عنوان مقاله [English]
Back ground and Objectives
Hairy root culture obtained via Agrobacterium rhizogenes-mediated genetic transformation is considered as an important strategy for in planta enhancement of secondary metabolites/or recombinant proteins production. Hair roots are characterized by high growth rates and genetic stability in comparison with the ordinary plant root systems. Low root biomass is one of the major 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 2 day at 25±2 ˚C. Leaf explants were washed and transferred to the MS culture medium supplemented with cefotaxime at sterile condition. The inoculated explants were kept in tissue culture room at 25±2 ˚C until hairy roots appeared. To optimize hairy roots formation, 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 compared in a factorial experiment based on completely randomized design with three replications, each replicate containing 10 explants. Fresh and dry weight of hairy root were recorded 30 days after hairy root formation.
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 variance of hairy roots dry and fresh weight data showed that nitrogen, phosphorus and type of culture media had a significant (P<0.05) effect on hairy roots biomass production. The highest accumulation of biomass (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.
The results of 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 ratio 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.