Elicitors on Atropine and Scopolamine Content

مقدمه: استفاده از Agrobacterium rhizogenes جهت ايجاد ريشه‌هاي مويين تراريخت، روش بسيار كارآمد براي افزايش متابوليت‌هاي ثانويه است. هدف: هدف از اين تحقيق كشت ريشه‌هاي مويين تراريخت به منظور افزايش توليد متابوليت‌هاي ثانويه در گياه تاتوره تماشايي است. روش بررسي: در اين تحقيق توليد ريشه‌هاي مويين با تلقيح ريز نمونه‌هاي برگ و كوتيلدون حاصل از گياهچه‌هاي 2 ماهه، توسط سويه‌هاي A4، A7 و 15834 اگروباكتريوم رايزوژنز به دو روش درشت تزريقي و غوطه‌وري انجام شد و به منظور القاي اليسيتور متيل جاسمونات در غلظت‌هاي 50 و 100 ميكرو مولار و ساليسيليك اسيد در غلظت‌هاي 1، 01/0 و 1/0 ميلي‌مولار در قالب طرح كاملاً تصادفي در سه تكرار كشت داده شدند و محتواي آتروپين و اسكوپولامين ريشه‌هاي مويين تراريخت تحت غلظت‌هاي مذكور توسط HPLC بررسي شد. نتايج: بيشترين و كمترين توليد ريشه مويين تراريخته به ترتيب مربوط به سويه A4 و سويه 15834 بود. بهترين ريز نمونه جهت توليد ريشه‌هاي مويين با تلقيح برگ در سويه A4 و كوتيلدون در سويه A7 گزارش شد. روش درشت تزريقي با بيشترين توليد ريشه مويين تراريخته به عنوان برترين روش شناسايي شد. اثر محرك‌هاي ساليسيليك اسيد در غلظت 1/0 ميلي مولار سبب افزايش 3/4 برابري محتواي آتروپين در ريشه‌هاي مويين تراريخته نسبت به نمونه شاهد در غلظت مذكور شد. همچنين نتايج نشان داد، استفاده از متيل جاسمونات در غلظت‌هاي بالاتر (100 ميكرو‌مولار) باعث كاهش محتواي آتروپين و اسكوپولامين مي‌شود. نتيجه‌گيري: اگروباكتريوم به عنوان يك روش مناسب جهت توليد ريشه‌هاي مويين و اليسيتورهاي هورموني به عنوان تيمار مناسب براي افزايش آلكالوييدهاي مورد نظر مي‌توانند ايفاي نقش كنند.

Journal of M dicinal Plantʹ > The Production of Hairy Roots from (Datura imwxia L.) by Agrobacterium rhizogenes and Effects of Salicylic Acid and Methyl Jasmonate Biological Elicitors on Atropine and Scopolamine Content Ghadcrmazi S (M.Sc. Student)ʹ, Masoud Tohidfar :\1 (Ph.D.(, Miri SM (Ph.0.)1 1- Department of Horticulture, Karaj Branch, Islamic Azad University, Karaj, Iran 2- Department of Biotechnology, Faculty of Engineering, Enl:rgy and Ne\V Technology, ShJhid Behe! > hti University, Tehran, Iran "ʹCorre JXmding author: Department of Biotechnology, Faculty of E!lgineering, Energy and New Te..:hnology, Shahid Bcheshti University, Tehran, Iran Tel: +98-2129903244. lʹ; x: +98-21-22431964 Em.:1il: M TohiJfar@shu.ac.ir Abstract Background: Using Agrobacterium rltizogenes due to create hairy roots, is a userul method to increase secondary metabolites many plants. Objective: Purpose of this research is to transgenic hairy roots culture, in order to produce secondary metabolites in Datura imwxia. Methods: Explants leaf and cotyledon of Datura imwxia were inoculated for two months with A7. A4 and 15834 strains of Agrobacterium rlthogenes; Furthermore injection and Immersion methods were used in this scrutiny. The presence ofT-DNA in transgenic hairy roots were confirmed by PCR. Transgenic hairy roots in liquid medium or l/2MS were cultured. In order to induct elicitors, methyl jasmonate in tow densities or SO tM and IOOJ1M, and salicylic acid in three densities or lmM, O.lmVI and 0.01 mVI were used randomly three times. Atropine and scopolamine content of transgenic hairy roots were examined by HPLC. Results: The highest and lowest rate of tr-dnsgenic hairy roots production was respectively related to the strains of A4 and 15834. Best explants for inoculation, lear with A4 strain and cotyledon with A7 strain, were reported. With highest production rate of hairy roots, Simple deposit using a syringes method was recognized as the best method of inoculation. The effect of salicylic acid at a density of 0.1 mM increases the content of atropine concentrations. Also the results showed that usage of Methyl jasmonate nt higher doses (100 tM) reduces the content or atropine and scopolamine. Conclusion: A. rltizogenes as an appropriate method to produce hairy roots and elicitors the best treatment for increase alkaloids. eywords: Datura imwxia, Atropine, Elicitors, Hairy roots Scopolamine Journal of M dicinal Plantʹ > The Production of Hairy Roots from (Datura imwxia L.) by Agrobacterium rhizogenes and Effects of Salicylic Acid and Methyl Jasmonate Biological Elicitors on Atropine and Scopolamine Content Ghadcrmazi S (M.Sc. Student)ʹ, Masoud Tohidfar :\1 (Ph.D.(, Miri SM (Ph.0.)1 1- Department of Horticulture, Karaj Branch, Islamic Azad University, Karaj, Iran 2- Department of Biotechnology, Faculty of Engineering, Enl:rgy and Ne\V Technology, ShJhid Behe! > hti University, Tehran, Iran "ʹCorre JXmding author: Department of Biotechnology, Faculty of E!lgineering, Energy and New Te..:hnology, Shahid Bcheshti University, Tehran, Iran Tel: +98-2129903244. lʹ; x: +98-21-22431964 Em.:1il: M TohiJfar@shu.ac.ir Abstract Background: Using Agrobacterium rltizogenes due to create hairy roots, is a userul method to increase secondary metabolites many plants. Objective: Purpose of this research is to transgenic hairy roots culture, in order to produce secondary metabolites in Datura imwxia. Methods: Explants leaf and cotyledon of Datura imwxia were inoculated for two months with A7. A4 and 15834 strains of Agrobacterium rlthogenes; Furthermore injection and Immersion methods were used in this scrutiny. The presence ofT-DNA in transgenic hairy roots were confirmed by PCR. Transgenic hairy roots in liquid medium or l/2MS were cultured. In order to induct elicitors, methyl jasmonate in tow densities or SO tM and IOOJ1M, and salicylic acid in three densities or lmM, O.lmVI and 0.01 mVI were used randomly three times. Atropine and scopolamine content of transgenic hairy roots were examined by HPLC. Results: The highest and lowest rate of tr-dnsgenic hairy roots production was respectively related to the strains of A4 and 15834. Best explants for inoculation, lear with A4 strain and cotyledon with A7 strain, were reported. With highest production rate of hairy roots, Simple deposit using a syringes method was recognized as the best method of inoculation. The effect of salicylic acid at a density of 0.1 mM increases the content of atropine concentrations. Also the results showed that usage of Methyl jasmonate nt higher doses (100 tM) reduces the content or atropine and scopolamine. Conclusion: A. rltizogenes as an appropriate method to produce hairy roots and elicitors the best treatment for increase alkaloids. eywords: Datura imwxia, Atropine, Elicitors, Hairy roots Scopolamine