Salt Stress Mitigation via the Foliar Application of Chitosan-Functionalized Selenium and Anatase Titanium Dioxide Nanoparticles in Stevia (Stevia rebaudiana Bertoni).

High salt levels are one of the significant and major limiting factors on crop yield and productivity. Out of the available attempts made against high salt levels, engineered nanoparticles (NPs) have been widely employed and considered as effective strategies in this regard. Of these NPs, titanium dioxide nanoparticles (TiO2 NPs) and selenium functionalized using chitosan nanoparticles (Cs-Se NPs) were applied for a quite number of plants, but their potential roles for alleviating the adverse effects of salinity on stevia remains unclear. Stevia (Stevia rebaudiana Bertoni) is one of the reputed medicinal plants due to their diterpenoid steviol glycosides (stevioside and rebaudioside A). For this reason, the current study was designed to investigate the potential of TiO2 NPs (0, 100 and 200 mg L-1) and Cs-Se NPs (0, 10 and 20 mg L-1) to alleviate salt stress (0, 50 and 100 mM NaCl) in stevia. The findings of the study revealed that salinity decreased the growth and photosynthetic traits but resulted in substantial cell damage through increasing H2O2 and MDA content, as well as electrolyte leakage (EL). However, the application of TiO2 NPs (100 mg L-1) and Cs-Se NPs (20 mg L-1) increased the growth, photosynthetic performance and activity of antioxidant enzymes, and decreased the contents of H2O2, MDA and EL under the saline conditions. In addition to the enhanced growth and physiological performance of the plant, the essential oil content was also increased with the treatments of TiO2 (100 mg L-1) and Cs-Se NPs (20 mg L-1). In addition, the tested NPs treatments increased the concentration of stevioside (in the non-saline condition and under salinity stress) and rebaudioside A (under the salinity conditions) in stevia plants. Overall, the current findings suggest that especially 100 mg L-1 TiO2 NPs and 20 mg L-1 Cs-Se could be considered as promising agents in combating high levels of salinity in the case of stevia.

Health-Promoting Compounds in Stevia: The Effect of Mycorrhizal Symbiosis, Phosphorus Supply and Harvest Time.

This work aimed to establish the synergic role of arbuscular mycorrhizal fungi (AMF) symbiosis, phosphorus (P) fertilization and harvest time on the contents of stevia secondary metabolites. Consequently, steviol glycosides (SVglys) concentration and profile, total phenols and flavonoids as well as antioxidant assays, have been assessed in inoculated and no-inoculated plants, grown with or without P supply and collected at different growth stages(69, 89 and 123 days after transplanting).The obtained results suggest that the synthesis of stevia secondary metabolites is induced and/or modulated by all the investigated variability factors. In particular, AMF symbiosis promoted total SVglys content and positively influenced the concentration of some minor compounds (steviolbioside, dulcoside A and rebaudioside B), indicating a clear effect of mycorrhizal inoculation on SVglys biosynthetic pathway. Interestingly, only the mycorrhizal plants were able to synthesize rebaudioside B. In addition, P supply provided the highest levels of total phenols and flavonoids at leaf level, together with the maximum in vitro antioxidant activities (FRAP and ORAC). Finally, the harvest time carried out during the full vegetative phase enhanced the entire composition of the phytocomplex (steviolbioside, dulcoside A, stevioside, rebaudioside A, B, C. total phenols and flavonoids). Moreover, polyphenols and SVglys appeared to be the main contributors to the in vitro antioxidant capacity, while only total phenols mostly contributed to the cellular antioxidant activity (CAA). These findings provide original information about the role played by AMF in association with P supply, in modulating the accumulation of bioactive compounds during stevia growth. At the cultivation level, the control of these preharvest factors, together with the most appropriate harvest time, can be used as tools for improving the nutraceutical value of raw material, with particular attention to its exploitation as functional ingredient for food and dietary supplements and cosmetics.

Optimization of regeneration and Agrobacterium-mediated transformation of Stevia (Stevia rebaudiana Bertoni): a commercially important natural sweetener plant.

Stevia rebaudiana Bertoni is a commercially important zero calorie natural-sweetener herb which produce sweet compounds known as steviol glycosides. Rising demands of steviol glycosides by food and beverage industries has led to an increase in its cultivation in various countries. Unfortunately, stevia cultivation faces 2-25% yield penalty due to weeds which further adds to its cultivation cost. To resolve this major challenge, Agrobacterium-mediated genetic transformation of in vitro derived stevia-nodal explants using herbicide resistance gene (bar) has been optimized, for the production of stable transgenic stevia plants. Several parameters including explant type, pre-incubation duration, acetosyringone (As) concentration, Agrobacterium cell density, Agro-inoculation duration, co-cultivation duration, selection regime and plant growth regulators (PGRs) combination and concentration, have been successfully optimized. Among the two types of explants used, nodal explants showed a higher regeneration response of 82.85%, with an average of 25 shoots/explant. The best PGRs combination and concentration for shoot-induction, shoot-elongation and root-induction was found to be 6-benzyladenine (1.0 mg l-1) + naphthalene acetic acid (0.5 mg l-1), gibberellic acid (1.0 mg l-1), and half-strength MS medium, respectively. The two-step selection (phosphinothricin) regime resulted in an average transformation efficiency of 40.48% with nodal explants. Molecular characterization of putative transformants through PCR, RT-PCR, qRT-PCR and Southern-blot hybridization confirmed the presence, stability, expression as well as copy number of bar gene respectively. Compared to the non-transgenic plants, the T0 transgenic plants successfully tolerated 8 mg l-1 glufosinate ammonium sprays. Thus, the optimized protocol can be useful for the introduction of other genes (inter-kingdom transfer) into stevia genome.

Melatonin promotes seed germination under salinity and enhances the biosynthesis of steviol glycosides in Stevia rebaudiana Bertoni leaves.

Melatonin (MEL) can act as a plant growth regulator and biostimulator in stressful situations. Using MEL in seed pretreatment also affects the future growth of plants. Therefore, this research investigated the effects of MEL on seed germination and seedling growth under NaCl in in vitro conditions. The additional effects of MEL on the accumulation of steviol glycosides (SGs) and on the expression of appropriate genes were also studied. Five µM of MEL was the best concentration for seed germination, while 20 µM exerted a positive impact on the biomass of stevia plantlets. NaCl significantly decreased seed germination, but MEL alleviated this effect when seeds were germinated in 50 mM of NaCl. Under salinity, the values of almost all morphological traits decreased as MEL concentration increased. The highest amounts of stevioside and rebaudioside A (Reb A) were observed as a result of treating seeds with 5 and 20 µM of MEL, respectively. When adding NaCl, positive impacts of MEL on the accumulation of both SGs were also observed. Expression analyses of the genes involved in SGs biosynthesis was explored in seeds and leaves, and the transcripts of key enzymes occurred in both the tissues. However, quantitative polymerase chain reaction (qPCR) analysis showed that all tested genes were upregulated in younger leaves, contrary to older ones. Also in younger, rather than older, leaves SG gene expression varied according to MEL concentration. This study, therefore, presents the promising potential of MEL for improving stevia seed germination under salinity conditions and for enhancing the production of SGs in stevia plants.

Engineered ZnO and CuO Nanoparticles Ameliorate Morphological and Biochemical Response in Tissue Culture Regenerants of Candyleaf (Stevia rebaudiana).

Sustainable production of secondary metabolites in medicinal plants by artificial culturing on the industrial scale has gained worldwide importance. Engineered nanoparticles (ENPs) play a pivotal role in the elicitation of compounds of medicinal value. This investigation explores the influence of ZnO and CuO ENPs on in vitro roots formation, non-enzymatic antioxidant activities, and production of steviol glycosides (SGs) in regenerants of Candyleaf, Stevia rebaudiana. ENPs were applied in 0, 2, 20, 200, and 2000 mg/L of concentration in the MS medium containing plant shoots. The percentage of rooting induced was 91% and 94% by applying ZnO ENPs (2 mg/L) and CuO ENPs (20 mg/L), respectively. Moreover, at 2 mg/L of ZnO and 20 mg/L of CuO ENPs, the high performance liquid chromatography studies determined the significantly greatest content of SGs; rebaudioside A (4.42 and 4.44) and stevioside (1.28 and 1.96). Phytochemical studies including total flavonoid content, total phenolic content, and 2,2-diphenyl-1-picryl hydrazyl-free radical scavenging activity were calculated highest by the regenerants grown in 2 mg/L of ZnO and 20 mg/L of CuO ENPs dosage. Both ZnO and CuO ENPs at 200 mg/L and 2000 mg/L of concentration induced adverse effects on plant biomass, antioxidant activities, and SGs content up to 1.22 and 1.77 for rebaudioside A and 0.21 and 0.25 for stevioside. Hence, the biochemical and morphophysiological responses of Candyleaf were elicited as a defense against ZnO and CuO ENPs applied under threshold limit. This artificial biotechnological technique holds great promise for continued production of natural antioxidants on commercial scale and our study has further strengthened this impact.

Exposure of stevia (Stevia rebaudiana B.) to silver nanoparticles in vitro: transport and accumulation.

The impact of nanotechnology in the field of agricultural sciences creates the need to study in greater detail the effect of products offering nanoparticles for application in plant species of agricultural interest. The objective of this study was to determine the response of stevia (Stevia rebaudiana B.) in vitro to different concentrations of AgNPs (silver nanoparticles), as well as to characterize and identify their absorption, translocation and accumulation mechanisms. Nodal segments of stevia grown in MS medium supplemented with AgNPs (0,12.5, 25, 50,100 and 200 mg L-1) were used. After 30 days of in vitro shoot proliferation, the number of shoots per explant, shoot length, chlorophyll content, dry matter content and the metallic silver (Ag) content of the plants were quantified. In addition, characterization, transport and accumulation of silver nanoparticles were performed by microscopic analysis. AgNPs were shown to be present in epidermal stem cells, within vascular bundles and in intermembrane spaces. In leaves, they were observed in ribs and stomata. The current and future use of AgNPs in agricultural sciences opens up the possibility of studying their effects on different plant species.

Effects of cytokinins, gibberellic acid 3, and gibberellic acid 4/7 on in vitro growth, morphological traits, and content of steviol glycosides in Stevia rebaudiana.

Steviol glycosides (SGs) and gibberellic acids share a part of their biosynthesis pathways. Despite the widespread studies on the effect of gibberellic acid 3 (GA3), the effect of gibberellic acid 4 and 7 (GA4/7) on Stevia rebaudiana has never been studied. This study aimed at a comparative evaluation of different hormone effects, i.e., 1 mg L-1 GA4/7, 1 mg L-1 GA3, or 0.5 mg L-1 kinetin and 0.5 mg L-1 6-benzylaminopurine (BAP) (KB 0.5), on in vitro propagation, growth, morphological properties, and content of SGs in leaf samples of stevia. In comparison with the control group (hormone-free), the treatments of KB 0.5 or GA3 produced the highest biomasses and largest leaf areas. The three hormonal treatments produced a similar number of leaves, the ratio of fresh to dry weight, and leaf length. GA4/7-treated explants produced the highest ratio of leaf area to leaf length. The effect of GA4/7 on shoot elongation was greater than that of the control or even GA3. While the effect of GA3 on rebaudioside-A (Reb-A) production was similar to that of the control (16.2 and 18.04 mg g-1, respectively), GA4/7 resulted in a lower amount of it (13.31 mg g-1). Except for GA4/7, which induced more stevioside accumulation, the treatments' effects were comparable to that of the control. The ratio of stevioside to Reb-A was the highest for GA4/7 (2.62), followed by GA3 (1.93), and then the two others. Sum of Reb-A and stevioside content was not changed by the use of any of the treatments.

Stimulation of secondary metabolites by copper and gold nanoparticles in submerge adventitious root cultures of Stevia rebaudiana (Bert.).

Nanotechnology is one of the advance technologies that almost found implications in every field of science. The importance is due to the unique properties of nanoparticles. In this study, bimetallic alloys of copper (Cu) and gold (Au) were tested in submerge root cultures of Stevia rebaudiana for production of biomass and secondary metabolites. A known amount of inoculum roots were submerged in liquid Murashige and Skoog medium containing combination of naphthalene acetic acid (NAA; 0.5 mg l-1) and different ratios of nanoparticles (NPs). NAA augmented medium was used as control. The addition of nanoparticles (30 µg l-1) stimulated biomass accumulation (1.447 g/flask) on 27th day of log phases. The maximum total phenolics content (TPC; 16.17 mg/g-DW) and total flavonoids content (TFC; 4.20 mg/g-DW) were displayed using AuCu-NPs (1:3) and NAA. The same combinations enhanced total phenolic production (TPP; 116 mg/L) and total flavonoid production (TFP; 29.5 mg/L) in submerged cultures. A strong correlation was observed between phenolics, flavonoids and dry biomass. Moreover, maximum 1, 1-diphenyl-2-picrylhydrazyl (DPPH) activity of 79% was displayed by addition of AuCu (1:3) nanoparticles. In conclusion, nanoparticles application has shown a positive effect in enhancing biomass and secondary metabolites production in adventitious root cultures of Stevia rebaudiana.

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.

Effect of nitrogen sources on gene expression of Stevia rebaudiana (Bertoni) under in vitro conditions.

Stevia rebaudiana (Bertoni) is a non-caloric sweetener belonging to Asteraceae family. Stevia compounds such as steviol glycosides (SGs) are 200 times sweeter than sugar. Stevioside and rebaudioside A are the two major steviol glycosides. Nitrogen is an essential element for plant growth and development. In this study the effects of nitrogen influenced by different concentrations of NH4NO3 (0, 825 and 1650 mg/l) and KNO3 (0, 950 and 1900 mg/l) is examined in MS medium. To analysis the UGT74G1 and UGT76G1 genes expression, involved in the synthesis of SGs, RT-qPCR technique was performed. Data showed that there were significant differences between all media. The shoot length, seedlings dry weight and leaf fresh weight of stevia increased with applying NH4NO3 along with KNO3. The highest expression of UGT74G1 gene, was observed in plantlets grown on MS medium with 0 mg/l NH4NO3 and 950 mg/l KNO3 (1.291 total lab unit) but the highest expression of UGT76G1 gene, was observed in plantlets grown on MS medium added by 1650 mg/l NH4NO3 +950 mg/l KNO3 (1.08 total lab unit). Moreover, the lowest value of UGT74G1 gene expression were revealed in MS medium added by 1650 mg/l NH4NO3 +0 mg/l KNO3 (0.80 total lab unit) and the lowest values of UGT76G1 gene expression seen in MS medium with 0 mg/l NH4NO3 +950 mg/l KNO3 (0.85 total lab unit) concentrations. The results of this study could be valuable in stevia breeding programs through glycosides biosynthesis pathways.

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.

Investigation of different concentrations of MS media effects on gene expression and steviol glycosides accumulation in Stevia rebaudiana Bertoni.

Stevia rebaudiana Bertoni is one of two species that contains steviol glycosides. Among steviol glycosides that extracted from leaves, stevioside and rebaudioside A are the two major and the sweetest glycosides that are about 200-300 times sweeter than sucrose with zero calories. The best method for stevia propagation is tissue culture. So, for investigation of nutrients in medium, we studied the effect of different concentrations of MS media (MS, 0.5 MS, 0.25 MS, 0 MS) on morphological traits, UGT74G1 and UGT76G1 genes expression and accumulation of steviol glycosides in stevia leaves. The best growth rate (0.472 mm/d) has occurred in plants grown in MS media. Also, the highest gene expression of UGT74G1 gene (1.000 Total lab unit) was seen under MS treatment. However, the highest expression level of UGT76G1 gene (1.701 Total lab unit) was observed at plants grown in 0 MS. The highest amount of both Stevioside and Rebaudioside A (14.23 and 8.12, respectively) were accumulated in plants under MS treatment. Obviously, dilution of MS media associated with decreasing in both expression of the intended genes and accumulation of steviol glycosides.

Metal oxides as a biostimulator for upregulation of genes involved in the biosynthesis of Rebaudioside- A.

Stevia rebaudiana Bertoni is a kind of perennial medicinal plant with sweetening properties which belongs to Asteraceae family. Its leaves with fundamental glycoside compounds consist of both a sugar part and a non-sugar sector. One of the glycoside compounds is Rebaudioside- A which has a greater importance in business. This experiment was conducted to evaluate the effects of Ag2O, CrO3, PbO, Fe2O3, BaO and TiO2 on the expression pattern of these genes in the Stevia rebaudiana. Rebaudioside- A biosynthesis was repeated 3 times with concentrations of 50, 100 and 200µM. Also, the results of the study pertaining to the expression pattern of these genes showed that metal oxides have led to an increase in the expression of the regulatory genes involved in biosynthesis of Rebaudioside- A. According to the expression profile, it was found that its effect on DXR, HDS, HDR, IDI and CPPS genes is more than other genes. The peak HPLC indicated for stevioside and Rebaudioside- A represents an increase in the production of this active ingredient under the influence of all treatments. In general, the expression profile of these genes and the results of HPLC show that whatever going to the end of the pathway of production of Rebaudioside- A, the activity of the enzymes increases under the influence of these treatments, and eventually a greater amount of Rebaudioside- A will be produced. This process shows that metal oxides will have a significant effect on the biosynthesis of Rebaudioside- A.

The Effects of different nitrogen doses on antioxidant and antimicrobial activity of Stevia (Stevia rebaudiana Bert.).

In this study, the efficiency of the different nitrogen doses (0, 5, 10, 15 and 20 kg ha-1) on biological activity levels (antioxidant and antimicrobial activity) of Stevia rebaudiana Bert. was investigated. In addition, methanol extracts were obtained by maceration method from different doses of fertilizer applied stevia. The components in methanol extracts of plants were determined by gas chromatography-mass spectrometry (GC-MS) method. Antimicrobial activities of stevia extracts were investigated by microdilution method. The antioxidant activity evaluated by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, ferric thiocyanate (FTC), thiobarbituric acid (TBA), reducing power, total phenol content (TPC), and total flavonoid content (TFC) methods. According to the results, the fertilizer doses effects on antimicrobial activity of stevia were not made much difference. But in antioxidant activity, there were some variations in the activity-dependent on fertilizer amount.

Callus induction and somatic embryogenesis in Stevia rebaudiana Bertoni as a medicinal plant.

Stevia rebaudiana (Bert.) from Asteraceae family is a useful medicinal plant that prevents and cures diabetes, blood pressure, weight gain and tooth decay. Due to self-incompatibility in stevia, somatic embryo investigation for artificial seed production is valuable in this plant. In order to evaluate the callus induction characteristics in stevia, a factorial experiment was laid out based on a completely randomized design with three replications. The factors included ten hormone combinations and control, two kinds of media (MS and B5) and two types of explants (leaf and internode). Callus induction characters including the percentage of callus formation, days to callus induction, fresh and dry callus weight were recorded. Analysis of variance showed significant differences (p<0.01) among hormone combinations, media and explant types as well as their interactions. The best treatment for callus induction with minimum time to callus formation was 1 mg/l NAA+1 mg/l BAP. The highest fresh and dry callus weight were obtained on B5 medium supplemented by 1 mg/l 2,4-D+1 mg/l BAP (in leaf explant) and 0.25 mg/l 2,4-D+ 0.1 mg/l BAP (in internode explant). These results can be used in suspension culture. To induce somatic embryogenesis in suspension culture, six hormone treatments were investigated. The highest somatic embryogenesis percentage was obtained in MS medium supplemented by 2 mg/l 2,4-D+ 0.5 mg/l NAA+0.5 mg/l BAP.

Effect of decapitation and exogenous application of gibberellic acid (GA3) and cytokinin (CK) on some physiological characteristics of stevia.

Stevia (Stevia rebaudiana B.) has auxiliary buds that often remain dormant for a long time and sometimes remain dormant until the plants change at the reproductive stage. This study was designed out to investigate whether decapitation and exogenous application of plant growth regulators enhance the productivity of stevia through breaking the apical dominance and increasing physiological characteristics. Experiment was carried out as a factorial in randomized complete block design with three replications. Factors were consisted two agricultural practices (Decapitation and No-decapitation) and eight foliar spray including without spray as control, water spray, GA3 (300, 600 and 900 µm) and CK (100, 200 and 400 µm). The results of the present investigation indicated a positive response on number of branches and leaves, leaves and stem fresh weight and total dry weight, in both harvests not only from the decapitation of apical buds but also from foliar application of CK (400 µM). Thus, it can be concluded that the decapitation practices in conjunction with foliar application of CK (400 µM) could be used to increase the dry-leaf yield of stevia. However, further studies are required to standardize the dose of CK (400 µM) to improve the yield and quality of stevia.

Assessment of the nitrogen fertilization effect on bioactive compounds of frozen fresh and dried samples of Stevia rebaudiana Bertoni.

The present study aims to assess the effect of different nitrogen (N) rates on the chemical composition and antioxidant properties of stevia frozen fresh and dried leaves, and to define the best growing conditions to maximize the levels of bioactive compounds. In general, processing affects more significantly the tocopherol and sugar contents than N fertilization. The most abundant sugars were xylose, arabinose + fructose and sucrose, presenting dried samples with higher contents than frozen fresh ones, while the latter better retained tocopherols than dry samples. Regarding phenolic compounds, greater levels were found in dried samples and in those fertilized with 25 kg N ha-1. Leaves from plants fertilized with 25 and 50 kg N ha-1 also evidenced higher antioxidant activity, which seemed to be influenced by the phenolic composition. In general, N fertilization provides an improvement in the chemical composition and bioactive potential of stevia leaves.

Effect of sucrose concentrations on Stevia rebaudiana Bertoni tissue culture and gene expression.

Stevia rebaudiana (Bert.) Bertoni is known as sweet plant which it contains a high level of steviol glycosides in the leaves.  This plant has been used from centuries ago as a sweetener for tea. One of the most important steviol glycosides is stevioside that is attractive for diabetic persons. Tissue culture is the only rapid process for the mass propagation of stevia. One of the most important factors in the medium is sucrose that is a necessary for plant growth. In the present study, we use nodal segments of the stem as explants in mediums with different sucrose concentration (50 mM, 100mM and 150mM). Several morphological traits were measured in a 28 day period. Results analysis showed a significant variation between treatments. The highest growth rate, rooting and leaf production was obtained in medium with 100mM sucrose. The correlation between measured traits was significant at the 0.01 level. To investigation of UGT74G1, UGT76G1, UGT85C2 and KS genes expression that are involved in the synthesis of SGs, RT- PCR was done with the housekeeping gene of as internal control. There were significant differences between all media. The results showed thatsucrose 100 mM containing media was more desirable than others for expression of UGT76G1 and UGT85C2 genes. Whereas, the best medium for expression of UGT74G1 was sucrose 150 mM and sucrose 50 mM for KS gene. Totally, it seems that sucrose at a concentration of 100 mMprovides the best condition for stevia growth and steviol glycosides production.

Effect of salinity on gene expression, morphological and biochemical characteristics of stevia rebaudiana Bertoni under in vitro conditions.

Stevia rebaudiana Bertoni is a famous medicinal plant for its low calorific value compounds which are named steviol glycosides (SGs) and they are 150-300 times sweeter than sugar. Among various SGs, stevioside and rebaudioside A considered to be the main sweetening compounds.  Soil salinity is one of the most essential stress in the world. Salinity affects the survival and yield of crops. In current study the effects of salinity and osmotic stress caused by different concentration of NaCl (0, 20, 40, 60 and 80 mM) on morphological traits, genes expressionand amount of both stevioside and rebaudioside Aunder in vitro conditions has been investigated. The morphological traits such as bud numbers, root numbers, shoot length (after 15 and 30 days) were evaluated. With increasing salinity, the values of all studied morphological traits decreased. To investigation of UGT74G1 and UGT76G1 genes expression that are involved in the synthesis of SGs, RT-PCR was done and there were significant differences between all media. The highest expression of both genes was observed in plantlets grown on MS media (with NaCl-free). Also, the lowest amounts of gene expression of the both genes were seen in MS+ 60 mM NaCl. Based on HPLC results, the highest amount of both stevioside and rebaudioside A were observed in plantlets grown in MS media (with NaCl-free). Finally, it can be concluded that stevia can survive under salt stress, but it has the best performance in the lower salinity.

Effect of KH2PO4 on gene expression, morphological and biochemical characteristics of stevia rebaudiana Bertoni under in vitro conditions.

Stevia rebaudiana is one of the most important biologically sourced and low-calorie sweeteners Bertoni that has a lot of steviol glycosides. Tissue culture is the best for propagation of stevia and micro nutrients can affect both morphological traits and steviol glycosides production. Therefore, the effect of different concentrations of KH2PO4on stevia growth factors and gene expression had been studied by tissue culture methods, RT-PCR and HPLC. According the results, bud numbers had increased significantly in MS + 0.034 mMKH2PO4 media and the highest measured length was seen in plants grown under MS + 0.034 mM KH2PO4 treatment. Also, the highest growth rate (1.396 mm/d) was observed in MS + 0.034 mMKH2PO4.The best concentration of KH2PO4 for expression of UGT74G1 was 0.00425mMand the best one for UGT76G1 expression was 0.017mM. Interestingly, the best media for both stevioside and rebaudioside A accumulation was 0.017mM KH2PO4containing media. There was positive correlation between the best media for gene expression and the best one for steviol glycosides production.