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.

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.

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.

High-yield Cultivation Techniques of Stevia rebaudiana(Bertoni) Hemsl. in Gannan Area of Jiangxi Province

In order to expand the production of Stevia rebaudiana( Bertoni) Hemsl.,the main points of high-yield cultivation techniques of a two-year-old stevia cultivar Shoutian No. 3 in Gannan area of Jiangxi Province were summarized: cutting and transplanting in early spring; fine management,applying more basic and additional fertilizers and retaining stubbles two times in summer,retaining roots for seeding in late autumn,and mulching membrane for safe overwintering in winter. This study will provide practical guidance for the planting of S. rebaudiana in Ganzhou,Jiangxi Province.

甜菊(Stevia rebaudiana)糖基转移酶基因的克隆及序列分析

利用与植物次生代谢产物糖基化相关的UDPG糖基转移酶的特征性保守区域 ,设计了同源简并引物 ,以甜菊基因组DNA为模板 ,PCR扩增获得甜菊糖基转移酶基因片段 .根据获得的基因片段设计引物GSTr和GSTf,分别与cDNA文库的T3 和T7通用引物扩增获得全长核苷酸序列的甜菊糖基转移酶基因 ,定名为sudpt 2 ,该基因全长 16 6 2bp ,包括poly(A)和一个 136 2bp的开放阅读框 ,编码 45 4个氨基酸 .与常见的糖基转移酶基因的相似性达 44 %～ 77% ,且具有UDPG糖基转移酶的特征性保守序列 .分子发育进化分析表明 。

Steviol Glycoside Content Dynamics during the Growth Cycle of Stevia rebaudiana Bert

The sweetener compounds in Stevia, steviol glycosides (SG), are mainly found in the leaves. The SG content depends on the plant’s growth stage and is usually highest just before flowering. In temperate areas, Stevia is a polyannual crop (5 - 7 years) with a vegetative period lasting from April-May until October, during which time the crop can be harvested two or three times. This research focuses on the need for knowledge about Stevia’s response to temperate climates in Southern Europe. Two field assays were established from June to October 2013 at two sites in Navarra (Spain). The biomass and content of the two major SG, stevioside (ST) and rebaudioside A (RA), were measured using High Performance Liquid Chromatography (HPLC) in 66 cloned plants, at different developmental stages. Although the results from the two sites showed different SG leaf content dynamics during the plant growth, the optimum harvest date at both sites coincided with the bud-flowering stage at the beginning of September (around 96 days after planting), when a ST yield of 27 g·m-2 was reached. These results show Stevia’s potential as a commercial crop for stevioside production in northern Spain.

Comparison of Chemical and Functional Properties of Stevia rebaudiana(Bertoni)Varieties Cultivated in Mexican Southeast

The leaf powders from two varieties of Stevia rebaudiana (Bertoni) cultivated in Yucatan, Mexico were analyzed for their proximate composition, dietary fiber composition and functional properties. The leaf powders were a good source of carbohydrates (64.06%-67.98%), protein (12.11%-15.05%), and crudefiber (5.92%-9.52%). Total dietary fiber content in the S. rebaudiana leaf powders were 28.61 (Morita II) and 29.12 (Criolla) g/100g sample, with most of this content represented by insoluble dietary fiber 87.79% (Morita II) and 70.02% (Criolla). Neutral detergent fiber (NDF), acid detergent fiber (ADF) and acid detergent lignin (ADL) were higher in Criolla (19.29%, 17.77%, 8.98%) than Morita II variety (18.11%, 14.16%, 2.28%). Hemicellulose and cellulose were higher in Morita II (3.96%, 11.78%) than criolla variety (1.51%, 8.79%). Functional properties of leaf powder from Morita II and Criolla were, water-holding capacity (2.87-4.07 g/g sample), oil-holding capacity (6.49-6.79 g/g sample), water-absorption capacity (3.41-3.44 g/g sample), water-adsorption capacity (0.25-0.28 g/g sample), and organic molecule absorption capacity (1.13-1.81 g/g sample). These suggest that S. rebaudiana (Bertoni) leaf powders may be used as dietary supplement or as food additive.

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.

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.

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.

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.

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.

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.

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.

Assessment of <i>Stevia rebaudiana</i>Bertoni Genotypes via Morpho-Agronomic Traits under Two Light Conditions

Stevia rebaudiana Bertoni, a non caloric natural sweetner is currently the available substitute to sugar. It is a genus of about 150 species of herbs and shrubs, a compositae and native to Paraguay. However, stevia is a short day plant with almost a constant day length 12 hours in Malaysia stevia generated to flower early. Therefore, the aim of the study was to investigate the effect of different day length on Stevia rebaudiana genotypes in Malaysia. The experiment was undertaken during December 2016 to April 2017 in the Faculty of Design and Architecture, Universiti Putra Malaysia, Serdang, Selangor, Malaysia. Eight stevia genotypes namely (CHV1, Morita2, MS012, MS012 (4x), SA18, TPU, and hybrids IIUM-F1 and UiTM-H2) were evaluated for the agronomic traits under two conditions: sun light (<12 hours) and simulated light (>14 hours) using a factorial experiment. The agronomic performance of Stevia rebaudiana was significantly in long day length (>14 hours) environment comparing with short day length (<12 hours) environment. Stevia plants under sun light start to flower after seven weeks, from the other hand stevia plants under simulated light did not flower. All stevia genotypes showed a high performance under long day length (>14 hours) environments.

High frequency multiplication of shoots using axillary buds for production of elite lines of <i>Stevia rebaudiana</i>

An efficient repeatable protocol has been developed for rapid clonal multiplication of an endemic, perennial herb Stevia rebaudiana using juvenile axillary buds from in vivo grown seedlings. MS basal medium supplemented with BAP (8.88 μM) and NAA (5.37 μM) was found to be the most suitable medium for initiation of shoots with 92% response. The repeated subculture resulted in high-frequency shoot multiplication (9.82 ± 1.93) at the average rate of 3-fold per subculture. The axenic multiple shoots were rooted ex-vitro on horticultural grate soilrite mix-peat:perlite:vermiculate (1:1:1 v/v) with half strength MS salts fortified with NAA (5.37 μM). The complete plantlets were successfully established in soil with 86% survival frequency. The statistical analysis of the data pertaining to multiple shoots and root formation revealed significant difference between and within the treatments.

Metal Salts Assisted Enzyme-Based Extraction of Stevioside from the Leaves of <i>Stevia rebaudiana</i>Bertoni

Stevioside, extracted from the leaves of Stevia rebaudiana Bertoni, is a natural, high intensity, lowcaloric sweetener with wide therapeutic activities. Conventional stevioside extraction methodologies involve the use of non-green solvents, supercritical fluids, microwaves, etc., however, all these processes are expensive, time-consuming and eco-unfriendly. Therefore an alternative process is desired for the isolation of stevioside. In this study, a novel enzyme-mediated extraction (EME) method has been developed. The dry stevia leaves were pre-treated with hydrolytic enzymes aided by transition metal salts (FeCl3). This was followed by pressurized hot water extraction (PHWE) to release stevioside. The crude extract was purified and clarified through multi-stage membrane filtration. The results confirm that metal salt-assisted cellulase pre-treatment enhanced the yield of stevioside to 72%, with 98% purity, which was higher in comparison to existing methods. Thus, the methodology developed establishes a simple, “green”, enzyme-mediated process for the efficient isolation of stevioside under economical and eco-friendly conditions.