Drying temperatures affect the qualitative-quantitative variation of aromatic profiling in Anethum graveolens L. ecotypes as an industrial-medicinal-vegetable plant.

Introduction: There are several factors that affect the quality and quantity of active ingredients and essential oil (EO) content, including pre and postharvest practices such as drying conditions. One of the most important factors in drying is temperature and then selective drying temperature (DT). In general, DT has a direct effect on the aromatic properties of Anethum graveolens. Methods: On this basis, the present study was conducted to evaluate the effects of different DTs on the aroma profile of A. graveolens ecotypes. Results and discussion: The results showed that different DTs, ecotypes, and their interaction significantly affect EO content and composition. The highest EO yield was obtained from the Parsabad ecotype (1.86%) followed by the Ardabil ecotype (1.4%), both at 40° C. More than 60 EO compounds were identified, mainly monoterpenes and sesquiterpenes, highlighting α-Phellandrene, Germacrene D, and Dill apiole as major components in all treatments. Besides α-Phellandrene, the major EO compounds at shad drying (ShD) were ß-Phellandrene and p-Cymene, while plant parts dried at 40° C showed l-Limonene and Limonene as the main constituents, and Dill apiole was detected in greater amounts in the samples dried at 60 °C. To determine the appropriate DT, simple and factorial based-ANOVA together multivariate analysis demonstrated significant differences in the compounds produced under different DTs. The results indicated that more EO compounds, mainly monoterpenes, were extracted at ShD than other DTs. On the other hand, the content and composition of sesquiterpenes increased significantly when DT was increased to 60 °C. From the genetic backgrounds point of view, the Parsabad ecotype (with 12 similar compounds) and Esfahan ecotype (with 10 similar compounds) were the most suitable ecotypes under all DTs in terms of EO compounds. Accordingly, the present study would help various industries to optimize specific DT(s) to obtain special EO compound(s) from different A. graveolens ecotypes based on commercial requirements.

Response of Satureja hortensis L. to gamma radiation and its impact on secondary metabolite content and biochemical characteristics.

PURPOSE: The current study investigated the effects of gamma irradiation on biochemical parameters and secondary metabolite accumulation in Summer Savory under field conditions. MATERIALS AND METHODS: The dry seeds of Summer Savory (with a moisture content of 12%) were exposed to gamma radiation at the doses of 20, 40, 60, 80, and 100 Gy. Non-irradiated seeds (0 Gy) were used as control. RESULTS: Our findings showed that gamma radiation at low doses (20-40 Gy) had no effect on biochemical parameters and secondary metabolites accumulation in S. hortensis. These parameters are steadily and significantly increased by raising gamma irradiation doses from 40 to 100 Gy. The highest amount of chlorophyll a and b, carotenoids, anthocyanin, and total phenolic and flavonoid content were observed in 80 and 100 Gy treatments. Plants exposed to 80 and 100 Gy treatments accumulated the maximum amounts of rosmarinic acid and caffeic acid, respectively. Furthermore, the analysis of S. hortensis essential oil revealed that gamma radiation significantly alters its components. Carvacrol, α-Pinene, and α-Thujene levels raised dramatically compared to control with an increase in gamma irradiation dose from 20 to 100 Gy, while Thymol and α-Terpinene levels lowered. CONCLUSIONS: Our results showed that treatment of Summer Savory seeds with gamma radiation at 80 and 100 Gy doses could significantly be raised biochemical parameters and secondary metabolites accumulation under field conditions. The current study showed that gamma irradiation could be used as a pre-sowing elicitor to improve the quantity and quality of phytochemicals in Summer Savory.

Genetic variation of Amaranthus retroflexus L. and Chenopodium album L. (Amaranthaceae) suggests multiple independent introductions into Iran.

Amaranthus retroflexus L. and Chenopodium album L. (Amaranthaceae) are weedy plants that cause severe ecological and economic damage. In this study, we collected DNA from three different countries and assessed genetic diversity using inter-simple sequence repeat (ISSR) markers. Our analysis shows both weed species have low genetic diversity within a population and high genetic diversity among populations, as well as a low value of gene flow among the populations. UPGMA clustering and principal coordinate analysis indicate four distinct groups for A. retroflexus L. and C. album L. exist. We detected significant isolation-by-distance for A. retroflexus L. and no significant correlation for C.album L. These conclusions are based data from 13 ISSR primers where the average percentage of polymorphism produced was 98.46% for A. retroflexus L. and 74.81% for C. album L.These data suggest that each population was independently introduced to the location from which it was sampled and these noxious weeds come armed with considerable genetic variability giving them the opportunity to manifest myriad traits that could be used to avoid management practices. Our results, albeit not definitive about this issue, do not support the native status of C. album L. in Iran.

Redroot Pigweed (Amaranthus retroflexus L.) and Lamb's Quarters (Chenopodium album L.) Populations Exhibit a High Degree of Morphological and Biochemical Diversity.

Amaranthus retroflexus L. and Chenopodium album L. are noxious weeds that have a cosmopolitan distribution. These species successfully invade and are adapted to a wide variety of diverse climates. In this paper, we evaluated the morphology and biochemistry of 16 populations of A. retroflexus L. and 17 populations of C. album L. Seeds from populations collected from Spain, France, and Iran were grown together at the experimental field of the agriculture research of University of Mohaghegh Ardabili, and a suite of morphological traits and biochemical traits were assessed. Among the populations of A. retroflexus L. and of C. album L. were observed significant differences for all the measured traits. The number of branches (BN) for A. retroflexus L. (12.22) and inflorescence length (FL; 14.34) for C. album L. were the two characteristics that exhibited the maximum coefficient of variation. Principal component analysis of these data identified four principal components for each species that explained 83.54 (A. retroflexus L.) and 88.98 (C. album L.) of the total variation. A dendrogram based on unweighted neighbor-joining method clustered all the A. retroflexus L. and C. album L. into two main clusters and four sub-clusters. Canonical correlation analysis (CCA) was used to evaluate relationships between climate classification of origin and traits. Similarly, the measured characteristics did not group along Köppen climate classification. Both analyses support the conclusion that A. retroflexus L. and C. album L. exhibit high levels of diversity despite similar environmental histories. Both species also exhibit a high diversity of the measured biochemical compounds indicating that they exhibit different metabolic profiles even when grown concurrently and sympatrically. Several of the biochemical constituents identified in our study could serve as effective indices for indirect selection of stresses resistance/tolerance of A. retroflexus L. and C. album L. The diversity of the morphological and biochemical traits observed among these populations illustrates how the unique selection pressures faced by each population can alter the biology of these plants. This understanding provides new insights to how these invasive plant species successfully colonize diverse ecosystems and suggests methods for their management under novel and changing environmental conditions.

Piriformospora indica based elicitation for overproduction of phenolic compounds by hairy root cultures of Ficus carica.

Ficus carica L. is an important source of phenolic and flavonoid compounds with valuable pharmaceutical application across various diseases. The current study was carried out to investigate the influence of Piriformospora indica elicitation on growth, production of phenolic compounds, antioxidant capacity, and expression level of flavonoid biosynthetic pathway genes in hairy root (HR) cultures of F. carica. The maximum improvement in accumulation of phenolic compounds was observed when HR culture of Ficus carica L. was exposed to 2% culture filtrate of P. indica for 72 h: gallic acid (80.5- fold), caffeic acid (26.2-fold), coumaric acid (4.5-fold), and cinnamic acid (60.1-fold), apigenin (27.6-fold) and rutin (5.7-fold). While the highest levels of chlorogenic acid (4.9-fold) and quercetin flavonoid (8.8-fold) were obtained after 48 h elicitation with culture filtrate and cell extract of P. indica at 6% (v/v), respectively. The analysis of biosynthetic genes revealed that the exposure to fungal elicitors resulted in up-regulation of phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), UDP-glucose flavonoid 3-O-glucosyltransferase (UFGT) and MYB3 transcription factor. This study shows the potential of P. indica as an efficacious elicitor for enhancing the secondary metabolites production by F. carica HRs.

Agrobacterium rhizogenes mediated transformation of Ficus carica L. for the efficient production of secondary metabolites.

BACKGROUND: Ficus carica L., an ancient source of food and medicines, is rich in valuable nutritional and secondary compounds with antioxidant, antimicrobial, and anticancer effects. The present study is the first attempt to examine hairy root (HR) induction of F. carica (Sabz and Siah) by inoculating the 3-week-old shoots and leaves with different strains of Agrobacterium rhizogenes and also to investigate methyl jasmonate (MeJA) elicitation of HRs to produce a fast and high-yield production method for secondary metabolites. RESULTS: The maximum transformation rate (100%) was achieved by inoculating the shoots with Agrobacterium rhizogenes strain A7. Siah HRs elicited with 100 and 200 µmol L-1 MeJA and Sabz HRs with 100 µmol L-1 MeJA showed the highest total phenolic content. The highest flavonoid content was 3.935 mg QE g-1 DW in Siah HRs treated with 200 µmol L-1 MeJA and 2.762 mg QE g-1 DW in Sabz HRs treated with 300 µmol L-1 MeJA. The 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity and ferric reducing antioxidant power (FRAP) value of HRs were affected by MeJA treatments. Methyl jasmonate elicitation also significantly enhanced the content of six phenolic acids (gallic acid, caffeic acid, chlorogenic acid, coumaric acid, rosmarinic acid, and cinnamic acid) and three flavonoids (rutin, quercetin, and apigenin). Thymol, a monoterpene phenol, was the main HR compound detected in gas chromatography mass spectrometry (GC-MS) analysis of the essential oils. CONCLUSION: Induction of HRs and elicitation of F. carica HRs by MeJA resulted in a significant increase in the production of important phenolic compounds and a significant increase in antioxidant capacity. © 2020 Society of Chemical Industry.

Root and shoot parts of strawberry: factories for production of functional human pro-insulin.

Diabetes, a disease caused by excessive blood sugar, is caused by the lack of insulin. For commercial production, insulin is made in bacteria or yeast by protein recombinant technology. The focus of this research is evaluating another resource and producing of recombinant insulin protein in as strawberry as this plant has high potential in production of pharmaceutical proteins. Strawberry is a suitable bioreactor for production of recombinant proteins especially edible vaccines. In this research, human pro-insulin gene was cloned in pCAMBIA1304 vector under CaMV35S promoter and NOS terminator. Agrobacterium tumefaciens LBA4404, AGL1, EHA105, EHA101, C58, C58 (pGV2260) and C58 (pGV3101) strains were used for transformation of pro-insulin gene into strawberry cv. Camarosa, Selva, Sarian Hybrid, Pajaro, Paros, Gaviota, Alpine. Additionally, Agrobacterium rhizogenes K599, R1000, A4 and MSU440 strains were utilized for gene transformation into hairy roots. PCR analysis indicated the presence of transformed human pro-insulin gene in the strawberry and hairy roots. Also, its transcription was confirmed using RT-PCR. Furthermore, the analysis of plants, fruits and hairy roots at the level of proteins using dot blot, ELISA, SDS-PAGE and ECL tests re-confirmed the expression of this protein in the transgenic plants as well as hairy roots. Protein purification of human pro-insulin from transgenic tissues was performed using affinity chromatography. Finally, the bioassay of recombinant pro-insulin was performed. The analysis of second generations of transgenic plants (T1) at DNA and protein levels was also performed as a complementary experiment. This study opens a new avenue in molecular farming of human pro-insulin through its mass production in roots and shoots of strawberry.

Dicistronic expression of human proinsulin-protein A fusion in tobacco chloroplast.

Different expression systems such as bacteria and mammalian cells have been used to produce pharmaceutical proteins. In recent years, the use of plants as bioreactors offers efficient and economical systems in recombinant protein production. Furthermore, because of the large number of plastid copies in plants, chloroplast engineering functions as an effective method to increase recombinant protein expression. Because the commercially available insulin for treatment does not contain C-peptide, which is of great importance for type 1 diabetic patients, the current study introduces the human proinsulin gene fused with protein A into the tobacco chloroplast genome using the biolistic method. To achieve homoplasmy, three rounds of selection and regeneration of transforming cells were performed on the medium that contained spectinomycin antibiotic and hormones. The PCR analysis indicated the presence of the proinsulin gene in transplastomic plants. The reverse-transcription PCR analysis confirmed the expression of the proinsulin-protein A fusion at the transcription level. Immunoblot assays of leaf-derived protein extracts confirmed that the target gene expression is up to 0.2% of the total soluble protein. Our study showed that protein A fusion is not as efficient as other reported fusions. The transplastomic plants were also confirmed for homoplasmy using Southern blot analysis.

Cloning, Transformation and Expression of Proinsulin Gene in Tomato (Lycopersicum esculentum Mill.).

BACKGROUND: Plants are among promising and suitable platform systems for production of recombinant biopharmaceutical proteins due to several features such as safety, no need for fermentation, inexpensive investment, and fast and easy scale-up. Human insulin is one of the most widely used medicines in the world. Up to now different expression systems including Escherichia coli, yeast and CHO have been exploited for producing recombinant human insulin and a variety of different recombinant insulin are extensively used. OBJECTIVES: This study reports on the transformation and expression of proinsulin gene in tomato plants for the first time in Iran. MATERIALS AND METHODS: This study reports the cloning, transformation and expression of proinsulin gene in tomato plants. Specific primers were designed and used for PCR amplification and cloning of the proinsulin gene in the plant expression vector pCAMBIA1304. The recombinant construct was transferred into Agrobacterium tumefaciens strain LBA4404, and used for Agrobacterium mediated stable transformation of tomato plants. Presence of the desired gene in transgenic lines was confirmed through colony PCR and sequencing. The expression of the protein in transgenic lines was confirmed by immunodot blot assay. RESULTS: The presence of the proinsulin gene in the genomic DNA of transgenic tomato was confirmed by PCR. Also total protein of transgenic tomato was extracted and the expression of proinsulin was detected using dotblot assay. CONCLUSIONS: This survey addresses the possibility of proinsulin gene transfer and expression in tomato transgenic lines. This study can be used as a basis for future researches to produce human proinsulin in tomato and other candidate plants.