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Volume 9, Issue 4 (Number 4 - 2007)
Abstract
Agrobacterium rhizogenes causes hairy root disease in plants. These hairy roots are ge-netically stable and grow rapidly. Transformed hairy roots of Hyoscyamus muticus in-duced by the bacterium can produce tropane alkaloids in trace amounts of intact plant tissues. In this research, in order to compare growth and biosynthetic stability of hairy roots with wild type ones, leaf and nodal segments of the plant were inoculated with the A. rhizogenes strains A4 and LBA9402. When hairy roots appeared, both the wild type and transformed roots were cultured in a liquid B5 medium. The amounts of tropane alka-loids in both the wild type and transformed roots were measured using HPLC. The growth rates of transformed roots were stable and their dry weights were up to four times higher than those of wild type roots. Alkaloid content was the same for both transformed roots and wild type ones. However, when hairy roots underwent an autonomous dediffer-entiation and produced callus, their hyoscyamine content decreased considerably, whereas not only did their scopolamine content, not decrease, but in some cases it in-creased dramatically. A callus producing hairy root clone, produced 2.72 mg/g scopola-mine which is a significantly high record for hairy roots of H. muticus.
M. Farsi , M. Mirzaei , J. Zolala ,
Volume 9, Issue 4 (Fall 2018)
Abstract
Aims: The production of recombinant proteins in transgenic plants (molecular farming) is considered a functional aspect of genetic engineering. Unlike animal and bacterial cell-based production systems, proteins produced by plants are very safe and have low production costs due to the absence of common pathogens in humans and animals. The aim of this study was the transient expression of recombinant PARS II endonuclease enzyme using agroinfiltration in tobacco.
Materials and Methods: In this experimental study, the possibility of producing a recombinant form of PARS II endonuclease was investigated, using transient expression system via Agrobacterium. The pBI-Pars expression construct (based on the binary vector pBI121) containing the full sequence of the PARS II encoder, upstream kozak, and a downstream 8x-His tag sequence, was infiltrated into Nicotiana tabacum leaves with Agroinfiltration method. After 72 hours, the expression of PARS II gene in agroinfiltrated leave samples was confirmed through Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) and protein Dot-blot, using Anti-His antibody at the levels of mRNA and protein, respectively.
Findings: The accuracy of the constructed expression construct was confirmed, and the results of Dot-blot by Anti-His antibody confirmed the expression of the recombinant PARS II protein, while no recombinant protein expression was observed in agroinfiltrated control plants with pBI121 construct. Significant amounts of recombinant PARS II nucleases were produced in tobacco leaves.
Conclusion: Agroinfiltration is an effective and short-term method for mass production of pure recombinant PARS II nucleases in tobacco.
