Rapeseed Transformation with aroA Bacterial Gene Containing P101S Mutation Confers Glyphosate Resistance.

Field weed infestations can cause serious problems and require regular and planned programs to control them. Glyphosate is a broad-spectrum herbicide that inactivates the 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) enzyme and causes plant death. It has been reported that the mutation of proline 101 to serine in EPSPS is one of the effective mutations to reduce the affinity of glyphosate to EPSPS enzyme. In this study, we investigated the effect of the bacterial P101S mutant aromatic acid (aroA) gene on glyphosate resistance in transgenic rapeseeds. For this purpose, the mutant gene was synthesized and cloned into the pUC18 and pBI121 vectors. The gene was transferred to rapeseed by the Agrobacterium-mediated method. In this experiment, three generations of transgenic plants (T0, T1, and T2) were studied under in vitro and in vivo conditions. After the treatment of 75 putative transgenic plants with 10 mM glyphosate in T0 generation, resistant plants were identified and their seeds were harvested. In the T1 generation, out of 200 cultivated plants, 141 showed resistance. After the plants were treated with herbicides and resistance was determined, the seeds were harvested when they mature. In the T2 generation, most plants (162 plants of 200) were resistant to glyphosate. Therefore, the inheritance of resistance followed Mendel's first law, which is a sign of the monogenic character of resistance. Purification and increasing the percentage of resistant plants will be carried out in the next generations. It is concluded that P101S mutation guarantees glyphosate resistance of rapeseed and is recommended to study it in other plants.

Impact of priming on seed germination, seedling growth and gene expression in common vetch under salinity stress.

Salinity is one of the most important abiotic stress factors that is expanding its influence because of global climate change and global warming. It causes gene expression changes, a reduction in seed germination and related characteristics, and poor seedling establishment in many crop plants by creating a lower osmotic potential in the seedbed and/or toxic ion effects in germinated seeds. In recent years, seed priming has been considered a promising strategy in modern stress management to protect plants against stress conditions. This study was conducted to elucidate the effects of osmopriming with polyethylene glycol 6000 (PEG-6000) on seed germination, seedling growth and gene expression in the common vetch (Vicia sativa L.) in different saline conditions. Common vetch seeds were primed with PEG-6000 solutions having different osmotic potentials (0.00, -0.50,  -0.75, -1.00, -1.25, and -1.50 MPa) for 12 hours. Control (un-primed) and primed seeds were germinated and seedlings were grown in different saline conditions (EC= zero, 4, 8 and 16 dS m-1). Furthermore, gene expression was compared in the primed seedlings in two different osmotic potentials (0.00 and -1.50 MPa) by microarray technology. Results demonstrated that germination percentage of common vetch seeds and seedling growth were diminished by high salinity. However, several priming treatments alleviated the adverse effects of high salinity on germination and early seedling growth of common vetch. The microarray showed that the expression of many genes in both stress and normal conditions was not significantly different.

In vitro antioxidant assessment, screening of enzyme inhibitory activities of methanol and water extracts and gene expression in Hypericum lydium.

Hypericum lydium Boiss. is a perennial plant of the Hypericaceae family, which has been used in particular to treat depression. The aim of this study was to determine in vitro antioxidant, antimicrobial activities, anticholinesterase (acetylcholinesterase (AChE)/butyrylcholinesterase (BChE)), antidiabetic activities (α-glucosidase/α-amylase) and Tyrosinase inhibitor activity of methanol and water extracts of H. lydium. Also, gene expression has been evaluated in the shoot and root by microarray technology. So, in general, the purpose of this study is to study the active molecules such as antioxidant, antimicrobial, antidiabetic, enzymes and genes in the plant, which is the first to be reported. The experiments were conducted in a completely randomized design with three replications. In addition, gene expression was compared in the shoot and root parts. Expression profiling was carried out by microarrays. According to the results, the highest chemical components were determined in methanol extract rather than water extract. There was a difference between the obtained components. While the highest antioxidant activity was determined from the methanol extract of plant herbs for DPPH Free Radical Scavenging Activity, antioxidant activity was the same in both methanol and water extracts using the ABTS method. The methanol extract demonstrated stronger anticholinesterase (AChE and BChE) and α-amylase inhibition activity. This study was complemented by the detection of antioxidant activity and some enzyme inhibition activity in the methanol extract. Microarray showed 10,784 genes had significantly different expression in root and shoot. There was a positive effect of methanol extract in respect of different activities compared to the water extract. Gene expression showed that the number of expressed genes in the root was greater than the shoot.

A simplified and optimized protocol for total DNA extraction from insect species: applicable for studying genetic diversity and PCR-based specimen identification via partial amplification of cytochrome oxidase I (COI) gene.

The efficient DNA extraction from insects has been suggested as a critical and main step affecting molecular entomology for taxonomic identification, the establishment of DNA barcoding library and analysis of genetic diversity relationship between insect populations. For successfully apply these molecular techniques, high-quantity and high-quality of the extracted DNA are required. Several protocols for efficient genomic DNA extraction from insects have been developed. In this research, we represent a rapid, reliable and cost-effective method that it is not reliant on poisonous and enzymatic reagents for DNA extraction from insect tissues. Results showed that high quantity and high-quality of the isolated DNA by this method is suitable and can be used directly for PCR, also is enough to do hundreds of molecular reactions. In conclusion, we described a fast, cost-effective, non-toxic and enzyme-free protocol for high yield genomic DNA extraction from green Lacewings (Chrysoperla carnea) tissues in basic equipment laboratories.

Effects of various glutamine concentrations on gene expression and steviol glycosides accumulation in Stevia rebaudiana Bertoni.

Stevia rebaudiana Bertoni is one of the most important biologically sourced and low-calorie sweeteners that contains a lots of Steviol glycosides. Tissue culture is the best method for propagation of stevia and micro nutrients can affect both morphological traits and steviol glycosides production. In the present study, we investigated the effect of different concentrations of glutamine (10, 20, 30 and 40 g/l) on expression of UGT74G1 and UGT76G1 genes and stevioside and rebaudioside A accumulation in the leaves of stevia under in vitro conditions. The highest level of expression for UGT74G1 (1.000 Total lab unit) was seen at plants grown in MS media without glutamine and the highest gene expression level for UGT76G1 (1.321 Total lab unit) was observed at plants grown in 2% glutamine. Based on HPLC results, the highest amount of stevioside (22.74) was accumulated in plants which were under 3% glutamine treatment and the lowest production level of stevioside (16.19) was resulted under MS (0 glutamine) medium. The highest rebaudioside A (12.19) accumulation was observed under 2% glutamine treatment and the lowest accumulation of rebaudioside A (8.41) was seen at plants grown in MS medium.

Effects of life cycle and leaves location on gene expression and glycoside biosynthesis pathway in Stevia rebaudiana Bertoni.

Stevia rebaudiana Bertoni is One of the most important biologically sourced and low-calorie sweeteners that known as "Sweet Weed". It contains steviol glycosides that they are about 200-300 times sweeter than sucrose. Tissue culture is the best method with high efficiency that can overcome to problems of traditional methods, and it is the most useful tools for studying stress tolerance mechanisms under in vitro conditions to obtain drought tolerance. In the present research, we investigated the impact of life cycle, leaves location and the harvesting time on expression of UGT74G1 and UGT76G1 as well as steviol glycosides accumulation. The highest gene expression of both UGT74G1 and UGT76G1 (207.677 and 208.396 Total Lab unit, respectively) was observed in young leaves in the second vegetative year. Also, the highest amount of stevioside accumulation (13.04) was due to the old leaves in vegetative stage which had significant differences with other effects whereas the lowest accumulation (7.47) was seen at young leaves at vegetative stage. Interestingly, the highest level of rebaudioside a production (15.74) was occurred at the young leaves at vegetative stage. There was significant differences between life cycle and leaves location on steviol glycoside production in stevia.

Effects of different concentrations of mannitol on gene expression in Stevia rebaudiana Bertoni.

Stevia rebaudiana Bertoni is one of the most important herbal sweetener plants from Asteracea family that have a lot of Steviol glycosides. Among different methods, tissue culture is the best way with high efficiency that is useful for studying stress tolerance mechanisms to obtain drought tolerance of stevia. For this purpose, different concentrations of mannitol (0, 10, 20, 30, 40, 50 mg/l) were used as various treatments in the culture medium of stevia. According to the results, the highest level of UGT85C2 gene expression (1.181 Total lab unit) was seen in plants grown under 30 mg/l mannitol treatment and the lowest level of this gene expression (0.603 Total lab unit) was observed under 40 mg/l mannitol treatment. However, the highest level of KO gene expression (1.323 Total lab unit) was observed under 20 mg/l mannitol. It shows stevia growth is affected by osmotic stress. Water deficiency has a negative impact on Stevia. However, the expression of genes had increased by particular mannitol concentrations. Actually, stevia can survive under various abiotic stresses.