Cell and Tissue Journal

Abstract

Abstract Aim: In this study, the effect of IAA on salt tolerance of tobacco plants (Nicotiana plumbaginifolia) was investigated under in vitro culture conditions.
Material and Methods: Sterile seeds of N. plumbaginifolia were cultured in MS medium. In vitro propagated seedlings under tissue culture conditions were treated with IAA (mg / l) and salinity (0, 100 and 200 mM) for 4 weeks. Physiological and biochemical parameters including H2O2, chlorophyll, soluble carbohydrate, protein and antioxidant enzyme activity were measured.
Results: Salt stress increased H2O2, proline, catalase, superoxide dismutase and ascorbate peroxidase, while reverse patterns were observed for photosynthetic pigments, soluble carbohydrates and also total protein.  In salt-treated seedlings, application of auxin increased proline, catalase and H2O2, but decreased photosynthetic pigmentation, soluble carbohydrates, total protein and some enzymes such as superoxide dismutase and ascorbate peroxidase.
Conclusion: Auxin treatment in salt-treated tobacco seedlings improved salinity tolerance by changes in some physiological parameters.
 
 

Abstract -
Aim: The purpose of this research was to design and prepare a suitable gene construct and transfer it to the tobacco plant and analysis of transgenic plants.
Material and methods: Seed-specific construct that containing Napin promoter, Ω sequence, GUS gene, and SAR sequence was prepared in pBI121 plasmid and proliferated in E.coli.. Then tobacco leaf explants were inoculated with LBA4404 agrobacterium strain by standard protocol. Selection of regenerated shoots also were performed in a selection media (co-culture medias + 25 mg/L Kan + 200 mg/L Cef). Transgenic plants were analyzed by PCR, RT-PCR and histochemical assay.
Results: Analysis of regenerated plantlets by using PCR and specific primers of nptII and GUS indicated that transfer of these genes to plantlets was successful. RT-PCR reaction results showed that nptII is transcribed in both tissues while GUS is transcribed only in the seed tissue. This finding was expected because Nos promoter (which controls the nptII transcription) is a constitutive and Napin is a seed specific promoter.. Expression and activity of Beta-glucuronidase enzyme in seeds of selected plants was confirmed by SDS-PAGE and histochemical assay.
Conclusion: The result of this research showed that designed gene construct was appropriate,  because Napin promotes the expression of GUS gene in the seeds and Omega sequences have also been effective in increasing of transgene expression.  In the fallowing, this construct can be used for the production of recombinant proteins by replacing valuable genes with GUS..
 

Abstract Aim: Movement of water across cellular membranes is facilitated by the presence of water channels named aquaporins. Plant plasma membrane intrinsic proteins (PIPs) fall into two groups, PIP1s and PIP2s that exhibit different water channel activities. So PIP1s are inactive, whereas PIP2s induce a marked increase in the membrane osmotic water permeability coefficient, p < /em>_f. This difference may be due to their subcellular localization. The aim of this research was to determine the subcellular localization of NtPIP1;1 and NtPIP2;1 in living tobacco cells.
Material and methods: In this experimental study, NtPIPs cDNA sequences were fused to green fluorescent protein gene and expressed transiently in tobacco mesophyll protoplasts. Then, cellular localization of aquaporins was investigated using fluorescent microscope under blue excitation wavelength.
Results: When expressed alone, NtPIP1;1 fusion protein was retained in the internal membrane structures, whereas NtPIP2;1 was found in membrane structure within the cell. To identify the internal structures containing NtPIP1;1 fusion proteins, NtPIP1;1-GFP was co-expressed in tobacco protoplasts with the YFP::HDEL protein, an ER marker. As shown, NtPIP1;1-GFP and YFP::HDEL colocalized.
Conclusion: NtPIP2;1 was transported to plasma membrane, but NtPIP1 was retained in the ER of tobacco protoplasts so showed no water transport activity.
 

Abstract Aim: Movement of water across cellular membranes is facilitated by the presence of water channels named aquaporins (AQPs). Mercurial compounds are potent blockers of plant and animal aquaporins. However some aquaporin isoforms such as NtAQP1 in tobacco are mercury-insensitive. The aim of this research was to determine the sensitivity of tobacco NtPIP2;1 to mercury.
Materials and methods: In this experimental study, after invitro transcription of NtPIP2;1 and cRNA synthesis, cRNA was injected to Xenopus oocytes by microinjection. Two days after incubation, oocytes were subjected to 1 mM mercury chloride for 10 min. Then, oocyte volume swelling assay in hypotonic medium was conducted and the membrane osmotic water permeability coefficient (Pf ) was determined.
Results: Pf for control and mercury chloride-treated oocytes expressing NtPIP2;1 was respectively calculated 0.99×10^-2 and 0.98×10^-2 cm s^-1 , which was not significant (P>0.05). Comparing the amino acid sequence of NtPIP2;1 with NtAQP1, a mercury-insensitive aquaporin, revealed the similar to  NtAQP1 replacement of cys with Thr in 233 position near the aquaporin pore in NtPIP2;1.
Conclusion: NtPIP2;1 encodes a mercury-insensitive aquaporin in tobacco which could be due to cysteine replacement with threonin near the aquaporin pore. 
 

Abstract Aim: Nicotiana tabacum contains nicotine, Which is used in cigarettes causing many health problems. Application of new technologies such as, plant tissue culture, genetic engineering in order to block the pathway of nicotin biosynthesis, and somaclinal variation  can be used as tools to produce plants with a lower level of nicotin. The aim of this research is to evaluate nicotine changes in tobacco plants mutated by T-DNA.
Material and methods: In this research transgenic plants including K4 carrying T DNA, plants carring Ri TDNA and regenerated plants from leaf and wild type plants (mother plants) were grown on MS medium containing canamycin. Leaves were harvested and dried, and extracted with hexan as internal standard. Then analysed using TLC and GC.
Results: The analysis of the extracts showed that nicotine content decreased in line k4 (containing T-DNA) and increased in Ri-TDNA (plant containing Ri-TDNA) and regenerated plants compared to non mutated plants.
Conclusion: Variation of nicotine content in mutated plants might be related to the activation or inhibition of the key enzymes involved in nicotin biosynthesis pathway.