Study on genetic diversity of some Iranian Pistachio (Pistacia vera L.) cultivars using random amplified polymorphic DNA (RAPD), inter sequence repeat (ISSR) and simple sequence repeat (SSR) markers: A comparative study

Iran has a rich and diverse pistachio germplasm and thereby, the diversity and number of Iranian pistachio cultivars is unique in the world. In this study, 31 pistachio cultivars and genotypes were characterized by random amplified polymorphic DNA (RAPD), inter sequence repeat (ISSR) and simple sequence repeat (SSR) markers. The general dendrogram constructed using the combined data of the three sets of molecular markers was to some extent similar to those obtained separately with each marker. The overall principle coordinate analysis (PCA) based on genetic similarity matrices showed that the first three eigenvectors accounted for 28.46% of the total molecular variation. Therefore, the PCA results confirmed the results of cluster analysis .In SSR population analysis, the four primers produced 11 alleles among 31 pistachio genotypes with an average value of 2.75 alleles. 100% polymorphism was observed at all of these loci. The low average polymorphic information content value of 0.4374 indicated the presence of high genetic similarity among genotypes and entails development of additional polymorphic SSR primers for effective characterization of Iranian pistachio cultivars/genotypes. According to the effective multiplex ratio and assay efficiency index, it was shown that RAPD markers were the most powerful to differentiate the genotypes followed by ISSR and SSR markers, respectively.Keywords: Pistacia vera, genetic diversity, clustering, population parameters

TuMV as an efficient transient vector for expressing heterologous proteins in Nicotiana tabacum and N. benthamiana

Nowadays the production of recombinant proteins such as drugs and commercial protein compounds in plants is called molecular farming. It has some benefits such as fast and large quantity production of recombinant proteins with low cost. In this research, the green fluorescent protein (GFP) was transiently expressed in two tobacco species via turnip mosaic virus (TuMV) derived vector, a virus which can infect a wide range of plant species. Florescence microscopy results indicated that TuMV could infect tobacco plants and accumulate GFP protein in plant leaves. In addition, RT-PCR, Dot-Blot and ELISA assays demonstrated the recombinant gene transcription, translation and stability. This is the first report of using TuMV-based viral vectors for producing recombinant proteins in tobacco. Optimized TuMV-based viral vectors could be used for producing recombinant proteins in tobacco

Biotechnological production of recombinant tissue plasminogen activator protein (reteplase) from transplastomic tobacco cell cultures

Transplastomic plants are a system of choice for the mass production of biopharmaceuticals due to the polyploidy of the plastid genome and the low risk of pollen-mediated outcrossing because of maternal inheritance. However, as field-grown plants, they can suffer contamination by agrochemicals and fertilizers, as well as fluctuations in yield due to climatic changes and infections. Tissue-type plasminogen activator (tPA), a protein used to treat heart attacks, converts plasminogen into plasmine, which digests fibrin and induces the dissolution of fibrin clots. Recently, we obtained transplastomic tobacco plants carrying the K2S gene encoding truncated human tPA (reteplase) with improved biological activity, and confirmed the presence of the target protein in the transgenic plant leaves. Considering the advantages of plant cell cultures for biopharmaceutical production, we established a cell line derived from the K2S tobacco plants. The active form of reteplase was quantified in cultures grown in light or darkness, with production 3-fold higher in light.This work has been supported by a grant from the Spanish Ministry of Science and Innovation (BIO2014-51861-R) and the Generalitat de Catalunya (2014SGR215). Diego Hidalgo is a predoctoral fellow of Mexican CONACyT

Quantitative Genetic Analysis Reveals Potential to Genetically Improve Fruit Yield and Drought Resistance Simultaneously in Coriander

Enhancing water use efficiency of coriander (Coriandrum sativum L.) is a major focus for coriander breeding to cope with drought stress. The purpose of this study was; (a) to identify the predominant mechanism(s) of drought resistance in coriander and (b) to evaluate the genetic control mechanism(s) of traits associated with drought resistance and higher fruit yield. To reach this purpose, 15 half-diallel hybrids of coriander and their six parents were evaluated under well-watered and water deficit stressed (WDS) in both glasshouse lysimetric and field conditions. The parents were selected for their different response to water deficit stress following preliminary experiments. Results revealed that the genetic control mechanism of fruit yield is complex, variable and highly affected by environment. The mode of inheritance and nature of gene action for percent assimilate partitioned to fruits were similar to those for flowering time in both well-watered and WDS conditions. A significant negative genetic linkage was found between fruit yield and percent assimilate partitioned to root, percent assimilate partitioned to shoot, root number, root diameter, root dry mass, root volume, and early flowering. Thus, to improve fruit yield under water deficit stress, selection of low values of these traits could be used. In contrast, a significant positive genetic linkage between fruit yield and percent assimilate partitioned to fruits, leaf relative water content and chlorophyll content indicate selection for high values of these traits. These secondary or surrogate traits could be selected during early segregating generations. The early ripening parent (P1; TN-59-230) contained effective genes involved in preferred percent assimilate partitioning to fruit and drought stress resistance. In conclusion, genetic improvement of fruit yield and drought resistance could be simultaneously gained in coriander when breeding for drought resistance

Reteplase Fc-fusions produced in N. benthamiana are able to dissolve blood clots ex vivo.

Thrombolytic and fibrinolytic therapies are effective treatments to dissolve blood clots in stroke therapy. Thrombolytic drugs activate plasminogen to its cleaved form plasmin, a proteolytic enzyme that breaks the crosslinks between fibrin molecules. The FDA-approved human tissue plasminogen activator Reteplase (rPA) is a non-glycosylated protein produced in E. coli. rPA is a deletion mutant of the wild-type Alteplase that benefits from an extended plasma half-life, reduced fibrin specificity and the ability to better penetrate into blood clots. Different methods have been proposed to improve the production of rPA. Here we show for the first time the transient expression in Nicotiana benthamiana of rPA fused to the immunoglobulin fragment crystallizable (Fc) domain on an IgG1, a strategy commonly used to improve the stability of therapeutic proteins. Despite our success on the expression and purification of dimeric rPA-Fc fusions, protein instability results in high amounts of Fc-derived degradation products. We hypothesize that the "Y"- shape of dimeric Fc fusions cause steric hindrance between protein domains and leads to physical instability. Indeed, mutations of critical residues in the Fc dimerization interface allowed the expression of fully stable rPA monomeric Fc-fusions. The ability of rPA-Fc to convert plasminogen into plasmin was demonstrated by plasminogen zymography and clot lysis assay shows that rPA-Fc is able to dissolve blood clots ex vivo. Finally, we addressed concerns with the plant-specific glycosylation by modulating rPA-Fc glycosylation towards serum-like structures including α2,6-sialylated and α1,6-core fucosylated N-glycans completely devoid of plant core fucose and xylose residues

Transfer of human proinsulin gene into Cucumber (Cucumis sativus L.) via agrobacterium method

Nowadays, approximately 5.8% in adult population around the world are suffering by diabetes. It can be caused by an increase in risk factors such as being overweight. Also it has been estimated that the number of patients will be doubled in near future and the demands for insulin hormone will be growing up by 3 to 4 % annually. Therefore, it’s necessary to develop new methods for hormone production with high rate of capacity in future. By advanced technology of transgenic DNA, the transgenic plants are introduced as an attractive system for expression and production of many kinds of pharmaceutical proteins. In this study, we investigated transfer of Human Proinsulin Gene into the Cucumber (Cucumissativus L.). Transgenic cucumber could be a great prospect for future source of eatable insulin pharmaceutical drugs to be taken by patients.Agrobacterium tumefaciensstrain LBA4404 carrying proinsulin genes with CaMV 35S promoter was used for the transformation purpose. The transgenic plants were analyzed by PCR, RT-PCR, SDS-PAGE, Dot blot and Electrochemiluminescence techniques. Production of proinsulin in cucumber could be a great prospect in molecular farming of human proinsulin

Challenge towards plant recombinant protein expression: instability in nuclear and chloroplast transformation

It is crucial to maintain the stability of transgene and its expression level. It seems the transformation method and the target organ can influence this instability. To this aim, two transformation systems, Agrobacterium-mediated and particle bombardment systems which have been applied to introduce tissue plasminogen activator (tPA) into nuclear and chloroplast respectively, have been compared to determine transformation efficiency and tPA expression and stability. The presence of tPA gene in transformants has been confirmed by PCR analysis. The gene expression in nuclear transformants and homoplasmy in transplastomic plants have been assayed by ELISA and southern blot, respectively. Some of the Agrobacterium-derived transformants have shown the heritability and stability of the integrated T-DNA harboring the transgene which encodes the tissue plasminogen activator and instability of its expression in T1 generation. Using Southern blot analysis of bombardment-mediated transformants has surprisingly led to detecting the inheritability of tPA. There are several factors lead to silencing of transgene in transgenic plants which should be considered. Possible reasons for these silencing are like vector designing, methylation, copy number, and genome rearrangement

Fruit yield, fatty and essential oils content genetics in coriander

Some regions of the world suffers of drought which affects plant behavior regarding biochemical and yield responses. This study aimed to estimate the general and specific combining abilities of coriander (Coriandrum sativum L.) by analyzing its fruit yield, essential oil content (EOC) and fatty oil content (FOC). To reach this aim, 15 half-diallel hybrids and their six parents, selected for their different response to water stress in fruit yield, essential oil and fatty oil content were evaluated under well-watered, moderate water-stressed and sever water-stressed conditions in the field and in glasshouse cultivation systems. Fruit yield in the field (FYF) and glasshouse (FYG), percent of de-hulled fruit, percent of hulls, EOC, essential oil yield (EOY), de-hulled fruit fatty oil content (DFFOC), hull fatty oil content (HFOC), fatty oil content (FOC) and fatty oil yield (FOY) were examined. Water treatment (WT), genotype and genotype\ua0×\ua0WT effects were significant (P\ua0≤\ua00.01) for all measured traits. For FYF, gene action was mostly additive while dominance was more important for FYG. Genotypes gained different EOC and FOC in different WTs. Genetic control of the EOC was affected by water stress and the portion of dominance in gene action increased as water stress progressed leading to completely dominant genetic control of EOC under severe water stress. For FOC and FOY genetic control was governed by dominant and over dominant gene nature in all WTs. Parents including P, P and P were indicated as promising hybrid contributors for high EOC, DFFOC and FOY. Similar genetic control mechanisms of the EOC, EOY, FOC and FOY suggests that improvement of essential oil content and fatty oil content could be simultaneously achieved in coriander

Effects of acclimation and diapause on the thermal tolerance of the two-spotted spider mite Tetranychus urticae

International audienceThe two-spotted spider mite, Tetranychus urticae, is a worldwide pest species that overwinters as diapausing females. Cold hardening is presumed to start during diapause development to ensure the successful overwintering of this species. To address this hypothesis, we compared cold tolerance between non-diapausing and diapausing females. We measured supercooling point (SCP) and survival to acute cold stress by exposing the mites at a range of sub-zerotemperatures (from 4 to 28 °C for 2 h). The mean SCPs of non-diapausing and diapausing females were 19.6+/-0.5 and 24.7+/-0.3 °C respectively ,and freezing killed the mites. Diapausing females were significantly more cold tolerant than non-diapausing ones, with LT50 of 19.7 and 13.3 °C, respectively. Further, we also examined the effects of cold acclimation (10 d at 0 or 5 °C) in non-diapausing and diapausing females. Our findings indicated that diapause decreased SCP significantly, while cold acclimation had no effect on the SCP except for non-diapausing females that were acclimated at 5 °C. Acclimation at 5 °C enhanced survival to acute cold stress in diapausing and non-diapausing females, with LT50 of 22.0 and 17.1 °C, respectively. Altogether, our results indicate that T. urticae is a chill tolerant species, and that diapause and cold acclimation elevate cold hardiness in this species