تأثیر فسفر و نیتروژن بر تولید ریشه‌های مویین در گیاه توتون (Nicotiana tobaccum) به‌عنوان یک گیاه مدل

نوع مقاله : علمی - پژوهشی

نویسندگان

1 دانش‌آموخته دکتری اصلاح نباتات، گروه مهندسی تولید و ژنتیک گیاهی، دانشکده کشاورزی، دانشگاه لرستان، خرم‌آباد، ایران

2 استاد، گروه مهندسی تولید و ژنتیک گیاهی، دانشکده کشاورزی، دانشگاه لرستان، خرم‌آباد، ایران

چکیده

چکیده
تولید ریشه مویین به کمک Agrobacterium rhizogenesیکی از مهم‌ترین روش‌های بیوتکنولوژی برای تولید متابولیت‌های ثانویه و پروتئین‌های نوترکیب است. یکی از چالش‌های مهم ریشه مویین، تولید کم زیست‌توده است. به منظوربهینه‌سازی محیط کشت ریشه مویین در گیاه توتون، تأثیر فاکتور نیتروژن با نسبت‌های مختلف نیترات به آمونیوم (NO3/NH4) در سه غلظت 19/20، 1/5/1/5و 1/5/30میلی‌مولار، میزان پتاسیم دی هیدروژن فسفات (KH2PO4) به‌عنوان منبع تأمین‌کننده فسفر(3، 6 و 12 میلی‌مولار) در محیط MS به‌صورت کشت جامد و مایع در قالب یک طرح کاملاً تصادفی در آزمایشگاه بیوتکنولوژی دانشکده کشاورزی دانشگاه لرستان در سال 1395 انجام شد. تراریخت بودن ریشه‌ها به کمک واکنش زنجیره‌ای پلیمراز (PCR) با آغازگر‌های اختصاصی ژن rolC تأیید شد. تجزیه واریانس داده‌های میزان وزن خشک و تر ریشه‌های مویین نشان داد که نیتروژن، فسفر و نوع محیط کشت روی میزان تولید زیست‌توده اثرات معنی‌دار (05/0P<) دارند. بیشترین زیست‌توده، مربوط به محیط کشت MS حاوی میزان بالای از نسبت نیترات به آمونیوم و12 میلی‌مولار پتاسیم دی هیدروژن فسفات با حداکثر وزن‌ تر و خشک بعد از یک ماه به‌ترتیب با 66/11 و 47/0 گرم بود. کمترین وزن خشک و وزن تر در محیطی با میزان کمتری (1/5/1/5 میلیمولار) از نسبت نیترات به آمونیوم و 6میلی‌مولار پتاسیم دی هیدروژن فسفات مشاهده شد. همچنین در محیط کشت MS مایع همراه با میزان بالای نسبت نیترات به آمونیوم بیشترین میزان تولید زیست‌توده حاصل شد. با توجه به اهمیت تولید بیشتر زیست‌توده، از روش القاء ریشه مویین این مطالعه می‌توان برای افزایش زیست‌توده برای تولید ترکیبات دارویی و صنعتی استفاده کرد.

کلیدواژه‌ها


عنوان مقاله [English]

The Effect of Phosphorus and Nitrogen on Hairy Roots Production in Nicotiana tobaccum as a Model Plant

نویسندگان [English]

  • Mitra Khademi 1
  • Farhad Nazarian-Firouzabadi 2
  • Ahmad ismaili 2
1 Ph.D. Graduate of Plant Breeding, Departmant of Production Engineering and Plant Genetics, Faculty of Agriculture, Lorestan University, Khorramabad, Iran
2 Professor, Departmant of Production Engineering and Plant Genetics, Faculty of Agriculture, Lorestan University, Khorramabad, Iran
چکیده [English]

Abstract
Background and Objectives
Hairy root culture obtained via Agrobacterium rhizogenes-mediated genetic transformation is considered an essential strategy for in planta enhancement of secondary metabolites/or recombinant protein production. High growth rates and genetic stability characterize hair roots compared to ordinary plant root systems. Low root biomass is one of the significant challenges in any hairy root establishment. To this end, the hairy root culture medium was supplemented with various nitrogen and phosphorus sources and combinations to increase the total biomass production.
 
Materials and Methods
Sterile leaf explants were excised from 10-day old tobacco plants and inoculated with Agrobacterium rhizogenes to induce hairy roots formation. Inoculated leaf explants were kept in dark conditions for two days at 25±2 ˚C. Leaf explants were washed and transferred to the MS culture medium supplemented with cefotaxime at sterile conditions. The inoculated explants were kept in the tissue culture room at 25±2 ˚C until hairy roots appeared. Different ratios of nitrate to ammonium (5.1/5.1, 19/20, and 30/5.1 mM) and three potassium dihydrogen phosphate (KH2PO4) as a source of phosphorus (3, 6, and 12 mM) in two types of solid and liquid culture media were used to optimize hairy roots formation and compared in a factorial experiment based on a completely randomized design with three replications, each containing ten explants. The fresh and dry weight of hairy root was recorded 30 days after hairy root formation.
 
Results
Transgenic hairy roots were confirmed by PCR analysis using the rolC gene-specific primers. No bacteria contamination was found following PCR analysis of transgenic hairy roots. Analysis of the variance of hairy roots dry and fresh weight data showed that nitrogen, phosphorus, and culture media had a significant (P<0.05) effect on hairy roots biomass production. The highest biomass accumulation (11.66 gr/FW and 0.47 gr/DW) was recorded in the media containing 30/5.1mM ratio of NO3/NH4 ratio and 12 mM of KH2PO4, respectively. The lowest dry and fresh weight were obtained when 5.1/5.1 mM of NO3/NH4 and 3 mM KH2PO4 were used.
 
Discussion
This study suggested that a higher NO3/NH4 ratio and KH2PO4 can lead to the highest hairy roots biomass production. Therefore, it is recommended to use a higher proportion of NO3/NH4 to produce more in planta biomass needed for pharmaceutical and industrial products. Furthermore, it can be concluded that it is possible to optimize the hairy root production if hairy roots are used to scale up the amount of metabolite/recombinant proteins production in tobacco.
 

کلیدواژه‌ها [English]

  • Agrobacterium rhizogenes
  • Biomass
  • Hairy root
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