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Background Stevia rebaudiana produces sweet-tasting steviol glycosides (SGs) in its leaves which can be used as natural sweeteners. Metabolic engineering of Stevia would offer an alternative approach to conventional breeding for enhanced production of SGs. However, an effective protocol for Stevia transformation is lacking. Results Here, we present an efficient and reproducible method for Agrobacterium -mediated transformation of Stevia. In our attempts to produce transgenic Stevia plants, we found that prolonged dark incubation is critical for increasing shoot regeneration. Etiolated shoots regenerated in the dark also facilitated subsequent visual selection of transformants by green fluorescent protein during Stevia transformation. Using this newly established transformation method, we overexpressed the Stevia 1-deoxy-d-xylulose-5-phosphate synthase 1 ( SrDXS1 ) and kaurenoic acid hydroxylase ( SrKAH ), both of which are required for SGs biosynthesis. Compared to control plants, the total SGs content in SrDXS1- and SrKAH- overexpressing transgenic lines were enhanced by up to 42–54% and 67–88%, respectively, showing a positive correlation with the expression levels of SrDXS1 and SrKAH . Furthermore, their overexpression did not stunt the growth and development of the transgenic Stevia plants. Conclusion This study represents a successful case of genetic manipulation of SGs biosynthetic pathway in Stevia and also demonstrates the potential of metabolic engineering towards producing Stevia with improved SGs yield.

Background The glucosyltransferase UGT76G1 from Stevia rebaudiana is a chameleon enzyme in the targeted biosynthesis of the next-generation premium stevia sweeteners, rebaudioside D (Reb D) and rebaudioside M (Reb M). These steviol glucosides carry five and six glucose units, respectively, and have low sweetness thresholds, high maximum sweet intensities and exhibit a greatly reduced lingering bitter taste compared to stevioside and rebaudioside A, the most abundant steviol glucosides in the leaves of Stevia rebaudiana . Results In the metabolic glycosylation grid leading to production of Reb D and Reb M, UGT76G1 was found to catalyze eight different reactions all involving 1,3-glucosylation of steviol C 13 - and C 19 -bound glucoses. Four of these reactions lead to Reb D and Reb M while the other four result in formation of side-products unwanted for production. In this work, side-product formation was reduced by targeted optimization of UGT76G1 towards 1,3 glucosylation of steviol glucosides that are already 1,2-diglucosylated. The optimization of UGT76G1 was based on homology modelling, which enabled identification of key target amino acids present in the substrate-binding pocket. These residues were then subjected to site-saturation mutagenesis and a mutant library containing a total of 1748 UGT76G1 variants was screened for increased accumulation of Reb D or M, as well as for decreased accumulation of side-products. This screen was performed in a Saccharomyces cerevisiae strain expressing all enzymes in the rebaudioside biosynthesis pathway except for UGT76G1. Conclusions Screening of the mutant library identified mutations with positive impact on the accumulation of Reb D and Reb M. The effect of the introduced mutations on other reactions in the metabolic grid was characterized. This screen made it possible to identify variants, such as UGT76G1 Thr146Gly and UGT76G1 His155Leu , which diminished accumulation of unwanted side-products and gave increased specific accumulation of the desired Reb D or Reb M sweeteners. This improvement in a key enzyme of the Stevia sweetener biosynthesis pathway represents a significant step towards the commercial production of next-generation stevia sweeteners.

Enhanced production of steviol glycosides (SGs) was observed in callus and suspension culture of Stevia rebaudiana treated with proline and polyethylene glycol (PEG). To study their effect, yellow-green and compact calli obtained from in vitro raised Stevia leaves were sub-cultured on MS medium supplemented with 2.0 mg l⁻¹ NAA and different concentrations of proline (2.5–10 mM) and PEG (2.5–10 %) for 2 weeks, and incubated at 24 ± 1 °C and 22.4 μmol m⁻² s⁻¹ light intensity provided by white fluorescent tubes for 16 h. Callus and suspension culture biomass (i.e. both fresh and dry weight content) was increased with 5 mM proline and 5 % PEG, while at further higher concentrations, they got reduced. Further, quantification of SGs content in callus (collected at 15th day) and suspension culture (collected at 10th and 15th day) treated with and without elicitors was analysed by HPLC. It was observed that chemical stress enhanced the production of SGs significantly. In callus, the content of SGs increased from 0.27 (control) to 1.09 and 1.83 % with 7.5 mM proline and 5 % PEG, respectively, which was about 4.0 and 7.0 times higher than control. However, in the case of suspension culture, the same concentrations of proline and polyethylene glycol enhanced the SG content from 1.36 (control) to 5.03 and 6.38 %, respectively, on 10th day which were 3.7 times and 4.7 times higher than control.

The ability to synthesize particular steviol glycosides (SvGls) was studied in Stevia rebaudiana Bertoni hairy roots (HR) grown in the light or in the dark under the influence of different osmotic active compounds. Manipulation of culture conditions led to changes in the morphology and growth rate of HR, as well as to an increase in oxidative stress manifested as an enhancement in endogenous hydrogen peroxide concentration in the cultured samples. The highest level of H2O2 was noted in HR cultured under light or in the medium with the highest osmotic potential. This correlated with the highest increase in the expression level of ent-kaurenoic acid hydroxylase, responsible for the redirection of metabolic route to SvGls biosynthesis pathway. An analysis of transcriptional activity of some UDPglucosyltransferase (UGT85c2, UGT74g1, UGT76g1) revealed that all of them were upregulated due to the manipulation of culture conditions. However, the level of their upregulation depended on the type of stress factor used in our experiment. Analysis of SvGls content revealed that HR grown under all applied conditions were able to synthesize and accumulate several SvGls but their concentration differed between the samples across the different conditions. The level of rebaudioside A concentration exceeded the content of stevioside in HR in all tested conditions. Concomitantly, the presence of some minor SvGls, such as steviolbioside and rebaudioside F, was confirmed only in HR cultured in the lowest osmotic potential of the medium while rebaudioside D was also detected in the samples cultured in the media supplemented with NaCl or PEG.Key Points● Several steviol glycosides are synthesized in hairy roots of S. rebaudiana.● Light or osmotic factors cause enhancement in oxidative stress level in hairy roots.● It correlates with a significant increase in the level of KAH expression.● UGTs expression and steviol glycosides content depends on culture conditions.

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.

Stevia rebaudiana (Bertoni), commonly known as stevia, is a sought-after natural sweetener, but its conventional propagation methods are slow and inefficient. This study aims to enhance the in vitro culture for stevia by investigating the impact of different Murashige and Skoog (MS) medium salt strengths and plant growth hormones on growth and rebaudioside A content. Apical bud-containing shoot segments were used as explants and cultured on various semi-solid and liquid MS media formulations, incorporating cytokinins (BAP and Kin), auxins (NAA and IAA), and different MS major salt concentrations (MS full, ½ MS, and ¼ MS). Assessments of shoot growth parameters, root formation, and HPLC analysis for rebaudioside A content were conducted. The optimal conditions for in vitro growth was found to be in the ¼ MS + Kin 3 mg/L + NAA 0.1 mg/L (semi-solid) medium, resulting in significantly improved shoot growth and enhanced 30.04% rebaudioside A content. Genetic fidelity of regenerated plants was confirmed using RAPD and ISSR markers. These findings offer valuable insights for optimizing in vitro propagation of stevia and potentially enhancing rebaudioside A content.

Two steviol glycosides containing quinovose were isolated from a biotransformation mixture of stevia extract derived from Stevia rebaudiana Bertoni leaves. These compounds were elucidated using comprehensive spectroscopic techniques, including nuclear magnetic resonance (NMR) and mass spectrometry (MS). These compounds were designated as Rebaudioside QM and the novel Rebaudioside 2QM. Based on structural similarity, we hypothesize that Stevioside E may serve as a biosynthetic precursor for Rebaudioside QM. Comprehensive LC-MS profiling also suggests potential precursors for Rebaudioside 2QM. Tentative biosynthetic pathways were proposed for both compounds. The presence of these unknown compounds further supports the notion that S. rebaudiana harbours a wide array of yet-undiscovered steviol glycosides, potentially driven by the inherent diversity of UDP-dependent glycosyltransferases (UGTs) within the plant itself. The discovery of Rebaudioside QM and Rebaudioside 2QM expands the known diversity of steviol glycosides and provides new insights into glycosylation patterns in S. rebaudiana, which may support the development and production of novel sweeteners with improved sensory and physicochemical properties.

The use of in vitro cultures in plant breeding allows for obtaining cultivars with improved properties. In the case of Stevia rebaudiana Bert., genotypes with an appropriate rebaudioside A/stevioside ratio are desirable. The use of indirect organogenesis allows for the induction of somaclonal variation, which, consequently, results in obtaining variability within the regenerants. The Murashige and Skoog medium containing 4.0 mg × dm−3 6-benzylaminopurine (BAP), 2.0 mg × dm−3 1-naphthaleneacetic acid (NAA), and 2.0 mg × dm−3 2,4-dichlorophenoxyacetic acid (2,4-D) resulted in obtaining plants that were biochemically and genetically diverse. The obtained regenerants were characterized by an increased content of rebaudioside A and a better rebaudioside A/stevioside ratio. Genetic analysis using SCoT (start-codon-targeted) markers showed their diversity at the molecular level. Moreover, this study showed that genotype multiplication through six subsequent re-cultures does not cause variability at the genotype level and does not affect the steviol glycoside profile. This study is the first report on obtaining genotypes with higher rebaudioside A content and a more attractive rebaudioside A to stevioside ratio through the use of in vitro cultures. The improved regenerants can be used as parents in hybridization programs or directly as valuable new genotypes.

Stevia (Stevia rebaudiana) has been employee for developing food products as a substitute for sucrose, low caloric, and natural sweetener. Different studies have evaluated the effect of this ingredient on the physicochemical, nutritional and technological properties of products; their application in dairy products affects some sensory characteristics such as taste; in the case of bread, cookies, and cakes modify the properties of the dough, altering attributes of the manufactured product, including color, texture, and flavor; also the use of stevia reduces gelling and affects the optical properties of the final product; and in beverages cases, the total substitution of sugar has led to bitter and unpleasant flavor. This review presents stevia general information and its employees in dairy, bakery, beverages, and jelly products.

Background The UDP-glucuronosyltransferase 91D2 ( SrUGT91D2 ) gene is a crucial element in the biosynthetic pathway of steviol glycosides (SGs) and is responsible for creating 1,2-β-D glucosidic bonds at the C19 and C13 positions. This process plays a vital role in the synthesis of rebaudioside M (RM) and rebaudioside D (RD). The promoter, which regulates gene expression, requires functional analysis to understand gene expression regulation. However, investigations into the function of the promoter of SrUGT91D2 ( pSrUGT91D2 ) have not been reported. Results The pSrUGT91D2 was isolated from six S. rebaudiana lines, and subsequent multiple sequence comparisons revealed the presence of a 26 bp inDel fragment ( pSrUGT91D2 -B1188 type) in lines GP, GX, 110, 1114, and B1188 but not in the pSrUGT91D2 of line 023 ( pSrUGT91D2 -023 type). Bioinformatics analysis revealed a prevalence of significant cis-regulatory elements (CREs) within the promoter sequences, including those responsive to abscisic acid, light, anaerobic conditions, auxin, drought, low temperature, and MeJA. To verify the activity of pSrUGT91D2 , the full-length promoter and a series of 5’ deletion fragments (P1–P7) and a 3’ deletion fragment (P8) from various lines were fused with the reporter β-glucuronidase (GUS) gene to construct the plant expression vector, pCAMBIA1300-pro∷GUS. The transcriptional activity of these genes was examined in tobacco leaves through transient transformation. GUS tissue staining analysis and enzyme activity assays demonstrated that both the full-length promoter and truncated pSrUGT91D2 were capable of initiating GUS expression in tobacco leaves. Interestingly, P8-pSrUGT91D2 -B1188 (containing the inDel segment, 301 bp) exhibited enhanced activity in driving GUS gene expression. Transient expression studies of P8-pSrUGT91D2 -B1188 and P8-pSrUGT91D2 -023 in response to exogenous hormones (abscisic acid and indole-3-acetic acid) and light indicated the necessity of the inDel region for P8 to exhibit transcriptional activity, as it displayed strong responsiveness to abscisic acid (ABA), indole-3-acetic acid (IAA), and light induction. Conclusions These findings contribute to a deeper understanding of the regulatory mechanism of the upstream region of the SrUGT91D2 gene and provide a theoretical basis for future studies on the interaction between CREs of pSrUGT91D2 and related transcription factors.