دکتر هوشنگ علیزاده

Cold stress is one of the major factors limiting rice (Oryza sativa L.) productivity and yield. Two rice cultivars were chosen for this study: a japonica rice, ‘Gerde’, and an indica rice, ‘Shiroodi’, representing tolerant and sensitive cultivars, respectively. In this work, the early physiological and molecular responses of these two cultivars to 4°C cold stress were investigated. We found that cold stress caused a significant decrease in the content of chlorophyll a, chlorophyll b, and carotenoids in both rice cultivars. However, the photosynthetic pigment content in the Shiroodi cultivar decreased to a greater extent, indicating its limited genetic capacity to protect its photosynthetic machinery. During cold stress, the amounts of hydrogen peroxide and malondialdehyde produced by the Shiroodi cultivar were significantly higher than those in Gerde, suggesting a lower capacity of Shiroodi to scavenge reactive oxygen species. The higher level of beta-glucosidase gene expression in the final hours of cold treatment in the Gerde cultivar indicates the involvement of this enzyme in elevating cellular levels of active abscisic acid. Moreover, the significant increase observed in the transcript level of phenylalanine-ammonia-lyase in Gerde indicates the induction of defense responses related to the phenylpropanoid pathway. Altogether, one can conclude that the higher cold tolerance of the Gerde cultivar is multifaceted, involving various factors such as its ability to protect its photosynthetic machinery, its greater capacity for cell protection against oxidative stress, and the expression modulation of genes involved in abscisic acid metabolism and secondary metabolism.

The Valerian plant (Valeriana officinalis) contains compounds that can be used to treat nerve disorders, epilepsy, and sleep disorders. Terpenes (sesquiterpenoids) are the major group of compounds in this plant, whose biosynthesis pathway is poorly understood. Due to the lack of genomic sequence, studies based on de novo transcriptome assembly are important to elucidate more genes of the terpenoid biosynthetic pathway in this valuable medicinal plant. To reconstruct de novo assembly of RNA-Seq data from previously published SRR data for V. officinalis, Trinity and rnaSPAdes were used in this study. The primary assemblies were then analyzed using additional tools, such as CAP3 and CD-HIT-EST, in an attempt to determine the most effective de novo assembly. Based on the results of quality assessment tools such as rnaQUST, SeqKit statistics, and BUSCO, the combination of CAP3 and CD-HIT tools successfully produced an optimal assembly for Trinity (T-CAP-CD). A total of 57.78% of T-CAP-CD assembly genes were annotated in three subgroups (BP, MF, CC) with the highly accurate plant-specific Hayai-Annotation Plants annotation tool. The reconstruction of the KEGG pathway revealed 30 ortholog genes in the terpenoid biosynthetic backbone pathway and 8 ortholog genes in the sesquiterpenoid biosynthesis pathway. By performing homology search of T-CAP-CD transcriptome against PlantTFDB v.5, the most frequent transcription factor families were identified as bHLH (9.5 %), NAC (7.3 %), and MYB (6.6 %), respectively.

Early vigor, stress resistance, uniformity, high yield, and breeder right protection have turned hybrid cultivar development into the most successful breeding strategy in rapeseed (Brassica napus L.). The goals of this research were to develop the first CMS based rapeseed hybrids in Iran and to evaluate their performance and fertility. The effect of different traits on yield increasing of hybrids was also studied. Seven CMS lines were crossed with R2000 (a restorer-line) and the seed yield of hybrids was tested using a randomized complete block design with three replications in 2019-2020 and 2020-2021. Three of hybrids with 4.64, 4.40, and 4.38 tons per hectare, showed considerable yield and were significantly different than Nilufar, Neptune, and Nima (3.44, 3.37, and 3.30 tons per hectare, respectively), as check cultivars. R2000×A8 demonstrated the highest heterosis (34.71%) to high-check cultivar. All hybrids obtained were normal in terms of fertility, having no male-sterile flowers. Based on the results of principal component analysis and cluster analysis, the simultaneous increase in the number of pods per plant and the thousand seed weight, together with crossing the more distant parents will lead to more high yielding hybrids. The promising hybrids with high yield and remarkable fertility could be released as new cultivars after passing adaptability and stability experiments.

Preparation of cell suspension is an important step in the study of plant cells which is often done by the culture of friable embryogenic callus. The primary objective of this research was to produce friable callus and to determine an optimum culture medium for the induction of friable callus in maize plant. Using two basic media, Murashige-Skoog and Chu, with different concentrations of 2, 4 Dichlorophenoxyacetic acid and Benzyl adenine hormones (overall 16 different culture media), callus induction was performed on maize mature embryo explants. Selection criteria including high friable callus induction index, high callus size index, high callus fresh weight index, and low regeneration index were measured, and the mean values were compared using analysis of variance. Considering each of these selection criteria alone, different optimum media for callus induction were introduced. In order to determine one optimum medium considering all the criteria, the analytical hierarchy method was utilized. The optimum medium in this study was MS supplemented with 1 mg/L 2, 4-D wherein the induction, weight, and size indices were high, and the regeneration index was low. In order to evaluate and verify the selected medium, the explants were cultivated in in silico- selected medium and the growth curves were plotted for callus fresh and dry weights. Several successive subculturing of the callus was performed on the selected medium wherein no regeneration was observed. The results suggested the analytical hierarchy approach can be used as a reliable decision-making method for optimum-medium selection for inducing maize friable callus.

To optimize tomato (Solanum Lycopersicum) plant tissue culture condition, the effect of 13 different hormonal combinations and two explants of cotyledon and hypocotyl on the regeneration rate of three Newton, Infinity and Defines cultivars were investigated. Experiments were conducted in a factorial based on a completely randomized design with three replications. The highest shoot number (11.9 shoots per explant) was obtained for Defines cultivar on MS medium containing the hormones of TDZ=0.68, NAA=0.1milligrams per liter and cotyledon explant. Plant defense responses against environmental factors are one of the inhibitor factors of explants regeneration in plant tissue culture. Calcium ion signaling is one of the primary events and, of course, the most important event in plant defense response against pathogens, Therefore, the effect of verapamil as a calcium channel blocker was investigated on the plant defense response. For this purpose, inoculation was performed using Agrobacterium tumefaciens strain LBA4404 and based on optimized tissue culture condition. The effect of verapamil in four concentrations of 0, 50, 100 and 200 milligrams per liter was investigated in a co-culture medium. Results showed that the use of verapamil in comparison with the control sample reduced the plant defense response. Also, the use of 50 milligrams per liter of verapamil increased the amount of regeneration.

Peroxidases are used in a wide range of biotechnological processes, most of which are carried out at high temperatures and high pH levels. Since most of the commonly used peroxidases are unstable and inactive in alkaline conditions and high temprature, it is necessary to find thermoalkalophilic peroxidases for practical purposes.

In this study, extracellular production of peroxidase in the native strain Bacillus tequilensis was studied. for this purpose, Enzyme activity was evaluated using two substrates 2,4-DCP and pyrogallol in bacterial liquid culture and the effect of culture time on enzyme production, as well as the effect of parameters such as pH and temperature on enzyme activity investigated. The relative purification of the enzyme was performed using ion exchange chromatography with sephadex DEAE A50 and the kinetic parameters of enzyme activity were evaluated. In this study, kinetic parameters such as Km and Vmax were calculated.

Measurement of enzyme activity at different times of culture indicated that the highest amount of peroxidase production was obtained 72 h after bacterial culture.

The most common cause of diarrhea is Shigella and no vaccine has been found so far. IpaD and B subunit of Shiga toxin proteins (STxB) play an important role in invasion, infection and pathogenesis caused by Shigella. On the other hand, Co1 ligand has introduced as one of the antigen delivery systems to M-cells in mucosal layer. In this study, nasal administration of antigen in mice, used to evaluation and comparison of immunogenicity of IpaD, IpaD-STxB and IpaD-Co1 proteins. The coding sequences of proteins cloned in pET28a vector and transformed to bacterial strain of E.coli BL21(DE3). Recombinant protein expression confirmed by using of Western bloting technique. The each one of the recombinant protein purified by affinity chromatography column and used for nasal administration in four times consecutively on mice. After the second nasal administration (first booster), a week after each administration, blood samples were collected and ELISA performed on serum. The ELISA results of serum titration showed that the highest amount of IgG has been produced against IpaD-STxB protein; and IpaD-Co1 was in next step. The immunogenicity was evaluated using active toxin E. coli O157:H7. The challenge results showed that immunized mice could endure 5 times the LD50 Shiga toxin E. coli O157: H7; but after injection of 10 times the LD50, the immunized mice by IpaD, IpaD-Co1 and IpaD-STxB were death after 24, 24 and 72 hours, respectively. According to these results recombinant protein IpaD-STxB founded to be more immunogenic than IpaD-Co1 against shiga toxin in mice.

Small and powerful promoters play a key role in recombinant protein expression in different hosts including Pichia pastoris. In the present study, expression of cellulase beta endoglucanase CMC3 gene was studied under the control of the alcohol oxidase 1 (AOX1) and methanol oxidase (MOX) promoters, with the methanol feeding, in the yeast Pichia pastoris. To avoid the gene dosage effect, colonies harbored single copy of target constructs were screened by Real-Time PCR method. The results of enzymatic assay, zymogaphy and SDS-PAGE for the culture of single copy harboring transformants showed powerful, comparable expression of CMC3 under the control of both MOX and AOX1 promoters. High-level expression of recombinant CMC3 under control of the MOX promoter indicated that this small, new powerful promoter could be considered as a promising tool for recombinant protein production in the yeast Pichia pastoris.

Abrin is a plant-derived glycoprotein toxin composed of two polypeptide chains, A chain and B chain. The A-chain (molecular weight 30 kDa) as a N-glycosidase is able to catalyzes the deadenylation of the 28S rRNA and thereby inactivate ribosomes. On the other hand, B-chain (molecular weight 35 kDa) is responsible for binding to the target cell and internalization of A-chain. In this study, an abrin A-chain (ABRaA) DNA was isolated from Abrus precatorius by PCR technique and then cloned into a T-vector. This isolated sequence with 753 bp in length was registered to NCBI GenBank with the accession number MF573784, as the first report of A chain abrin DNA. In continue molecular structures and biochemicalcharacteristics of ABRaA were analyzed. The secondary and three dimensional analysis of the polypeptide structure with 28.07 kDa indicated that ABRaA is similar to the RIP-II family of plant proteins. The bacterial expression of ABRaA carried out in BL21 (DE3) strain and subsequently confirmed by Western blot.

Insulin is a hormone exclusively produced by pancreatic beta cells. The production of recombinant proteins in microorganisms has some disadvantages such as high cost, the possibility of contamination with toxic proteins, and costly purification steps; so, the production of recombinant proteins in plants can be investigated. Development of transient expression system based on a deleted version of Cowpea mosaic virus RNA-2, Cowpea Mosaic Virus-Hyper Translatable (CPMV-HT), has provided the extremely high-level and rapid production of proteins without viral replication. In this study, two constructions were prepared; pBI121-Proinsulin-Zera (pBI-ProZ) containing human proinsulin gene and Zera (N-terminal proline-rich domain of γ-zein) and pCAMBIA1304-Proinsulin-Extensin (pCAMBIA-ProE) containing CPMV-HT expression system for improvement of translation of human proinsulin gene and carrot extensin signal peptide. Both structures were transiently transferred in to lettuce and alfalfa leaves using Agrobacterium tumefasiens pv. C58. Statistical analysis of this study was conducted on the concentration of produced proinsulin per each gram of transgenic leaf using factorial arrangement of treatment in a complete randomized design. Gene expression was confirmed in transcription level using reverse transcription polymerase chain reaction (RT-PCR) method and in translation level using enzyme-linked immunosorbent assay (ELISA) and Dot blot assay. Protein accumulation for pCAMBIA-ProE and pBI-ProZ constructs were 6.82 and 4.32 ng/g in recombinant alfalfa leaves and 6.6 and 3.8 ng/g in recombinant lettuce leaves, respectively. Results showed that the expression of proinsulin in pCAMBIA-ProE contains CPMV-HT expression system was more than pBI-ProZ.

Atend of 2010 year, about 34 million people living with HIV. With current therapy AIDS is still not curable. Griffithsin (GRFT) is a potent anti-HIV protein that binds to HIV and inhibits cell to cell transmission of virus. In this study, first GRFT gene sequence was optimized. Then pBIgR-KD and pBIgR-ZR vector containing GRFT gene plus N termination of Zera SP ZERA and endoplasmic reticulum retention signal peptide (SEKDEL) was constructed. To obtaining transformants, young leaf explants of Nicotiana tobacco cv. Samsun were transformed with agrobacterium LBA4404 strain containing pBIgR-KD and and pBIgR-ZR. Formed shoots on MS medium containing 1 mg/l BA and 0/1 mg/l NAA and 100 mg/l kanamycin were transferred to root inducing medium (½ MS hormone free) containing 50 mg/l Kn. PCR using GRFT and 35S primers and sothern blot analysis showed succefull integration of GRFT transgene in tobacco genome. RT-PCR and real time PCR showed expression of GRFT gene at transcriptome level with high differences of expression transgenic lines. A band with corresponding size (27 KD for dimer GRFT protein) was detected based on SDS-PAGE analysis. Western blot analysis with anti-GRFT antibody confirmed production of GRFT protein as monomer (13 KD) and dimer (27 KD). Based on ELISA data, targeting to protein bodies using Zera signal peptide resulted in higher amount of recombinant GRFT in comparison to ER targeting via KDEL signal peptide. Based on our results high level of recombinant protein expression is achievable by optimizing of gene codon, selection of suitable host plant and application of suitable promoter and signal peptides.

The production of pharmaceutical compounds having role in treating diseases like AIDS is necessary. In this regard, griffithsin (GRFT) (an algae lectin) can inhibit HIV entry to the cell at picomolar concentration. To production of recombinant GRFT protein, alfalfa, soybean and lettuce were used as hosts for transient expression. Effect of KDEL, signal peptide of carrot extensin and SP subunit of ZERA signal peptide that target protein to endoplasmic reticulum, apoplastic space and space around the cell wall, respectively was studied. 72 hours after agroinfiltration, qRT- PCR analysis showed high potential of lettuce to express of GRFT at transcript level while alfalfa had lowest transcription level. ELISA result using polyclonal anti-mouse anti-GRFT antibody confirmed that targeting of GRFT to apoplast space of soybean led to highest amount of GRFT. Lettuce despite of high level of gene expression, showed low level of GRFT accumulation which may be due to posttranscriptional and post translational modifications. Our results suggest using of soybean leaf tissue for fast and reliable production of pharmaceutical using transient expression.

ransgenic plastids offer unique advantages in plant biotechnology, including high-level foreign protein expression and reducing environmental contamination risks. Here, the development of a specific plastid vector for lettuce, (Lactuca sativa L. TN-96-39), was described using of rbcl and accD as target fragments, Prrn promoter, psbA terminator and a gene cassette for antibiotic resistance. The species-specific vector for plastid transformation to Lactuca sativa, called pCL 96-39 (TN-96-39 (Iranian line) chloroplast plasmid), has been sequenced. Plastid transformation was optimized by biolistic bombardment. Transfomants were confirmed by PCR and also histochemical test for GUS protein production. Beta-glucoronidase production was confirmed by SDS-PAGE electrophoresis in transformants. This system will open up new possibilities for the efficient production of edible vaccines, pharmaceuticals, and antibodies in plants.

As regards the kind of use and production of biomass, lettuce benefits from a potential of acting as a bioreactor in producing recombinant proteins as well as edible vaccines. For the propose, one needs to optimize tissue culture and gene transformation in Lactuca sativa. Seeds of two cultivars TN-96-39 and TN-96-41 were disinfected in a solution of 2.5% sodium hypochlorite containing 0.1% Tween 20 for 25 minutes. This was followed by three rinses of sterile distilled water. The seeds were then germinated in Petri dishes on filter paper soaked with sterile distilled water. The cultures were continuously exposed to white fluorescent light at a constant temperature of 25oC, for a minimum of 16 hours. When aseptically germinated seedlings were 48 to 72 hours old, cotyledons were excised near the cotiledonary node, and cut into either 6 or 8 pieces to expand the already existing wound on the explants for callus growth. The most suitable growth regulators for callus production and embryogenesis were 0.05 mg/l NAA, and 0.2 mg/l BA. The most appropriate growth regulator for direct regeneration and proliferation was found out as 0.05 mg/l NAA, 0.4 mg/l BA. To produce roots, shoots were transferred to MS medium culturing 0.2 mg/l of NAA. Strong root bearing plantlets were planted in pots to produce seeds. Transgenic plants of lettuce cultivars (TN-96-39, TN-96-41) were produced by use of Agrobacterium tumefaciens vectors containing the β- glucuronidase (GUS) reporter gene and the nptII gene for kanamycin resistance as a selectable marker. High frequency of transformation based on kanamycin resistance and GUS expression, was obtained with 72-h-old cotyledon explants cocultivated for 48 h with Agrobacterium tumefaciens strain LBA4404 & 2 minute dive for infection at cultivar TN-96-39. Progenies of T0 and T1 plants along with PCR & assay demonstrated linked monogenic segregation for kanamycin resistance and for GUS activity.

Environmental stresses such as drought and salinity cause to reduce productivity of crops down to 50%. Harmful effects of salinity stress involve, derangement in membranes activity, increase of toxic metabolites, prevention of mineral element uptake and photosynthesis, producing Reactive oxygen species (ROS) and finally, cells and whole plant dead. In this study, effects of short term salinity stress on proteome pattern of bread wheat leaf (Triticum aestivum L.) were examined. Seeds of the two varieties of wheat; salt resistant (Roshan variety) and salt sensitive (Falat variety), planted in agreenhouse and at the tilling stage, plants were exposed to 100 and 200 mM NaCl solutions. The samplings were done after 3,6,12 and 24 h of stress treatment by removing seedling leaves. Proteins extracted from wheat leaves were separated by two-dimensional gel electrophoresis. Result of analysis using Melanie software showed that, 86 spots in resistant variety significantly different from control and in this variety 78% of proteins had up expression and 13.5% had down expression. In sensitive variety 94spots significantly different from control. In this variety 25% of proteins had up expression and 58.5% had down expression. Our results showed that the increasing of protein expression was corresponded with genes that responded to salinity.