Maria Beihaghi; Abdolreza Bagheri; Seyyed Hassan Marashi; Mojtaba Sankian; Afsaneh sadat Farsad
Abstract
Introduction: Plant tissue culture is a collection of techniques used to maintain or grow plant cells, tissues or organs under sterile conditions on a nutrient culture medium of known composition and widely used to produce clones of a plant in a method known as micropropagation. Plant research often ...
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Introduction: Plant tissue culture is a collection of techniques used to maintain or grow plant cells, tissues or organs under sterile conditions on a nutrient culture medium of known composition and widely used to produce clones of a plant in a method known as micropropagation. Plant research often involves growing new plants in a controlled environment. These may be plants that we have genetically altered in some way or may be plants of which we need many copies all exactly alike. These things can be accomplished through tissue culture of small tissue pieces from the plant of interest. These small pieces may come from a single mother plant or they may be the result of genetic transformation of single plant cells which are then encouraged to grow and to ultimately develop into a whole plant. Tissue culture techniques are often used for commercial production of plants as well as for plant research. Tobacco (Nicotiana tabacum L.) is one of the most important model plants used in the physiologic, genetic and tissue culture studies. The manipulation of tobacco genetic structure requires an efficient technique of gene transferring and regeneration. Whereas, the tobacco plant is a very effective bioreactor in the production of recombinant proteins, in this research we optimized the best tissue culture system and also, genetic transformation process of this plant.
Materials and Methods: Our plant tissue culture protocols, Include helpful information for Murashige and Skoog media, plant growth regulators, plant growth hormones, plant transformation systems, and other products for plant tissue culture. For this purpose, different concentrations of sucrose and 4 combinations of growth regulators (BAP and NAA) on callus induction, direct shoot regeneration and rooting were examined in a factorial experiment based on completely randomized design with 3 replications. The sensitivity of tobacco explants to kanamycin was examined through the cultivation of them on the selective medium with different concentrations of antibiotic. For genetic transformation, agrobacterium tumifacious (GV3101) harboring plasmid pBI121 was used and the transgenic plants were confirmed by PCR analysis.
Results and Discussion: The results of variance analysis and the means comparison showed that the best medium for callus induction was M1 (0.1 mg/l NAA and 1 mg/l BAP) with 15 g/l sucrose in the leaf explants, while the most direct shoot regeneration rate was obtained on the M1 medium with 30 g/l sucrose concentration. High-frequency of rooting was also influenced by 0/1 mg/l NAA and 60 g/l sucrose. So, supplementing the medium with NAA and BAP at different concentrations facilitated induction of multiple shoots from explants. NAA was proved to be the best and the number of shoots increased with increase in the concentration up to (0.1 mg/l), and exceeding this concentration resulted in decline in percent response as well as number of shoots was recorded shoot regeneration. The concentration of BAP was further increased a linear increase in the number of shoots was observed up to an optimal level (1 mg/l). Beyond the optimal concentration (1 mg/l), a decrease in the response as well as number of shoots was recorded due to profuse basal callusing. The effect of cytokinins on multiple shoot regeneration, higher concentrations of NAA found to be inhibitory for shoot regeneration because of huge callusing which hampered the growth and development of new shoots. Also different concentrations of sucrose have a different effect on the shoots and callus. The concentration of sucrose had significant effect on direct shoot regeneration. The main effect of sucrose concentration, concentration of 30 grams per liter, compared with a concentration of 15 grams per liter had the highest direct shoot regeneration. Concentration of 50 mg/l kanamycin could completely prevent the regeneration of untransformed explants so was used in the selective culture medium. Subsequently, the presence of nptII gene (798 bp) in the transgenic plants was confirmed and the transformation efficiency obtained by using the agrobacterium-mediated transformation was more than 95%.
Conclusions In present research, an efficient in vitro regeneration protocol has been developed for tobacco, where different factors including the age of the explant and plant growth regulators were optimized for maximum propagation of tobacco. The results showed that regeneration and transformation method described here is highly efficient and fast for the introduction of any foreign gene directly in tobacco plant.
Mohsen Mahmoodnia; Mohammad Farsi; Seyyed Hassan Marashi; Parviz Ebadi
Abstract
Investigation of the cultivated tomato plant as a plant ideal system along with the drought resistant wild species can be useful to a better understanding of the mechanisms of drought resistance and improvement of tomato plants. To investigate the effect of drought stress on leaf Relative Water Content ...
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Investigation of the cultivated tomato plant as a plant ideal system along with the drought resistant wild species can be useful to a better understanding of the mechanisms of drought resistance and improvement of tomato plants. To investigate the effect of drought stress on leaf Relative Water Content (RWC), electrolyte leakage and photosynthetic parameters in four species of tomato (a cultivated species and three wild species) at two levels of irrigation (field capacity and 40% field capacity) and four time periods (before stress, 10 days after stress, 20 days after stress and recovery) a factorial experiment based on Completely Randomized Design (CRD) was used. Among the tested species, cultivated species showed the highest decrease in RWC. Electrolyte leakage was significantly increased in stress conditions. The rate of photosynthesis and chlorophyll fluorescence yield in the two drought resistant wild species increased in stress conditions unlike of cultivated species, probably because of more adaptation of these species with low water conditions. The results showed that each of these species according to their own mechanism for dealing with drought stress, in one or more properties are superior to other species. it is therefore suggested that the different species for future studies (molecular) with different characteristics to be used.
Somayeh Heydari; Seyyed Hassan Marashi; Mohammad Farsi; Amin Mirshamsi
Abstract
Abstract
Seedless barberry (Berberis vulgaris L. var. asperma) is one of the few crops that is cultured only in Iran and southern parts of Khorasan provinces. The Origin of this variety is unknown and there has not been any study aiming to identify phylogenic relationships of this plant with other species ...
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Abstract
Seedless barberry (Berberis vulgaris L. var. asperma) is one of the few crops that is cultured only in Iran and southern parts of Khorasan provinces. The Origin of this variety is unknown and there has not been any study aiming to identify phylogenic relationships of this plant with other species existing in Iran. In this study, AFLP markers based on four primer combinations (EcoRI/Tru1I) were used to evaluate genetic variation and Phylogenic relationship among 30 different samples of wild and cultured barberry belonging to Khorasan provinces namely: Shomali (north), Razavi and Jonubi (south), together with 2 species of ornamental barberry and one sample Mahonia aquifolium. Data resulted from cluster analysis, showed that these two genera (Berberis and Mahonia) form 2 completely distinct groups with a significant genetic distance. These results can clarify the ambiguity of separation procedure between Mahonia and Berberis genera. Heterozygosity index, Principal co-ordinates analysis (PCoA), Fst Index and analysis of molecular variance (AMOVA) revealed significant difference among wild barberry populations existing in Khorasan provinces; so that, as was expected, observed variation within cultured barberry population was very low and near zero. The results also showed that Berberis integerrima is the predominant species in Khorasan provinces. Therefore further molecular and morphological investigations aiming better understanding of the relationships between species and genera of Berberis family looks necessary.
Key words: Berberis, AFLP, Genetic diversity
Bahman Hosseini; Haleh Hashemi; Farajollah Shahriari; Seyyed Hassan Marashi
Abstract
Abstract
Benzylisoquinoline alkaloids are a diverse group of nitrogenous compounds which is found in about 20% of plant species. Isolation of effective genes involved in morphine biosynthesis of opium poppy is very important in the production of specific which can be achieved using metabolic engineering ...
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Abstract
Benzylisoquinoline alkaloids are a diverse group of nitrogenous compounds which is found in about 20% of plant species. Isolation of effective genes involved in morphine biosynthesis of opium poppy is very important in the production of specific which can be achieved using metabolic engineering techniques. In this biosynthesis pathway, the key enzyme SAT is Involved in the conversion of salutaridinol 7-O-acetyltransferase (EC 2.3.1.150) to salutaridinol-7-O-acetate, which is the immediate precursor of thebaine. In this project, the gene encoding this enzyme was isolated using primers which were designed on the basis of the gene sequence available on data banks (NCBI) for papaver somniferum. This gene IS then cloned in expression vectors under the Control of Camv 355 promoter. The result of this cloning was confirmed using different molecular methods such as enzyme digestion and PCR. Agro infiltration method was also used for transient expression of SAT gene. The result of evaluation showed that morphine and codeine were only Produced in the leaves of transgenic plants containing SAT transgen.
Keywords: Benzylisoquinoline alkaloids, Salutaridinol 7-O-acetyltransferase, opium poppy
