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The Role of virulence gene inducing factors in the improvement of in planta transformation of wheat (Triticum aestivum) | ||
Iranian Journal of Genetics and Plant Breeding | ||
دوره 9، شماره 2 - شماره پیاپی 18، دی 2020، صفحه 60-71 اصل مقاله (787.02 K) | ||
نوع مقاله: Research paper | ||
شناسه دیجیتال (DOI): 10.30479/ijgpb.2021.15234.1294 | ||
نویسندگان | ||
Mohsen Imanzadeh؛ Mohammad Mehdi Sohani* ؛ Ali Alami؛ Reza Shirzadian-Khoramabad | ||
Biotechnology Department, Faculty of Agricultural Sciences, University of Guilan, P. O. Box: 41635-1314, Rasht, Iran. | ||
تاریخ دریافت: 23 اسفند 1399، تاریخ بازنگری: 03 شهریور 1400، تاریخ پذیرش: 23 شهریور 1400 | ||
چکیده | ||
Agrobacterium tumefaciens-based plant transformation is a natural and ideal way of introducing genes into plant genomes. The system is based primarily on tissue culture techniques, including cell differentiation and plant regeneration, which might produce somatic cell mutations. In the present research, wheat plants were transformed by a non-tissue culture approach. Accordingly, mature embryos were inoculated with A. tumefaciens at an early stage of germination. A variety of different treatments, such as Agrobacterium strains (EHA105 and LBA4404 harboring pCAMBIAl105.1R), levels of glucose, concentrations of acetosyringone (0, 50, 100 and 200 µM), and types of wheat cultivars (Azar2, Alvand, and Sardari) were studied. The transformation efficiency was determined by using the number of resistant leaves to hygromycin, histochemical GUS analysis of leaf tissues, as well as PCR analysis of three transgenes located within the T-DNA region. The results showed that the maximum efficiency was obtained when 200 µM acetosyringone was used in combination with 15% glucose in the induction medium. In addition, the relative expression analysis of virulence genes by qRT-PCR revealed that VirB2 and VirD2 were significantly up-regulated when 200 µM acetosyringone and 15% glucose were used as inducers and carbon source, respectively. Therefore, Vir genes inducing factors as well as a three-step culture of Agrobacterium should be considered as major elements for any wheat transformation protocol. The putative transgenic T1 seeds were soaked and germinated in hygromycin solution, and the seedlings were further analyzed with the aforementioned methods. Based on the results, a modified Agrobacterium-mediated transformation protocol is presented. The current results suggest an inexpensive, quick and efficient approach for wheat genome transformation. | ||
کلیدواژهها | ||
Acetosyringone؛ glucose؛ Histochemical GUS assay؛ Hygromycin resistance؛ Real-Time PCR | ||
عنوان مقاله [English] | ||
نقش عوامل القا کننده ژن های بیماریزا در بهبود تراریزش به روش in planta در گندم (Triticum aestivum) | ||
نویسندگان [English] | ||
محسن ایمان زاده؛ محمد مهدی سوهانی؛ علی اعلمی؛ رضا شیرزادیان خرم آباد | ||
گروه بیوتکنولوژی، دانشکده کشاورزی، دانشگاه گیلان، گیلان، ایران، کد پستی: 41635-1314. | ||
چکیده [English] | ||
تراریزش بهواسطه Agrobacterium، یکی از راههای طبیعی و ایدهآل ورود ژن به ژنوم گیاه است، اما عمدتاً نیاز به استفاده از تکنیک کشت بافت است که در آن تمایز سلولها و باززایی گیاه اتفاق میافتد که همراه با جهش های سوماتیکی و عمدتاً نامطلوب همراه است. در این پژوهش، ارقام گندم به روش in planta و مستقل از کشت بافت تراریزش شدند. آزمایش با دو سویه Agrobacterium به نامهای EHA105 و LBA4404 حاوی ناقل pCAMBIAl105.1، سطوح مختلف استوسرینگون (0، 50، 100 و 200 (µM، دو سطح گلوکز بهعنوان منبع کربن، سه رقم گندم (آذر2، الوند و سرداری) در دو تکرار بهصورت آزمایش فاکتوریل در قالب طرح کاملاً تصادفی انجام شد. بر این اساس، جنین بالغ در مرحلة مشخص با Agrobacterium مایه زنی شد. کارایی تراریزش با آزمون مقاومت بافتهای برگی به آنتیبیوتیک هایگرومایسین، آزمون هیستوشیمیایی GUS و آزمون PCR با استفاده از سه تراژن ناحیه T-DNA بررسی شد. بالاترین کارایی تراریزش در هر دو سویه Agrobacterium در سطح µM 200 استوسرینگون و 15% گلوکز در داخل محیط القایی بدست آمد. با بررسی بیان ژنهای vir و با استفاده از تکنیکqRT-PCR نیز افزایش بیان ژنهای virB2 و virD2 در سطح µM 200 استوسرینگون و 15% گلوکز را در مقایسه با شاهد بدون القاگرهای فوق تأیید کرد. بهمنظور آنالیز نسل T1، گیاهان احتمالا تراریخت در حضور آنتی بیوتیک هایگرومایسین جوانهدار شده و برای تأیید تراریزش نسل T1از همان آزمونهای نسل T0 استفاده و کارایی در این نسل تعیین شد. نتایج جدید نشان داد که روش بالا نسبتاً ارزان، سریع و کارا در انتقال تراژن به ژنوم گیاهان گندم و سایر غلات است. | ||
کلیدواژهها [English] | ||
استوسیرینگون, گلوکز, آزمون هیستوشیمیایی گاس, مقاومت به هایگرومایسین, ریل تایم PCR | ||
مراجع | ||
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