تأثير فسفر و نيتروژن بر توليد ريشه‌هاي مويين در گياه توتون (Nicotiana tobaccum) به‌عنوان يك گياه مدل

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

توليد ريشه مويين به كمك Agrobacterium rhizogenes يكي از مهم‌ترين روش‌هاي بيوتكنولوژي براي توليد متابوليت‌هاي ثانويه و پروتئين‌هاي نوتركيب است. يكي از چالش‌هاي مهم ريشه مويين، توليد كم زيست‌توده است. به منظوربهينه‌سازي محيط كشت ريشه مويين در گياه توتون، تأثير فاكتور نيتروژن با نسبت‌هاي مختلف نيترات به آمونيوم (NO3/NH4) در سه غلظت 20/19، 1/5/1/5و 1/5/30ميلي‌مولار، ميزان پتاسيم دي هيدروژن فسفات (KH2PO4) به‌عنوان منبع تأمين‌كننده فسفر(3، 6 و 12 ميلي‌مولار) در محيط MS به‌صورت كشت جامد و مايع در قالب يك طرح كاملاً تصادفي در آزمايشگاه بيوتكنولوژي دانشكده كشاورزي دانشگاه لرستان در سال 1395 انجام شد. تراريخت بودن ريشه‌ها به كمك واكنش زنجيره‌اي پليمراز (PCR) با آغازگر‌هاي اختصاصي ژن rolC تأييد شد. تجزيه واريانس داده‌هاي ميزان وزن خشك و تر ريشه‌هاي مويين نشان داد كه نيتروژن، فسفر و نوع محيط كشت روي ميزان توليد زيست‌توده اثرات معني‌دار (P<0.05) دارند. بيشترين زيست‌توده، مربوط به محيط كشت MS حاوي ميزان بالاي از نسبت نيترات به آمونيوم و12 ميلي‌مولار پتاسيم دي هيدروژن فسفات با حداكثر وزن‌ تر و خشك بعد از يك ماه به‌ترتيب با 11/66 و 0/47 گرم بود. كمترين وزن خشك و وزن تر در محيطي با ميزان كمتري (1/5/1/5 ميلي‌مولار) از نسبت نيترات به آمونيوم و 6ميلي‌مولار پتاسيم دي هيدروژن فسفات مشاهده شد. همچنين در محيط كشت MS مايع همراه با ميزان بالاي نسبت نيترات به آمونيوم بيشترين ميزان توليد زيست‌توده حاصل شد. با توجه به اهميت توليد بيشتر زيست‌توده، از روش القاء ريشه مويين اين مطالعه مي‌توان براي افزايش زيست‌توده براي توليد تركيبات دارويي و صنعتي استفاده كرد.

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.