بهینه‌سازی و ارائه دستورالعمل استخراج و باززایی پروتوپلاست سیب‌زمینی و تراریختی آن

نوع مقاله : مقاله پژوهشی

نویسندگان

1 دانشجوی دکتری رشته بیوتکنولوژی، دانشکده کشاورزی دانشگاه شهید مدنی آذربایجان

2 دانشیار گروه بیوتکنولوژی دانشگاه شهید مدنی آذربایجان، تبریز، ایران.

چکیده

هدف: ویژگی‌های منحصر بفرد پروتوپلاست‌های گیاهی، آن‌ها را به ابزاری قدرتمند برای محققین، جهت مطالعه تغییرات ژنتیکی سلولی تبدیل کرده است. مهمترین پیش­نیاز استفاده از پروتوپلاست‌ها، توانایی استخراج ساده در مقادیر بالا، کشت و باززایی آن‌ها جهت تشکیل کلونی سلولی و تولید گیاه است. معمول‌ترین روش استخراج پروتوپلاست از بافت‌های گیاهی، روش آنزیمی است. غلظت و ترکیب آنزیم‌ها و مراحل جداسازی و کشت، از عوامل کلیدی استخراج پروتوپلاست­ می‌باشد. هدف این تحقیق، بررسی اثر فاکتورهای مذکور بر روی پروتوپلاست‌های گیاهان درون‌شیشه­ای سیب‌زمینی (Solanum tuberosum L.) و معرفی روشی کارا و مناسب برای استخراج، تراریختی و کالوس‌زایی/باززایی از این گیاه است.
مواد و روش‌ها: برای جداسازی پروتوپلاست سیب‌زمینی رقم رایج تجاری آگریا، تیمارهای مختلف غلظت آنزیم‌های سلولاز و مسروزیم، مدت نگهداری در محلول آنزیمی و مراحل جداسازی بر روی بافت­ برگ، ساقه و بخش‌های هوایی گیاهچه‌های درون‌شیشه­ای و اثر غلظت هورمن‌های مختلف برکشت و باززایی پروتوپلاست مورد بررسی قرار گرفت. پس از جداسازی، کارآیی تراریختی پروتوپلاست‌ها به‌واسطه­ی PEG 4000 با استفاده از ناقل pBin61-GFP ارزیابی شد.
نتایج: بهترین نتایج از ریزنمونه‌های برگی و بالاترین تعداد پروتوپلاست (106 × 2 پروتوپلاست بر میلی­لیتر)، پس از 16 ساعت نگهداری در محلول آنزیمی (حاوی 1 درصد سلولاز و 2/0 درصد مسروزیم) از بافت‌های مزوفیل برگ حاصل شد. کارآیی تراریختی پروتوپلاست‌ها به­واسطه­ی PEG با غلظت 40 درصد و مدت زمان 30 دقیقه ،50 درصد بود. بهترین تیمار محیط کشت برای القای کالوس‌زایی، محیط MS حاوی 5 میلی‌گرم بر لیتر NAA و 1/0 میلی‌گرم بر لیتر BAP بدست آمد که در این تیمار هورمونی، 100 درصد پروتوپلاست‌ها بعد از حدود یک ماه کالوس تشکیل دادند. بهترین تیمار باززایی از کالوس ترکیب هورمونی 2 میلی‌گرم برلیتر Zeatin با 1/0 میلی‌گرم بر لیتر GA3 به­همراه 01/0 میلی‌گرم بر لیتر NAA با راندمان 86 درصد بود.
نتیجه‌گیری: بر اساس نتایج حاصل، دستورالعمل استخراج پروتوپلاست و انتقال ژن توصیف شده در این تحقیق برای مهندسی ژنتیک، انتقال و بیان ژن در گیاه سیب‌زمینی کارآیی لازم را دارد.

کلیدواژه‌ها


عنوان مقاله [English]

Optimization and presentation of a protocol for protoplast isolation, regeneration and gene transfer in potato

نویسندگان [English]

  • Esmat Ashaar Ghadim 1
  • Maghsoud Pazhouhandeh 2
  • Mohammad Ahmadabadi 2
1 Ph.D. Student, Biotechnology Department, Faculty of Agriculture, Azerbaijan Shahid Madani University, Tabriz, Iran.
2 Associate Professor, Biotechnology Department, Faculty of Agriculture, Azarbaijan Shahid Madani University, Tabriz, Iran.
چکیده [English]

Objective
The unique characteristics of plant protoplasts have made them a powerful tool for researchers to study the genetic modification of plant cells. The most important prerequisite for the use of protoplasts is the ability to extract them easily and in large quantities, and to cultivate and regenerate them for cell colony formation and plant production. The most common method of extracting protoplasts from plant tissues is the enzymatic method. The concentration and composition of enzymes and isolation and culture steps are key factors in protoplast extraction. This study aimed to investigate the effect of these factors on protoplasts of in vitro potatoes (Solanum tuberosum L.) and to develop an efficient and suitable method for their extraction, transfection, callus formation, and regeneration.
 
Materials and methods
For protoplast isolation from commercial and common Agria cultivars of potato, different treatments of Cellulase and Macerozyme enzymes concentration, incubation time in an enzyme solution, and isolation steps on leaf tissue, stem, and aerial part of in vitro plantlets were investigated. The effects of different hormone concentrations on the culture and regeneration of protoplasts were studied. After protoplast isolation, the efficiency of transfection by PEG 4000 using pBin61-GFP plasmid was evaluated.
 
Results
The best results were obtained from leaf explants and the highest number of protoplasts (2×106 protoplasts per ml) were extracted from leaf mesophilic tissues after 16 hours incubation in an enzyme solution (1% Cellulase and 0.2% Macerozyme). The transfection efficiency of protoplasts by PEG with 40% concentration and duration of 30 minutes was 50%. The MS medium containing 5 mg/L NAA and 0.1 mg/L BAP was selected as the best culture medium for callus induction. In this hormonal treatment, 100% of callus induction from protoplasts was obtained after about one month. The best medium for plant regeneration and shooting from callus was the combination of 2 mg/L Zeatin with 0.1 mg/L GA3 and 0.01 mg/L NAA with 86% efficiency.
 
Conclusions
Based on the results, the described protocol for protoplast extraction and gene transfer in this study provides the necessary steps for genetic engineering, transformation, and gene expression in the potato plant.

کلیدواژه‌ها [English]

  • Cellulose
  • Genetic engineering
  • Macerozyme
  • Polyethylene glycol
  • Protoplast
 
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