mohammadali malboobi

RNA silencing-based antiviral breeding is a promising strategy for developing virus-resistant plants.

This study employed viral sense, anti-sense, and hairpin constructs to induce resistance against beet curly top virus (BCTV) and beet curly top Iran virus (BCTIV).

For this purpose, a 120-bp conserved sequence of Rep- and C2-BCTV and a 222-bp conserved sequence of CP-, Reg-, and MP-BCTIV were selected for construct production. The efficiency of constructs was investigated in transient expression in Nicotiana benthamiana and sugar beet plants and stable expression in N. benthamiana.

In transient expression, all designed constructs induced effective resistance to BCTV and BCTIV; the hairpin constructs were more effective against both viruses. The stability of the achieved resistance by hairpin constructs was also confirmed in the T1 generation of transgenic plants.

This study showed that employing conserved coding sequences of BCTVs leads to effective resistance against BCTVs infection. The lack of protein production from transgene and degradation of its transcript due to the gene silencing mechanism makes this method safe for biosecurity. In stable transformation, the inheritance of induced resistance against BCTVs was confirmed in the T1 generation. These advantages make this mechanism commercially useful for the production of resistant plants to viruses.

Breeding crops with a higher ability in using soil minerals is one of the biotechnology researchers’ goals. Genetic engineering methods provide considerable advances in crop breeding by transferring and creating desired traits for further production under normal or stress conditions. In these procedures, Design of efficient gene constructs is of particular importance and requires promoters with proper function to specifically express the gene of interest in the target tissue and at the appropriate time to develop desired traits such as tolerance to biotic and abiotic stresses or other aims. Specific expression of phosphate-transporter genes in the roots and their induced levels in phosphate deficiency shows the potential of this gene-family promoters utilization in transgenic plants, particularly for the use in phosphate absorption from soil. Bioinformatics analysis showed that the 1826-bp promoter fragment of AtPHT1;1 gene carries several motifs leading to root-specific expression in Arabidopsis thaliana. The expression of a secretory acid phosphatase gene, AtPAP17, as a reporter gene in rapeseed transgenic plants indicated that the AtPHT1;1 promoter retains its root-specific criteria in rapeseed such that it could be used as a regulatory region for the specific expression of desired genes in transgenic rapeseed plant roots.

Genetic engineering is a powerful technology of the present century that has revolutionized the agricultural, health, pharmaceutical and food industries worldwide. It is important to identify changes caused by transgenes that may be attributed to unintended traits in the risk assessment of genetically modified (GM) crops. Rhizomania, which is caused by beet necrotic yellow vein virus (BNYVV) infection, is considered to be a significant constraint in order to produce sugar beet worldwide. The resistance of transgenic sugar beet plants to the BNYVV was previously developed through RNA silencing by expression of hairpin RNA (hpRNA) structures. In the present study, the RNA sequencing (RNA-seq) analysis was performed in order to evaluate the transcriptional changes of an event of transgenic sugar beet plants, named 219-T3:S3-13.2 (S3), with the non-transgenic parental plants grown in virus-infected soil. The results of the present study indicate that there are only 0.9% differentially expressed genes (DEGs) at significant levels. The functional analysis shows alterations of transcription in lipids, amino acids, and carbohydrates metabolisms, cellular processes (autophagy), hormone signal transduction, and biosynthesis of secondary metabolites in the transgenic event, which are related to stress-adaption for which most of the genes were up-regulated. All in all, we conclude that the presence of the transgenes does not have substantial effects on the plant gene expression patterns. This work also indicates that RNA-seq analysis can be useful to evaluate the unintended effects and risk assessment of GM sugar beet plants.

Due to the development and increasing cultivation of genetically engineered plants as an integral part of modern agriculture, the biosafety of soil microorganisms, essential elements of soil fertility, quality, and stability, has been discussed. To reveal the effect of transgenic plants on soil bio-diversity, it is necessary to compare the microflora of transgenic and parental plants. In this study, second-generation GM sugar beet seeds rendered resistant to the propagation of Necrotic yellow vein virus of beets (BNYVV) via gene silencing in a field trial were compared to their parental plant by analyzing 16S rDNA metagenomes with the use of the Illumina MiSeq platform. Analysis of the alpha and beta diversity found some influence on bacterial communication of rhizosphere between non-transgenic and transgenic (including 211S3, 219S3, 228S3, and 231S6 cultivars) sugar beets. Based on the results of the research on alpha diversity, the transgenic 211S3 and 219 S3 cultivars showed a lower average than the control sample in the Cho, phylogenetic diversity, Cho1 bias correction, and the number of OTUs indexes; The transgenic 231S6 cultivar showed a significantly higher mean than the control sample in the Simpsons index. Also, in the study of beta diversity based on the Bray-Curtis distance algorithm, all 211S3, 219S3, 228S3, and 231S6 cultivars and control samples were positioned in one group. According to the Unweighted UniFrac distance algorithm, 219S3 and 231S6 cultivars were put together in one group, and 211S3 and 228S3 cultivars were classified into one group. A comparison of the bacterial genera showed a noteworthy reduction in relative abundance. While a few genera showed a significant decrease in terms of overall abundance, other genera that stabilize molecular nitrogen and motivate plant growth, such as Agrobacterium, Devosia, Mesorhizobium, Burkholderia, and Bradyrhizobium, showed a significant decrease compared to the control cultivar in all transgenic beets.

Despite the significant benefits of transgenic crops, there is a general concern that if these crops, like their non-transgenic counterparts, are safe for human and animal nutrition. One way to investigate the food safety of these crops is to compare the molecular properties of transgenic crops with their natural parents as well as crops resulted from conventional breeding which have a history of safe use. This research pursued to determine whether the set of changes that occurred in the two transgenic events of rhizomania-resistant sugar beet is comparable to the parental non-transgenic plants and was within the range of natural changes? Whether genetic engineering led to the generation of compounds that are not safe for humans and animals? Investigations have shown that proteomic alterations in both transgenic lines were minimal and within the range of natural variations. Metabolomic changes in a selected S6 event were very low while there were remarkable alterations in the metabolome of S3 event, although these were within the range of natural deviations too.

Phosphate fixation by chemical fertilizers, in addition to being costly, carries environmental risks. Today, fertile phosphate biofertilizer 2 contains two types of phosphate-solubilizing bacteria, decomposes insoluble phosphorus compounds and thus absorbable for the plant. The aim of this study was to evaluate the effect of fertile biofertilizers 2 Pseudomounas putida (Strain P13) and Pantoea aggilomerans (Strain P5) (only once at the beginning of the growing season) and potassium phosphate in three concentrations (0, 1.2 mM, 5 mM) (Until the end of the growing season,once a week with irrigation) on some developmental characteristics of Arabidopsis thaliana in greenhouse conditions, 15 hours of light, temperature 25 ° C, light intensity of 6000 lux was done in a randomized design. After cell-histology, slides were observed under a light microscope. The results showed a significant reduction in wood vascular density in the stems of plants treated with 5 mM and 1.2 mM potassium phosphate compared to the other two treatments. Also, 5 mM potassium phosphate increased the tunica layers and the special wall thickness of the tetrads. Cell proliferation of ovule primordium, pre-embryo, and suspensor was observed in plants treated with biofertilizer and 5 mM phosphate. Treatment with biofertilizer showed the highest average number of seeds produced. The application of fertile biofertilizer 2 in this experiment, with effects similar to potassium phosphate, increased the quantitative and qualitative yield of Arabidopsis. Biofertilizer can be effective as a safe alternative to increase soil productivity and plant growth in sustainable agriculture and minimize environmental pollution.

Phosphorus (P) is one of the most vital elements for all living organisms, which acts as a constituent of essential biomolecules and metabolic reactions. Plants to cope with phosphate (Pi) deficiency have developed different adaptive mechanisms, including induction of acid phosphatases genes, Pi transporters and release of organic acids in rhizosphere as well. Under conditions of phosphorus deficiency stress, maintaining cellular phosphorus homeostasis depends on the interactions between these components. In the present study, the relationship between gene expression pattern of three families of phosphate transporters PHT1, PHT2 and PHO1 in Arabidopsis thaliana in interaction with AtPAP26 as the dominant purple acid phosphatase among 29 identified PAPs in A. thaliana genome were studied under Pi-deficient and Pi-sufficient conditions (MS culture medium). For this purpose, three different genotypes including the wild type (Wt) plants Col-0 as the control, mutated homozygote plants with T-DNA fragment insertion (Mu26), and the homozygous overexpress plants (OE26) for AtPAP26 gene were used in all experiments. Our results showed a significant reduction in the expression level of PHT1, PHT2 and PHO1 genes, as well as the phosphatase activity in Mu26 plants compared to the Wt ones under -P condition. Under +P conditions, 58% and 42% incensement were observed in total and free phosphate content. In addition, the dry weight biomass increased 3- folds in the mutant plants compared to the wild type, respectively. On the other hand, the results showed the improvement of morphological and physiological characteristics in OE26 plants compared to Wt plants in both environmental conditions. Our result demonstrated that maintaining cellular phosphorus homeostasis in Arabidopsis plant requires the interaction of important phosphatases such as AtPAP26 with phosphate transporter in Pi stress conditions. These findings can be use in production of plants with the ability to absorb the minimum amount of phosphorus for plant growth under phosphorus stress.

Iron and zinc are two essential micro-nutrients for plant growth and development. Therefore, isolation of siderophores-producing and zinc-solubilizing rhizobacteria involved in bio-availability of these elements is of great interest.

In this study, soil samples collected from slightly alkaline soil types were screened for high levels of siderophore secretion and zinc solubilization.

Among positive colonies, three isolates, named F21A, F37 and F38, were able to secrete siderophore at high levels, ranged between 200 and 300 µM/liter. A close association was observed between siderophore production capability and growth rate as an indicator of active metabolism. Siderophore production was closely correlated with the level of zinc ion released into the medium as well. All three siderophore producing isolates were able to withstand temperature as high as 37°C, high concentration of NaCl (up to 2.5%) and a wide range of initial pH from 6 to 9 while hydrolyzing Zn compounds actively. One of the isolates, F21A, tolerated the presence of 200 mgl-1 of zinc. Biochemical and molecular characteristics are indicative that these isolates are Pseudomonas japonica. As experienced in a greenhouse experiment, inoculation with the F21A and F37 isolates significantly increase the plants height, fresh and dry weight of corn with compared to control.

These findings demonstrated that the potential of P. japonica strains as plants growth promoting rhizobacteria (PGPR) in iron and zinc deficient soils.

Overexpression of known genes encoding key phosphate (Pi)-metabolizing enzymes, such as acid phosphatases (APases), is presumed to help plants with Pi availability and absorption as they are mostly exposed to suboptimal environmental conditions for this vital element.
In this study, the overexpression effect of AtPAP26, one of the main contributors in retrieving Pi from intracellular and extracellular compounds, was evaluated from various viewes in tobacco plant.
As a heterologous expression system, the encoding cDNA sequence of AtPAP26 was transferred into tobacco plants.
A high growth rate of the transgenic lines was observed which could be due to an increased APase activity, leading to the high total phosphorus as well as the free Pi content of the transgenic plants. Interestingly, a significant increased activity of the other APases was also noticed, indicating a networking among them. These were accompanied by less branched and short primary roots and a decreased lateral root numbers grown in Pi-starvation condition compared to the wild type seedlings. Besides, a delayed germination and dwarf phenotype indicates the possible reduction in gibberellic acid biosynthesis in the transgenic lines.
Such transgenic plants are of interest not only for increased yield but also for the reduced need for chemical fertilizers and removal of excessive Pi accumulation in soils as a consequence of fertilizers’ or poultry wastes’ over-usage.

Low-phosphorus (P) stress as a key factor limiting plant growth and production is common in most agricultural soils. Most of the soil-applied phosphate will be rapidly immobilized and most of annually applied phosphate fertilizers are fixed in the soil in organic forms by adsorption, sedimentation and transformation. However, excess P application may lead to contamination of water sources by enriching of water bodies with nutrients that cause eutrophication. Thus understanding the mechanisms that are used by plants to cope with low-P stress will be supportive to develop more competent breeding and genetic engineering schemes for generating improved phosphorus efficient crops. To cope with P deficiency and maintenance of phosphate homeostasis, plants have developed different adaptive mechanisms, including alterations in root morphology, recycling of inorganic phosphate (Pi) and induction of acid phosphatases (APases). To establish these strategies, numerous genes are involved in alternative metabolism pathways that are regulated by complex Pi signaling networks. In this review, we intend to summarize current advances in research on the mechanisms of P efficient crops and its breeding strategies, with a particular emphasis on APase and root architecture roles in response to low-P stress.

Aeluropus lagopoides is a salt and drought tolerant grass from Poaceae family, distributed widely in arid regions. There is almost no information about the genetics or genome of this close relative of wheat that stands harsh conditions of deserts. The main aim of this research was to isolation and characterization of salt and drought inducible genes from A. lagopoides by Differential Display Amplified fragment length polymorphism (DD-AFLP) method. In this research A. lagopoides was grown under salt or drought conditions and after modifying the DD-AFLP method several fragments were isolated and after nomination their induction was studied by reverse northern blotting. DD-AFLP led to the improvement of a non-radioactive method for which many parameters were optimized. Having screened approximately 1600 transcript-derived fragments, 1.4 percent of them showed varied expression levels in response to high salt or drought treatments. The relative abundance of twenty one selected differentially expressed fragments was inspected by reverse northern blotting that affirmed the potential of this applied method. Sequence comparisons revealed that some of the isolated genes are involved in osmotic adjustment, regulation of transcription, cation transportation and stress responses. These data clearly show that the modified DD-AFLP method was a successful and reliable approach for the isolation of differentially expressed genes.

Feverfew (Tanacetum partheniumL. Schulz Bip.) is a medicinal herb belonging to the Asteraceae which recently raised researcher’s attention due to its medicinal value and pharmacological activities¡ especially as a migraine prophylaxis agent and also for treatment of cancer. Parthenolide has a sesquiterpene lactone structure which is most likely synthesized through the mevalonic acid (MVA) pathway. Recently¡ it has been shown that there is cross a talk between the MVA and the MEP pathways through IDP (Iosopentenyl diphosphate) exchange as a precursor for the biosynthesis of different terpenes; hence¡ parthenolide biosynthesis could be affected by the MEP pathway as well. In the present work¡ the relative gene expression of two key genes of the MVA pathway¡ including 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) and germacrene A synthase (GAS) and two key genes of the MEP pathway including 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) and hydroxy-2-methyl-2-(E)-butenyl4-diphosphate reductase (HDR) were examined in leaves derived from the vegetative or generative phases¡ also in flowers using real time PCR. The results of our study showed that expression of these genes depend on the growth stage and genotype. Moreover¡ in which in the flowers and leaves derived from vegetative phase in different genotypes only the TpGAS gene expression showed a significant difference¡ while in the leaves derived of generative phase the relative gene expression showed a significant difference for TpHMGR¡ TpGAS and TpDXR.

Azotobacter is a diazotroph bacterium reported to possess various plant growth-promoting characteristics. The aim of this study was to isolate Azotobacter strains capable of fixing nitrogen and effectively hydrolyzing both organic and inorganic Pi compounds. In this study, soil samples collected from a diverse range of slightly alkaline soil types were screened for Azotobacter isolates. The inorganic and organic phosphate solubilization potentials of twenty competent phosphate solubilizing Azotobacter isolates were assessed. Variations were noted in the solubilization potentials. Three isolates, identified as Azotobactervinelandii strains O2, O4 and O6, were able to fix atmospheric N2 effectively. The nitrogenase activity of these isolates ranged between 158.6 and 326.4 C2H4h-1vial-1 (ethylene). Bacterial growth rates and phosphate solubilization activities were measured quantitatively under various environmental conditions. A close association was evident between phosphate solubilizing ability and growth rate as an indicator of active metabolism. All three phosphate solubilizing bacteria (PSB) were able to withstand temperature as high as 45°C, high concentration of NaCl (upto 5%) and a wide range of initial pH from 5 to 10 while hydrolyzing phosphate compounds actively.Azotobactervinelandii strains O2, O4 and O6 are superior candidates for biofertilizers that may result in the reduction of chemical nitrogen and phosphate fertilizers leading to increase crop production.

World population has already exceeded 6.5 billion, out of which about 850 million (13 percent) are undernourished. With the current growth rate, the world community faces even a greater challenge of hunger and food security as the estimated the population will catch 9 billion by the year 2050 with doubled needs for food. At the same time, preservation of biodiversity, stopping deforestation and reduced environmental footprint caused by agricultural practice are the main concerns towards sustainable agriculture. The progressive adoption of genetically modified organisms (GMOs) including GM crops and trees can make a decisive contribution to improve harvest and alleviate hunger and poverty. In addition to the environmental benefits, the introduced GMOs can improve water use efficiency and reduce the need for fossil based fuels and pesticide application and reduce thousands of tons of emissions of green house gases. In this respect, several social issues are still of concern. On one hand, many beneficial advantages of GMOs have encouraged a wide spectrum of large or small farmers to cultivate transgenic plants which is translated to food security and job opportunities. On the other hand, while ideological debates have hindered, or even ceased, technology provisions in developing and underdeveloped countries, GMO seed and food productions are monopolized by a quite small number of transnational companies. For instance, seeds that were previously available at low or no cost, mainly through public institutes, international entities or seed exchange among farmers, would be offered at higher prices due to exclusive right of producers and additional cost of patent royalties. Cartagena protocol is going to regulate the relationship among countries aiming at preservation of world biodiversity. Although the developing countries outnumbered developed nations that cultivate transgenic plants in recent years, current statistic shows that around 84 percent of GMO crops are cultivated by only four countries, USA, Brazil, Argentina and Canada. Scientist and scholars, particularly in Islamic states, as well as decision makers are the major responsible bodies that must take roles for the current and future situations. Despite the fact that most of scientists or scholars are not in navigation positions, however, they can discuss socio-economic issues and raise public awareness in order to harmonize their efforts towards proper utilization of biotech products in their society and towards a reliable point for food security and safety.

Phytic acid is the dominant form of phosphorous in plant seeds. However, phytic acid cannot be utilized by animals and causes them serious phosphate deficiency. Utilization of phytase as an animal feed additive can affect liberation of phosphor and its mineral availability. In this study, heterologous expression of the A. niger phyA gene was investigated in the methylotrophic yeasts P. pastoris and H.polymorpha and its expressed active recombinant phytase biochemical and biophysical properties were studies and compared to native A.niger phytase. The phyA gene sequence of A.niger was designed and expression of synthetic genes was highly successfully in active form in both yeast hosts. Size of the secreted recombinant phytases, due to heavy glycosylation, varied from 50 to 65 KDa. Expressed extracellular recombinant phytase samples were purified and biochemical tests on properties demonstrated that both recombinant phytase samples had similar pH profiles with pH optima determinations of pH 2.5, pH.5.5 as acidphosphatase and optimum temperature of 60 and 50 °C respectively in P. pastoris and H.polymorpha. This study concludes that recombinant P. pastoris and H. polymorpha phytases could fulfil a series of predefined industrial quality criteria for application as animal feed supplement.

Phosphorus (P) is one of the most vital elements for all living organisms which acts as aconstituent of essential biomolecules such as nucleic acids, phospholipids, and phosphosugars, and as a major contributor to almost all metabolic reactions including photosynthesis, respiration, and energy delivery. It is one of the most needed nutrients for plant growth and development. Despite high levels of P in the soil, plants absorb it only in the soluble inorganic form of free phosphate ion (Pi) which is scarce in soil. Therefore, there has been a large demand for Pi fertilizers to secure crop yields, yet its deposition in soil and gradual run-off into water reservoirs lead to chains of events that cause irreversible damages to ecosystems. Researches, including genome-wide data analyses, have revealed interesting molecular aspects of plant adaptive strategies to deal with low Pi concentrations in soil. These include the higher expression of acid phosphatases and Pi transporters as well as the secretion of organic acids in the rhizosphere that maintain cellular Pi homeostasis in order to keep metabolic reactionsrunning. Describing the cycle of Pi exchange between physical and biological worlds, the extent to which current agricultural practices are disturbing the cycle, the necessity of introducing lessdestructive methods of providing Pi, and alternative measures and solutions for sustainable agriculture will be discussed in this review.

Global status of sugar beet transformation for enhanced biotic stress tolerance is reviewed. Biosafety concerns related to deliberate environmental release and commercialization of genetically modified (GM) sugar beet are discussed. Status of production of GM sugar beet in Iran is also reviewed. A case study of enhanced insect tolerance in sugar beet is presented. A cry1Ab gene under the control of two different PEPC and CaMV35 promoters was transferred to sugar beet using biolistic transformation method. Insect bioassays for T0, T1 and F1 generations against 3 different insect pests (Prodenia, Caradrina and Agrotis) were conducted. Results show significant enhanced tolerance among T0, T1 and F1 progenies against the tested insect pests in comparison to their non-transgenic counterpart.

Drought stress is a major constraint to rice (Oryza sativa) production, particularly at the reproductive stage. One of the most important reasons for the effect of drought is the inhibition of peduncle elongation, resulting in panicle retention inside the flag leaf sheath so that the unexserted spikelets remain sterile. This research was focused on studying expression patterns of OsVP1 transcription factor encoding gene during drought-stress and abscisic acid -induced growth arrest at peduncles. The expression of OsVP1 gene was significantly up-regulated during the growth arrest induced by either drought stress or ABA and down-regulated again by re-watering or ABA removal and restoring the growth in the peduncles. OsVP1 transcripts were mostly observed in the young immature tissues and the expression levels were decreased from division zone to maturation zone; however it was always increased by drought stress. In the divisional zone of the peduncle, transcripts of this gene was observed in almost all cells and accumulated more in the nuclei. Therefore, based on the achieved results and that OsVP1 encodes a transcription factor, this gene probably affects the expression of many other genes, directly or indirectly, involved in drought stress and ABA induced growth arrest.

Phosphatase (APase) enzymes including phytases have broad applications in diagnostic kits, poultry feeds, biofertilizers and plant nutrition. Because of high levels of sequence diversity among phosphatases, an efficient functional screening method is a crucial requirement for the isolation of the encoding genes. This study reports a functional cloning screening method for the isolation of APase-encoding genes from bacterial genomic libraries in a medium containing a chromogenic substrate. The method was optimized to distinguish the desired signal from the background chromosomal APase activity. This screening method led to the isolation of two novel APase-encoding genes from Pseudomonas putida with no similarities to the known genes in the databases, indicating successful implementation of the developed method.

The objective of this research is to identify Arabidopsis thaliana genes encoding acid phosphatases induced by phosphate starvation. Multiple alignments of eukaryotic acid phosphatase amino acid sequences led to the classification of these proteins into four groups including purple acid phosphatases (PAPs). Specific primers were degenerated and designed based on conserved sequences of PAPs isolated from plants, fungi and animals. RNA profiles of Pi-fed and Pi-starved A. thaliana roots were compared with two methods established for gene-family-directed differential display of pap genes. Having analyzed the differentially displayed fragments, seven pap encoding cDNA clones were isolated. One of the clones was a transsplicing product of two genes that encodes an acid phosphatase carrying a zinc-finger domain. Six other clones were predicted to encode secretory phosphatases. Reverse-northern blotting and semi-quantitative RT-PCR revealed distinct expression patterns for each gene under diverse environmental conditions such as Pi starvation, high-salt concentration, cold shock, nitrogen and sulfur deprivation. The presented data can provide some clues for dissecting the possible roles of PAPs in Pi acquisition.

Two Arabidopsis thaliana genes, psr9.2 and psr9.4 appeared to be highly similar to a phosphate-starved induced gene, psr9, isolated from Brassica nigra suspension cells. Sequence analysis classified the encoded polypeptides as members of leucine-rich repeat (LRR) proteins superfamily. The sequence of psr9 proteins comprise a unique N-terminal region encompassing a coiled-coil structure proceeding eleven LRRs along the C-terminal. Expression pattern analysis showed the responsiveness of these genes to various environmental conditions. Although both psr9 genes, psr9.2 and psr9.4, are expressed throughout the plant, the expression of psr9.2 was higher in the root whereas psr9.4 expression was prominent in the shoot. The expression levels were increased proportional to the duration of phosphate deprivation treatment. Plants exposed to cold temperature expressed both genes at high levels in both roots and shoots. In contrast, heat shock increased the expression levels of both genes in the shoots while reducing it in roots. High-salt treatment upregulated the expression of psr9 genes only in the roots. These data may suggest distinct roles for psr9 genes during plant response to various environmental conditions.Keywords: psr9; Cold stress; High salt stress; Phosphate starvation