Isolation and functional analysis of SrMYB1,a direct transcriptional repressor of SrUGT76G1 in Stevia rebaudiana

SrUGT76G1,the most well-studied diterpene glycosyltransferase in Stevia rebaudiana,is key to the biosynthesis of economically important steviol glycosides(SGs).However,the molecular regulatory mechanism of SrUGT76G1 has rarely been explored.In this study,we identified a MYB transcription factor,SrMYB1,using a yeast one-hybrid screening assay.SrMYB1 belongs to the typical R2R3-type MYB protein and is specifically localized in the nucleus with strong transactivation activity.The transcript of SrMYB1 is predominantly accumulated in flowers,but is also present at a lower level in leaves.Yeast one-hybrid and electrophoretic mobility shift assays verified that SrMYB1 binds directly to the MYB binding sites in the F4-3 fragment(+50–(–141))of the SrUGT76G1 promoter.Furthermore,we found that SrMYB1 could significantly repress the expression of SrUGT76G1 in both epidermal cells of tobacco leaves and stevia callus.Taken together,our results demonstrate that SrMYB1 is an essential upstream regulator of SrUGT76G1 and provide novel insight into the regulatory network for the SGs metabolic pathway in S.rebaudiana.

Characterization of yogurt goat milk added Aloe vera and Stevia rebaudiana with functional potential

The search for functional foods with health benefits has increased,and yogurt represents one of these products due to their properties,such as,source of probiotics that favour the gastrointestinal system,contains vitamins,minerals and improves the action of enzymes.Present work investigated bromatological,microbiological,sensorial properties and shelf-life of different yogurt goat milk formulations with Aloe vera and natural sweeteners.The goat yogurt with 99.40%A.vera pulp and 0.60%S.rebaudiana presented the best nutritional composition,being suitable for human consumption representing an innovative product with a great functional potential.The synergy between the ingredients used in the yogurt processing resulted in an innovative product to the market.Goat milk has a lower cost per liter in comparison with cow milk,Aloe vera is accessible due to its fast-vegetative propagation,Stevia Rebaudiana has a great popularity for being a natural sweetener with no calories,and all these products are produced in México.

Biological Effects of Stevia rebaudianum Induced by Carbon Ion Implantation

The biological effects during seed germination were investigated after the dry seeds of Stevia rebaudianum Bertoni were implanted with carbon ion beam of 75 keV and 10 14 ions/cm 2. The results showed that the germination rate of carbon ion implanted seeds was slightly higher than that of the control, but the survival rate of the treated seedlings, on the contrary, was lower than that of the control (P<0.02), while the height of the treated seedlings was significantly higher than that of the control (P<0.01). On the 4th day after germination, the leaf cell wall in the treated group was thick, some high electron_dense substance deposited in the enlarged plasmodesma; Cell membrane creased with high electron_dense granules deposited on it. The plasma membrane protruded towards cell wall, and the granules shifted via plasmodesma or deposited onto cell wall. These phenomena may be related to the conveyance of implanted ions across cell wall, or be related to the accumulation of callose. In addition, the implantation of carbon ions could increase the lamellae of the chloroplast and cause high development of the chloroplast which sometimes contained two plastid centers in an individual chloroplast. Also, the highly developed cristae, abundant mitochondria and typical crystalloid structure in microbody could be found. All these results indicated that the anabolic and catabolic activities in the seedlings implanted with carbon ions before germination were obviously more active than those in the controls.

Steviaside Containing Plant as Deconstractive (Degradative) Agent for Persistent Organic Pollutants

Steviaside containing plant extracts have been used for degradation of persistent chloroorganic pesticides. Reactions between DDT and Steviaside or sum of extractive substances isolated from ground up part of plant Stevia were studied to give of less toxic DDE. Herein researches on studying interaction sum of polysaccharides of Stevia with DDT in various ratios resulted also. The GC-MS and GLC methods were used for analyzing degradation degree of pesticides and to determine obtained compounds. Treat HCCH by water extract of Stevia basically formed tetrachlorocyclo-hexadiene (HCH) with 86.9% yield and in particularly formed of tri-, tetrachlorobenzenes. The HCH formed in 79.7% on treat pesticide by 80% Steviaside. Degradation of HCCH and DDT by water extract of Stevia in a presence of Ana-basine in a ratio of 2:1:1 occur to degrade of HCCH up to 70-80%, and DDT on 25% - 30%.

NMR Spectral Analysis and Hydrolysis Studies of Rebaudioside N, a Minor Steviol Glycoside of <i>Stevia rebaudiana</i>Bertoni

The complete proton and carbon NMR spectral assignments of a diterpene glycoside isolated from the commercial extract of the leaves of Stevia rebaudiana Bertoni, 13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] entkaur-16-en-19-oic acid-[(2-O-α-L-rhamnopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl) ester] (1);also known as rebaudioside N, was achieved by the extensive 1D and 2D NMR (1H and 13C, COSY, HMQC, HMBC) as well as mass spectral data. Further, hydrolysis studies were performed on rebaudioside N using acid and enzymatic studies to identify aglycone and sugar residues in its structure.

TDZ-Induced High Frequency Plant Regeneration through Direct Shoot Organogenesis in <i>Stevia rebaudiana</i>Bertoni: An Important Medicinal Plant and a Natural Sweetener

An efficient high frequency plant regeneration protocol through direct organogenesis was developed for Sevia rebaudiana Bert. Nodal segments containing axillary buds were used as an explant and inoculated on Murashige and Skoog’s (MS) medium containing 3% (w/v) sucrose, 0.8% (w/v) agar supplemented with various concentrations of benzyladenine (BA), kinetin (Kn) and thidiazuron (TDZ) ranging from 1.00 to 9.00 μM. Maximum multiple shoots (96%) were obtained in MS medium supplemented with 1.0 μM TDZ with an average of 60 shoots per culture, having an average shoot length of 6.0 cm. The best in vitro root induction (89%) was achieved on half strength MS medium without any growth regulator with an average of 24 roots per culture and root length of7 cm. The rooted plantlets were successfully established in soil and grown to maturity at the survival rate of 95% in the indoor grow room. High-performance liquid chromatography was used to assess the stability in chemical profile and quantification of stevioside and rebaudioside A content of in vitro propagated S. rebaudiana plants and compared with their mother plant at the peak vegetative stage. Our results show no significant differences (p in vitro propagated plants. Furthermore, fully developed in vitro propagated S. rebaudiana plants were also compared with mother plant for their gas and water vapour exchange characteristics and leaf anatomy. The results show that in vitro propagated and hardened plants of S. rebaudiana are morphologically as well as functionally comparable to each other and to their mother plant.

Morphological Changes and Antioxidant Activity of <i>Stevia rebaudiana</i>under Water Stress

Stevia rebaudiana, a herbaceous perennial shrub contains steviol glycosides, as an alternative source of sugar for diabetic patients. Water being an integral part plays a vital role in the maintenance of plant life. Availability of water is one of the limiting factors determining plant distribution and survival in natural ecosystem. The objective of this study was to investigate the ability of tolerance of Stevia plants to water stress. Potted plants of Stevia were subjected to different levels of water regimes (100 ml, 200 ml, 300 ml) per day, whereas control plants were watered daily with about 400 ml water. Plant height, leaf area, electrolyte leakage and antioxidant enzyme activity (peroxidase and catalase) were assayed during the experimental period. All these parameters were severely affected under water stress condition. Stress treatment caused an increase in electrolyte leakage compared to control. Plant height decreased under severe stress condition whereas a sharp increase in antioxidant enzyme activity was observed in stressed plants as compared to untreated control plants. Our experiment emphasizes the importance of proper watering schedule for the cultivation of Stevia as an agricultural crop to meet the challenges for sugar and energy crisis.

Reversed-Phase HPLC Analysis of Steviol Glycosides Isolated from Stevia rebaudiana Bertoni

High Performance Liquid Chromatography (HPLC) experiments have been performed on nine steviol glycosides namely rebaudioside A, steviolbioside, stevioside, rubusoside, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside F, and dulcoside A isolated from the leaves of Stevia rebaudiana using Reversed-Phase (RP) column. Using RP-HPLC method, the individual retention times for nine naturally occurring ent-kaurane diterpene glycosides of S. rebaudiana reported in JECFA have been determined at four different temperatures: 20℃, 40℃, 60℃, and 79℃. Also, calculated the relative retention times of the eight steviol glycosides steviolbioside, stevioside, rubusoside, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside F, and dulcoside A against the major steviol glycoside rebaudioside A. HPLC results suggested that temperatures 40℃ and 60℃ would be ideal conditions for better separation of steviol glycosides.

Spore production in the solid-state fermentation of stevia residue by Trichoderma guizhouense and its effects on corn growth

Trichoderma is an important and widely used plant growth-promoting fungus(PGPF).In this study,stevia residue amended with amino acids hydrolyzed from animal carcasses was used for the production of Trichoderma guizhouense NJAU 4742 by solid-state fermentation,and then its potential to promote corn plant growth was evaluated in combination with chemical fertilizer(CF)or organic fertilizer(OF).The highest spore number of 7×10^(9) CFU g^(–1) fresh weight was obtained under the following optimal parameters:material ratio of 50%(stevia residue:rice bran=1:1),pH value of 3.0(amended with 6.67%amino acids),initial moisture content of 60%,inoculum size of 10%,material thickness of 3 cm and an incubation time of 4 days.The aboveground corn plant biomass obtained with T.guizhouense applied alone and with CF treatments were slightly higher than those of no fertilizer control and CF treatments,respectively.However,T.guizhouense applied with OF significantly(P<0.05)increased aboveground biomass compared to OF and yielded the highest aboveground biomass among all the treatments.Moreover,T.guizhouense applications primarily influenced the fungal bulk soil community composition,among which three OTUs(OTU_(2) and OTU_(9) classified as Chaetomium,and OTU_(4)classified as Trichoderma)were stimulated in both bulk and rhizosphere soil.Notably,a specific OTU_(3)(Phymatotrichopsis)was only stimulated by T.guizhouense applied with OF,possibly leading to high soil productivity.These results show that it is feasible to employ stevia residue in the eco-friendly fermentation of T.guizhouense,which is strongly suggested for enhancing OF applications.

<i>In Vitro</i>Evaluation of Some Traits in <i>Stevia rebaudiana</i>(Bertoni) under Drought Stress and Their Relationship on Stevioside Content

The experiment was conducted to evaluate the differences of plant growth as well as sweetener material of two types of Stevia rebaudiana under conditions of absence and presence of drought stress. In vitro, the experiment was conducted as drought traits using four levels of polyethylene glycol (0.0, 10,000, 20,000, 30,000 ppm). It was observed that survival % and No. of shoots should be considered as the important factors associated with stevioside content in stevia plant. According to path coefficient analysis and positive direct effect of studied traits, survival % recorded the highest value (0.5386), whereas No. of shoots recorded the highest value (– 0.8827) as negative direct effect in related to stevioside content. The results showed that all traits were affected, especially under 30,000 ppm level of polyethylene glycol. Generally, the most of studies traits exhibited a recorded clearly difference between the two types and used drought levels on stevia plants. Therefore, selection based on survival % would be more effective to improving stevioside content of stevia plants in drought stress conditions.

Isolation and Structural Characterization of a New Minor Penta β-D-Glucopyranosyl Diterpene from Stevia rebaudiana Bertoni

From the commercial extract of the leaves of Stevia rebaudiana Bertoni, a new minor ent-kaurane diterpene glycoside having five β-D-glucopyranosyl units has been isolated. The chemical structure of the new compound was characterized as 13-[(2-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid-(2-O-β-D-glucopyranosyl-6-O-β-D-glucopyranosyl-β-D-glucopyranosyl) ester (1) on the basis of extensive 1D (1H & 13C) and 2D NMR (TOCSY, HMQC, and HMBC), and High Resolution (HR) mass spectroscopic data as well as hydrolysis studies.

Potassium deficiency inhibits steviol glycosides synthesis by limiting leaf sugar metabolism in stevia(Stevia rebaudiana Bertoni)plants

The steviol glycosides(SGs)in stevia(Stevia rebaudiana Bertoni)leaves are becoming increasingly valuable due to its high sweetness but low calorific value,which is driving the development of stevia commercial cultivation.Optimizing fertilization management can effectively increase SGs productivity,but knowledge on the relationship between potassium(K)fertilization and SGs production is still lacking.In this study,pot experiments were conducted in order to investigate the effect of K deficiency on SGs synthesis in stevia leaves,as well as the underlying mechanisms.Our results showed that when compared with standard K fertilization,K deficiency treatment has no significant effect on the biomass of stevia plant grown in a given soil with high K contents.However,K deficiency critically decreased leaf SGs contents as well as the expression of SGs synthesis-related genes.The contents of different sugar components decreased and the activities of sugar metabolism-related enzymes were inhibited under the K deficiency condition.Moreover,spraying sucrose on the leaves of stevia seedlings diminished the inhibitory effect caused by K deficiency.Our results also revealed the significant positive correlations between sucrose,glucose and SGs contents.Overall,our results suggest that K deficiency would suppress the synthesis of SGs in stevia leaves,and this effect may be mediated by the leaf sugar metabolism.Our findings provide new insights into the improvement of SGs production potential.

A review on current conventional and biotechnical approaches to enhance biosynthesis of steviol glycosides in Stevia rebaudiana

Stevia rebaudiana Bertoni is commonly called stevia and mostly found in the north east regions of South America.It is an herbaceous and shrubby plant belonging to the Asteraceae family.Stevia is considered as a natural sweetener and a commercially important plant worldwide.The leaves of S.rebaudiana contain steviol glycosides(SGs)which are highly potent and non-caloric sweeteners.The sweetening property of S.rebaudiana is contributed to the presence of these high potency,calorie free steviol glycosides.SGs are considerably suitable for replacing sucrose and other artificial sweetening agents which are used in different industries and pharmaceuticals.SGs amount in the plant mostly varies from 8%to 10%,and the enhancement of SGs is always in demand.These glycosides have the potential to become healthier alternatives to other table sugars for having desirable taste and zero calories.SGs are almost 300 times sweeter than sucrose.Being used as alternative sugar intensifier the commercial value of this plant in biopharmaceutical,food and beverages industries and in international market is increasing day by day.SGs have made stevia an important part of the medicinal world as well as the food and beverage industry,but the limited production of plant material is not fulfilling the higher global market demand.Therefore,researchers are working worldwide to increase the production of important SGs through the intercession of different biotechnological approaches in S.rebaudiana.This review aims to describe the emerging biotechnological strategies and approaches to understand,stimulate and enhance biosynthesis of secondary metabolites in stevia.Conventional and biotechnological methods for the production of steviol glycosides have been briefly reviewed and discussed.

Impact of Using Stevia on Physicochemical, Sensory, Rheology and Glycemic Index of Soft Ice Cream

The aim of this study was to formulate and develop a low calorie and low glycemic index (GI) of soft ice cream by using mixture of sucrose and Stevia. Five different formulations of ice cream were produced by using different proportions of sucrose and Stevia. Physicochemical characteristics, hedonic sensory evaluations and glycemic index determination of products were carried out by following conventional methods. Replacement of sucrose with Stevia resulted in a significantly lower viscosity and brix with a higher overrun and melting rate in a dose dependent manner. Total replacing of sucrose with Stevia resulted in significant reduction in caloric value from 143.03 to 105.25 Kcal and GI from 79.06 ± 4.0 to 72.18 ± 5.27 as compared to those of sucrose based formulation (p 0.05) indicating a 37.78% and 6.88% reduction, respectively. TB had the best sensory acceptance among all the treatments. We concluded that substitution of sucrose with Stevia may be a choice to produce low caloric and GI ice creams. However, using mixture of the two sweeteners improves sensory acceptance of the formulations.

Neurotropic effects of aspartame, stevia and sucralose on memory retention and on the histology of the hippocampus of the ICR mice(Mus musculus)

Objective: To identify the effects of the consumption of non-nutritive sweeteners on memory retention and on the histology of the hippocampus.Methods: In this study, 20 mice were used to determine if there is an effect of consuming the maximum allowable dose of the non-nutritive sweeteners on the memory retention and on the histology of the hippocampus. The mice were distributed into four groups and the treatments were given via oral gavage: Group 1(water), Group 2(aspartame: 1 000 mg/kg), Group 3(stevia: 1 000 mg/kg) and Group 4(sucralose:16 000 mg/kg). Treatments were administered to the different experimental groups for 32 days, after which memory retention was tested using the two-day water maze protocol.After the tests, the mice were sacrificed and the brain was analyzed histologically for neurotrophic effects.Results: Based on the results of the two-day water maze protocol, there were no differences between the non-nutritive sweeteners and the control group. However, stevia showed high cellular apoptosis followed by aspartame, sucralose and control group.Conclusions: There was no significant effect on the memory of the mice. It showed histologically however, that stevia had a significant neurotropic effect compared to the other sweeteners.

Rapid HPLC Method for Determination of Rebaudioside D in Leaves of Stevia rebaudiana Bertoni Grown in the Southeast of Mexico

Stevia leaves contain glycosides on which biological activity and sweetening capacity has been reported. Besides the main glycosides—stevioside and rebaudioside A—there are minor glycosides that may contribute to the activity and thus it is important to quantify them. Rebaudioside D is one of the minor glycoside present in S. rebaudiana leaves and there are no reports of a validated method to quantify it. Therefore a simple and sensitive high performance liquid chromatography (HPLC) method was validated for the determination of rebaudioside D in leaves of Stevia rebaudiana B. grown in the southeast of México. HPLC method was performed using a C18 column (250 mm × 4.6 mm, 5 μm) and UV detector set at 210 nm. The mobile phase consisted of 32:68 (v/v) mixture of acetonitrile and sodium phosphate buffer (10 mmol/L, pH 2.6), set to a flow rate of 1.0 ml/min. The calculated parameters were: sensitivity, linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy and precision. The retention time of rebaudioside D was found to be 3.47 min ± 0.04 (S.D.). The calibration curves were linear over the working range (25 - 150 μg/ml), with correlation coefficient ≥0.99 and determination coefficient ≥0.98. The calculated limit of detection (LOD) was 8.53 μg/ml, while the limit of quantitation (LOQ) was 25.85 μg/ml. The percent recoveries of fortified samples were 100% ± 10% and precision relative standard deviation was ≤2.79%. The criteria of validation showed accuracy, linearity, and precision;therefore the method is suitable for quantitative analysis of rebaudioside D in Stevia rebaudiana leaves. Rebaudioside D content (g/100g) in Morita II and Criolla varieties grown in the southeast of Mexico were 0.43 and 0.46, respectively with no significant differences (p > 0.05) between them.

Influence of Additives on Enhanced in Vitro Shoot Multiplication of <i>Stevia rebaudiana</i>(Bert.)—An Important Anti Diabetic Medicinal Plant

The present study was designed to develop an efficient protocol for micro propagation of S. rebaudiana from nodal explants and study the influence of additives on enhancement of shoot proliferation. A two-step protocol has been standardized in which, first step comprising growth hormones concentration is optimized and it was found that MS medium supplemented with 2.0 mg/l BAP + 0.5 mg/l Kin + 0.1 mg/l NAA turned out to be the best treatment for shoot induction. In the second step, the best treatment for shoot induction was fortified with different growth additives for further shoot proliferation. Among the different types of additives used, casein hydrolysate at 0.05% (w/v) was found to be most effective, resulted with maximum of 15.0 shoots. 90% regeneration frequency and shoot length of 6.0 cm were recorded per explant. Thus, the procedure described is a quick and reliable method which could be applied for efficient large scale propagation, genetic transformation assays and secondary metabolite production of Stevia.

IR Spectral Analysis of Diterpene Glycosides Isolated from <i>Stevia rebaudiana</i>

The objective is to identify the Infra-Red (IR) spectral analysis of the diterpene glycosides present in the commercial extracts of Stevia rebaudiana was achieved by PerkinElmer Spectrum 400 Fourier Transform (FT) spectrometer employing a PerkinElmer Universal Attenuated Total Reflection (ATR) accessory. Using this technique the IR spectral pattern of 15 steviol glycosides which belongs to three different classes of ent-kaurane diterpene glycosides namely ent-13-hydroxykaur-16-en-19-oic acid, ent-13-hydroxykaur-15-en-19-oic acid, and 13-methyl-16-oxo-17-norent- kauran-19-oic acid were identified. From the wave numbers found for all 15 steviol glycosides, it was observed that that though there are differences in the number of sugar units, nature of sugar units, and their attachments;there are not any notable differences in the IR values.

Enzymatic Synthesis and Structural Characterization of Rebaudioside D3, a Minor Steviol Glycoside of <i>Stevia rebaudiana</i>Bertoni

Rebaudioside D3, a novel steviol glycoside, is produced by specific UDP-glycosyltransferase of rebaudioside E, a minor steviol glycoside of Stevia rebaudiana Bertoni. The complete proton and carbon NMR spectral assignments of rebaudioside D3, 13-[(2-O-β-D-glucopyranosyl-6-O-β-D-glucopyranosyl-β-D-glucopyranosyl) oxy] ent-kaur-16-en-19-oic acid-(2-O-β-D-glucopyranosyl-β-D-glucopyranosyl) ester, was achieved by the extensive 1D and 2D NMR (1H and 13C, TOCSY, HMQC, HMBC) as well as mass spectral data. Further, hydrolysis studies were performed on rebaudioside D3 using acid and enzymatic studies to identify aglycone and sugar residues in its structure. Rebaudioside D3 is detected in the commercial extract of the leaves of Stevia rebaudiana by LC-MS analysis, suggesting rebaudioside D3 is a natural steviol glycoside.

Molecular Analysis of Genetic Fidelity in Micropropagated Plants of <i>Stevia rebaudiana</i>Bert. Using ISSR Marker

Inter-simple sequence repeat (ISSR) markers were used to evaluate the genetic fidelity of in vitro propagated and hardened plants of Stevia rebaudiana Bert. Nodal segments containing axillary buds were used as explant and inoculated on Murashige and Skoog’s (MS) medium containing 3% (w/v) sucrose, 0.8% (w/v) agar supplemented with various concentrations of benzyladenine (BA), kinetin (Kn) and thidiazuron (TDZ) ranging from 0.20 to 2.00 mg·L-1. Maximum multiple shoots (93%) were obtained in MS medium supplemented with 0.20 mg L-1 TDZ. The best in vitro root induction (87%) was achieved on half strength MS medium without any growth regulator. The rooted plantlets were successfully established in soil and grown to maturity at the survival rate of 96% in the indoor grow room. For ISSR analysis, total genomic DNA was extracted from 20 mg fresh leaves of mother and randomly selected in vitro propagated plants. Out of? fifteen arbitrary primers tested, each produced clear and scorable amplification products ranged in size from about 216 bp in UBC 811 to 1917 bp in (GGGGT)3M with an average of 4.5 products per primer. A total of 45 bands (number of plantlets analyzed multiplied by number of bands with all primers) were generated by the ISSR method. All the ISSR profiles from micropropagated plants were monomorphic and comparable to mother plants, confirming the genetic stability among micropropagated plants and mother plant. Chemical analysis, using high-performance liquid chromatography (HPLC), was done to further confirm the existence of qualitative and quantitative differences in the major secondary metabolites (rebaudioside A, stevioside and steviolbioside) between the mother plant and in vitro propagated plants. Our results clearly show similar chemical profiles and insignificant differences in the major secondary metabolites between the two types of plants. These results suggest that the micropropagation protocol followed in this study is appropriate and applicable for clonal mass propagation of true-to-type elite Stevia rebaudiana plants.