ترانسفورماسیون پایدار و آسان جنین بالغ برنج (Oryza sativa L. var. indica) با استفاده از روش In planta

نوع مقاله: علمی پژوهشی

نویسندگان

1 دانشگاه گیلان

2 عضو هیات علمی دانشگاه گیلان

چکیده

ترانسفورماسیون ژنتیکی برنج با استفاده از اگروباکتریوم (Agrobacterium tumefaciens) رویکردی مطلوب، ضروری و قدرتمند برای انتقال ژن به گیاهان زراعی می‌باشد. در این پژوهش به منظور ترانسفورماسیون گیاهان برنج از روش in planta استفاده شد. بر این اساس، بذور برنج به مدت دو روز خیسانده شدند و سپس سمت مریستم انتهایی بذر که جنین بالغ برنج وجود دارد در مراحل اولیه جوانه‌زنی با سوزن آغشته به محلول اگروباکتریوم زخم زنی و تلقیح -شد. آزمایش با دو سویه‌ی اگروباکتریوم ( EHA105و LBA4404) حاوی پلاسمید pCAMBIAl105.1R، سه غلظت استوسیرینگون (Mμ200، 100 و 0)، سه رقم برنج (هاشمی، حسنی و غریب) و دو روش وکیوم و بدون وکیوم به صورت فاکتوریل و در قالب طرح کاملاً تصادفی در سه تکرار انجام شد. کارایی ترانسفورماسیون با استفاده از آزمون مقاومت بافت‌های برگی به آنتی‌بیوتیک هایگرومایسین، آزمون بافت شیمیایی GUS و واکنش PCR با حداقل سه ژن مختلف بررسی شد. در نهایت، سویه‌ی EHA105 در رقم هاشمی، با حضور Mμ100 استوسرینگون همراه با استفاده از وکیوم بالاترین کارایی ترانسفورماسیون (46/37%) را نشان دادند. پایداری ترانس‌ژن در نسل بعد بررسی و بدین منظور 60 گیاه از نسل T1نیز با استفاده از سه تست اشاره شده آزمون و ترانسفورماسیون بودن 21% آنها تأیید شد.

کلیدواژه‌ها

موضوعات


عنوان مقاله [English]

A stable and simple transformation of rice (Oryza sativa L. ssp indica) mature embryo using in planta method

نویسندگان [English]

  • S Nasr-Ramzi 1
  • M Sohani 1
  • H Hasani 2
  • J Asghari 2
چکیده [English]

Agrobacterium-mediated transformation technique is a powerful and essential tool for genetic transformation and transgenic rice plant production. In this study an in planta transformation method was used for rice plants transformation. Therefore, rice seeds were socked for two days and the mature rice embryos was inoculated by means of an Agrobacterium coated needle. An experiment with factorial design including two strains of Agrobacterium tumefaciens (EHA105 and LBA4404) harboring pCAMBIAl105.1R, three levels of Acetosyringone (0, 100 and 200 Mµ), three cultivars of rice (Hashemi, Hasani and Gharib) and two treatments of vacuum and no vacuum operation was carried out in a Completely Randomized Design with three replications. Integration of the transgene into the genome of putative transgenic rice plants were confirmed using resistance of leaf tissues to Hygromycin, the histiochemical GUS assay and PCR with at least three different genes. Accordingly, EHA105 strain and Hashemi cultivar in the presence of 100µM acetosyringone using vacuum in a vir genes induction medium had a significant transformation efficiency (37.46%). The success of transformation was further confirmed by analysis of T1 generation with 21% transgenic.

کلیدواژه‌ها [English]

  • "Agrobacterium tumefaciens"
  • "Acetosyrmgone"
  • "Virulence genes"
  • Oryza sativa L. var. indica

 

 

 

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