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بررسی تأثیر عامل‌های مؤثر در میزان تشکیل پروتوپلاست در قارچ Fusarium oxysporum با هدف استفاده در امتزاج پروتوپلاست

نوع مقاله : مقاله پژوهشی

نویسندگان

1 دانشجوی دکتری، گروه گیاه‌پزشکی، دانشکدۀ کشاورزی دانشگاه زنجان

2 استاد، گروه گیاهپزشکی، پردیس کشاورزی و منابع طبیعی دانشگاه تهران، کرج

چکیده

امتزاج پروتوپلاست یکی از سیستم­های تراریختی مؤثر در ایجاد تغییرهای ژنتیکی به منظور بهبود عملکرد میکروارگانیسم (ریزجانداران) است که امروزه در جهت رسیدن به یک عامل بیوکنترل (مهار زیستی) موفق در کنترل علف هرزهای مختلف استفاده می­شود. در روش­های معمول تراریختی قارچ­های رشته­ای مانند گونه‌های فوزاریوم، تهیۀ پروتوپلاست نخستین گام است. در این پژوهش، پس از جداسازی قارچ Fusarium oxysporum (عامل پژمردگی آوندی) از گیاه‌ گل جالیز Orobanche sp.،  بررسی‌های لازم برای شناسایی ریخت‌شناسی و مولکولی آن انجام شد. سپس تأثیر عامل‌های مختلف مانند تأثیر نوع محیط کشت در مقدار اسپور تولید شده، بهترین زمان برای جوانه­زنی بیشینه‌ای اسپورها، غلظت­های مختلف آنزیم­های تجزیه‌کنندۀ دیوارۀ سلولی، زمان در معرض قرارگیری با آنزیم در تعداد پروتوپلاست­های تولیدشده و نیز نوع و غلظت تثبیت‌کنندۀ اسمزی در ایجاد و میزان زنده­مانی پروتوپلاست‌ها ارزیابی شد. نتایج به‌دست‌آمده از تجزیۀ آماری داده­ها، محیط Spezieller Nährstoffarmer Agar بهترین محیط کشت و زمان 16 ساعت را بهترین زمان برای ایجاد و جوانه­زنی اسپورها معرفی کرد. همچنین ترکیب آنزیمی  Glucanex (15 Mm) و Driselase (20 Mm) در زمان پنج ساعت با فاصلۀ معنی­داری از دیگر تیمارها بیشترین پروتوپلاست را تولید کرده و تثبیت‌کنندۀ KCl (6/0 مولار) نیز به عنوان بهترین تثبیت‌کننده معرفی شد.

کلیدواژه‌ها


عنوان مقاله [English]

Analysis of the effect of effective factors in protoplast preparation of Fusarium oxysporum for using in protoplast fusion

نویسندگان [English]

  • Afshin Rostami 1
  • Hossein Saremi 2
1 Ph. D. Candidate, Department of Plant Protection, Faculty of Agriculture, University of Zanjan, Zanjan, Iran
2 Professor, Department of Plant Protection, College of Agirculture & Natural Resources, University of Tehran, Karaj, Iran
چکیده [English]

Protoplast fusion as a transgenic system is an effective technique improving microorganism performances by making genetic changes. Today, exploitation of this technique has beenbeneficial in biocontrol of weeds. For filamentous fungi such as Fusarium species, protoplast preparation is the first step in the common genetic transformation process. For this objective, the Fusarium oxysporum isolates were isolated from Bromrape (Orobanche sp.) and were identified by morphological and molecular characteristics. Thereafter, the effects of various factors including medium and incubation time in spore preparation were evaluated. Furthermore, the effect of some factors on protoplast preparation and longevity including enzyme concentrations, type and concentration of osmotic stabilizers and lytic incubation times were also assessed. The results of this study demonstrated that Spezieller Nährstoffarmer Agarwas the best medium to prepare spores and the best incubation time for spore germination was 16 hours. In addition, concentrations of 15mM for Glucanex and20mM for Driselase and 5 hours incubation time were the best lytic enzyme complex and incubation time period, respectively. The KCl at the concentration of 0.6M was the best osmotic stabilizer in protoplast preparation and longevity.

کلیدواژه‌ها [English]

  • Fusarium oxysporum
  • optimization
  • Osmotic stabilizer
  • Protoplast
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