Impact of nitrogen supply on growth,steviol glycosides and photosynthesis in Stevia rebaudiana Bertoni

This work investigated the agronomic, physiological and biochemical response of Stevia rebaudiana Bertoni grown under different nitrogen (N) rates. A pot trial in open air conditions was set up in 2012 with the aim to evaluate the effect of four N rates on the biometric and productive characteristics, steviol glycoside (SG) content as well as on leaf gas exchanges, chlorophyll fluorescence, photosynthetic pigments, Rubisco activity and N use efficiency. N deficiency caused a decrease in leaf N content, chlorophylls and photosynthetic CO₂ assimilation, resulting in a lower dry matter accumulation as well as in reduced SG production. The application of 150 kg N ha^? 1 seems to be the most effective treatment to improve rebaudioside A (Reb A) content, Reb A/stevioside ratio, photosynthetic CO₂ assimilation, stomatal conductance, N use efficiency, ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco) and PSII efficiency. The results demonstrate that by using an appropriate N rate it is possible to modulate the SG biosynthesis, with a significant increase in the Reb A content and in the ratio between Reb A and stevioside. This finding is of great relevance in order to obtain a raw material designed to meet consumer needs and bio-industry requirements for high-quality, Reb A content, and safe and environmentally friendly products.     

The positive role of steviol glycosides in stevia (Stevia rebaudiana Bertoni) under drought stress condition

Steviol glycosides (SVglys) are a group of diterpenoids mainly present in the leaves of stevia (Stevia rebaudiana Bertoni). An experiment was conducted to find the functional role of SVglys compounds in stevia affected by drought stress. In this study, a liquid blend of SVglys (200 ppm) was sprayed on stevia plants grown in well-watered (90% field capacity) and drought-stress conditions (45% field capacity) and then the morphological traits and metabolites were evaluated. It was observed that leaf losses caused by drought stress were stopped through external application of SVglys and consequently the harvest index of stevia was increased. Metabolite analysis of stevia leaves showed that the total SVglys content was significantly decreased due to drought stress, but was compensated by external application of SVglys. Among the SVglys, Rebaudioside A responded more to external SVglys. A slight promotion in total antioxidant activity of stevia leaves was observed when external SVglys was applied. The glucose availability in stevia leaves was increased by external application of SVglys but only in well-watered plants. According to our findings, it can be concluded that in stevia, SVglys may have a positive function in drought stress tolerance by exerting a protective role under such conditions.     

Utilisation of steviol glycosides from Stevia rebaudiana (Bertoni) by lactobacilli and bifidobacteria in in vitro conditions

In the current study, eight strains of bifidobacteria and seven strains of lactobacilli were tested for their ability to grow in the presence of rebaudioside A and steviol glycosides from the sweetener Natusweet M001 originating from herb Stevia rebaudiana (Bertoni). Stevia is gaining popularity as a natural, non-caloric sugar substitute, and recently, it was allowed as a food additive by European Union too. Utilisation of steviol glycosides by intestinal microbiota suggests that they might have potential prebiotic effect. Based on the evaluation of bacterial density and pH values in our in vitro study, it was found that lactobacilli and bifidobacteria tested were able to utilise steviol glycosides as a carbon source only to a very limited extent. All strains tested showed significantly lower change in the absorbance A₅₄₀ (P?<?0.05) and pH decrease of the growth media as compared with the positive controls (medium containing glucose as a carbon source and de Man Rogosa Sharpe broth). We concluded that a suggested prebiotic effect was not confirmed either in the case of rebaudioside A or in the case of the sweetener Natusweet M001 containing a mixture of steviol glycosides.     

A comprehensive analysis of fifteen genes of steviol glycosides biosynthesis pathway in Stevia rebaudiana (Bertoni)

Stevia [Stevia rebuaidana (Bertoni); family: Asteraceae] is known to yield diterpenoid steviol glycosides (SGs), which are about 300 times sweeter than sugar. The present work analyzed the expression of various genes of the SGs biosynthesis pathway in different organs of the plant in relation to the SGs content. Of the various genes of the pathway, SrDXS, SrDXR, SrCPPS, SrKS, SrKO and three glucosyltransferases namely SrUGT85C2, SrUGT74G1 and SrUGT76G1 were reported from stevia. Here, we report cloning of seven additional full-length cDNA sequences namely, SrMCT, SrCMK, SrMDS, SrHDS, SrHDR, SrIDI and SrGGDPS followed by expression analysis of all the fifteen genes vis-à-vis SGs content analysis. SGs content was highest in the leaf at 3rd node position (node position with reference to the apical leaf as the first leaf) as compared to the leaves at other node positions. Except for SrDXR and SrKO, gene expression was maximum in leaf at 1st node and minimum in leaf at 5th node. The expression of SrKO was highest in leaf at 3rd node while in case of SrDXR expression showed an increase up to 3rd leaf and decrease thereafter. SGs accumulated maximum in leaf tissue followed by stem and root, and similar was the pattern of expression of all the fifteen genes. The genes responded to the modulators of the terpenopids biosynthesis. Gibberellin (GA₃) treatment up-regulated the expression of SrMCT, SrCMK, SrMDS and SrUGT74G1, whereas methyl jasmonate and kinetin treatment down-regulated the expression of all the fifteen genes of the pathway.     

Study on the bioactivity of steviol and isosteviol in stevia (Stevia rebaudiana Bertoni)

Steviol and isosteviol are diterpene metabolites that are produced from a shared biosynthesis pathway with gibberellins in stevia leaves. These compounds share a chemically similar structure with gibberellins. An experiment was conducted to find the bioactivity properties of steviol and isosteviol in stevia. During vegetative growth, external solutions of steviol and isosteviol (1.6 × 10^?5 and 1 × 10^?5 M, respectively) were sprayed on stevia shoots and then the stevia growth and metabolites were assessed. The results showed that the stevia leaf growth was reduced by external application of steviol. The leaf growth was not affected by external isosteviol while the stevia height and stem dry weight significantly increased. The antioxidant capacity and steviol glycosides content were increased by external steviol. Our results implicated that the isosteviol bioactivity is similar to that of gibberellin hormone. On the other hand, steviol induces the antioxidant system and stimulates the steviol glycosides biosynthesis in stevia leaves.     

Study of gene expression and steviol glycosides accumulation in Stevia rebaudiana Bertoni under various mannitol concentrations

Molecular Biology Reports - Stevia rebaudiana produces sweet steviol glycosides that are 300 times sweeter than sugar and have the beneficial effects on human health including anti-hyperglycaemic....     

Iron nano modulated growth and biosynthesis of steviol glycosides in Stevia rebaudiana

Plant Cell, Tissue and Organ Culture (PCTOC) - Two East Asian grape species Vitis amurensis and Vitis coignetiae have been investigated for their increased cold and drought tolerance and high level...     

Elevated antioxidant potential of chlorocholine chloride-treated in vitro grown Stevia rebaudiana Bertoni

Medicinal plants contain a plethora of secondary metabolites, most of which are bioactive in nature. The role of a popular plant growth retardant CCC has been investigated to explore its impact on secondary metabolite production, particularly phenols and flavonoids from in vitro grown Stevia rebaudiana. CCC stimulated the production of total phenols and flavonoids in calli and leaves. Moreover, this elevated level of phenols and flavonoids was correlated with the antioxidant potential of the tissue extracts. Methanolic extracts from CCC-treated calli and leaves showed significant increment in antioxidant activity as determined by standard DPPH, ABTS, and hydroxyl radical scavenging assays. No significant antiproliferative effect of methanolic extracts from different tissue was noticed against THP-1 monocyte (ATCC-TIB202), Hela cell (ATCC-CCL2) lines endorses the issue of clinical safety of the extracts.     

A beta-glucosidase gene from Stevia rebaudiana may be involved in the steviol glycosides catabolic pathway

Molecular Biology Reports - We herein report the preparation of a full-length raucaffricine-O-beta-d-glucosidase gene of stevia rebaudiana Bertoni (named SrRG1, GenBank accession number MK920450)....     

Photoperiod and elicitors increase steviol glycosides,phenolics, and flavonoid contents in root cultures of Stevia rebaudiana

The establishment of green root cultures of Stevia rebaudiana Bertoni, and the effect of elicitors such as hydrogen peroxide (H₂O₂) and methyl jasmonate (MeJA), is shown in the present study. Stevioside, rebaudioside A, and the isomers steviol/isosteviol were identified through DFI-ESI-IT-MSⁿ and UPLC-TOFMS spectrometric systems, in combination with solid-phase extraction. The accumulation of steviol glycosides increased by 2.4 times (compared to the control value of 22.35 μgSG per gDW), with the addition of 250 μM H₂O₂. The non-enzymatic antioxidant response, which resulted from production of phenolic and flavonoid compounds, was modified based on the elicitor and the dose used. The maximum accumulation of flavonoids was induced on the third day with the addition of H₂O₂ (250 or 500 μM), and with MeJA (250 or 500 μM); the increase was observed on the fifth day. The enzymatic antioxidant response of the catalase and peroxidase from the roots under elicitation confirmed the stress conditions.

     

Production,maintenance and plant regeneration from cell suspension cultures of Stevia rebaudiana (Bert.) Bertoni

A method is described for producing and maintaining Stevia rebaudiana suspensions and regeneration of plants from calli derived from cell suspensions. Suspension cultures composed of isolated cells (ca. 10%) and cellular aggregates (5–100 cells) were obtained in 20–30 days by using friable callus as the initial inoculum in liquid medi with BA (0.5 mg/l)+2,4-D (1.0 mg/l), and periodic filtering (100–500 m sieves) with 6–7 days interval between subcultures. Cultures derived from actively growing calli are mainly diploid (2n=22) whereas those derived from senescent calli showed a wide variation in chromosome number (55–200). Stock cell suspensions which had been maintained for 3 years were plated on basal LS agar medium with BA (0.5 mg/l)+2,4D (0.5 mg/l) to form callus. Calli originating from predominantly 2n cell suspensions when transferred to medium with K (2.0 mg/l)+NAA (0.02 mg/l) were able to form buds. Shoot elongation and further rooting of isolated shoots was better on LS medium devoid of growth regulators. Variation in rooting capacity, plant vigour, morphological characters and chromosome number was found amongst regenerated plants.Abbreviations BA Benzylaminopurine - 2,4-D 2,4 - Dichlorophenoxyacetic acid - GA₃ Gibberellic acid - IAA Indoleacetic acid - IBA Indolebutyric acid - K Kinetin - LS Linsmaier & Skoog     

Selection of suitable propagation method for consistent plantlets production in Stevia rebaudiana (Bertoni)

Stevia rebaudiana (Bert.) is an emerging sugar alternative and anti-diabetic plant in Pakistan. That is why people did not know the exact time of propagation. The main objective of the present study was to establish feasible propagation methods for healthy biomass production. In the present study, seed germination, stem cuttings and micropropagation were investigated for higher productivity. Fresh seeds showed better germination (25.51–40%) but lost viability after a few days of storage. In order to improve the germination percentage, seeds were irradiated with 2.5, 5.0, 7.5 and 10 Gy gamma doses. But gamma irradiation did not show any significant change in seed germination. A great variation in survival of stem cutting was observed in each month of 2012. October and November were found the most suitable months for stem cutting survival (60%). In order to enhance survival, stem cuttings were also dipped in different plant growth regulators (PGRs) solution. Only indole butyric acid (IBA; 1000 ppm) treated cutting showed a higher survival (33%) than control (11.1%). Furthermore, simple and feasible indirect regeneration system was established from leaf explants. Best callus induction (84.6%) was observed on MS-medium augmented with 6-benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid (2,4-D; 2.0 mg l⁻¹). For the first time, we obtained the highest number of shoots (106) on a medium containing BA (1.5 mg l⁻¹) and gibberellic acid (GA₃; 0.5 mg l⁻¹). Plantlets were successfully acclimatized in plastic pots. The current results preferred micropropagation (85%) over seed germination (25.51–40%) and stem cutting (60%).     

Glucosylation of Steviol and Steviol-Glucosides in Extracts from Stevia rebaudiana Bertoni

To evaluate and characterize stevioside biosynthetic pathway in Stevia rebaudiana Bertoni cv Houten, two enzyme fractions that catalyze glucosylation of steviol (ent-13-hydroxy kaur-16-en-19-oic acid) and steviol-glucosides (steviol-13-O-glucopyranoside, steviolbioside and stevioside), utilizing UDP-glucose as the glucose donor, were prepared from the soluble extracts of S. rebaudiana leaves. Enzyme fraction I, passed through DEAE-Toyopearl equilibrated with 50 millimolar K-phosphate pH 7.5, catalyzed the glucosylation to steviol and 19-O-methylsteviol, but not to iso-steviol and 13-O-methylsteviol, indicating that 13-hydroxyl group of the steviol skeleton is glucosylated first from UDP-glucose to produce steviol-13-O-glucopyranoside. Enzyme fraction II, eluted from the DEAE-Toyopearl column with 0.15 molar KCI, catalyzed the glucose transfer from UDP-glucose to steviol-13-O-glucopyranoside, steviolbioside and stevioside, but not to rubusoside (13, 19-di-O-glucopyranoside) and rebaudioside A. The reaction products glucosylated from steviol-13-O-glucopyranoside, steviolbioside and stevioside were identified to be steviolbioside, stevioside and rebaudioside A, respectively. These results indicate that in the steviol-glucoside biosynthetic pathway, steviol-13-O-glucopyranoside produced from the steviol glucosylation is successively glucosylated to steviolbioside, then to stevioside producing rebaudioside A.     

Efficient regeneration for enhanced steviol glycosides production in Stevia rebaudiana (Bertoni)

An efficient method of regeneration for antidiabetic plant (Stevia rebaudiana) has been established for healthy biomass and main steviol glycosides (SGs) production, using different PGRs and agar concentrations. Higher callus induction (93.3%) was recorded when leaf explants were placed on an MS medium supplemented with 3.5 gL⁻¹ agar and 2.0 mgL⁻¹ 2,4-D. The addition of 7.0 gL⁻¹ agar and BA (1.0, 2.0 and 4.0 mgL⁻¹) significantly (P < 0.01) influences shooting response (100%). A maximum mean shoot length (13.03 cm) and 28 shoots per explant were observed on a medium containing 1.0 mgL⁻¹ BA. However, the maximum number of leaves (132.67) was encouraged by the addition of BA (1.0 mgL⁻¹) and Kin (1.0 mgL⁻¹). Lower agar (3.5 gL⁻¹), IAA (2.0 mgL⁻¹), and NAA (2.0 mgL⁻¹) concentrations significantly influence the rooting percent (100%), the mean root length (2.9 cm), and the number of roots per plantlet (26.3). These plantlets were successfully acclimatized in the soil. The BA (3.0 mgL⁻¹) in combination with Kin (3.0 mgL⁻¹) and 3.5 gL⁻¹ agar increases dulcoside-A content (Dul-A; 71.8 μg/g-DW) in shoots compared to control (50.81 μg/g-DW). Similar PGRs with 7.0 gL⁻¹ significantly increases the production of steviosides (Stev. 82.48 μg/g-DW). A higher rebaudioside-A content (Reb-A; 12.35 μg/g-DW) was observed in shoots that underwent the addition of BA (1.0 mgL⁻¹) and 7.0 gL⁻¹ agar than in control (07.39 μg/g-DW). Hereby, we developed an efficient and cost-effective method for regeneration and major SGs production, which could be helpful for future studies on this species.     

Stimulation of steviol glycoside accumulation in Stevia rebaudiana by red LED light

The aim of this study was to determine whether steviol glycoside accumulation is under phytochrome control. The results indicate that Stevia rebaudiana Bertoni plants grown under short-day conditions showed precocious flowering and stagnation of steviol glycoside accumulation. Long night interruption by red LED light stimulated and sustained the vegetative growth as well as the accumulation of steviol glycosides in the leaves. After 7 weeks of treatment, steviol glycoside content was about two-fold higher in LED-treated plants than in the short-day control group. The effects of red LED light were measured both in a greenhouse and in a phytotron, irrespective of cultivar-specific differences. Therefore, it can be concluded that a mid-night interruption by red LED light during short photoperiods provides an easy and inexpensive method to increase vegetative leaf biomass production with an increased steviol glycoside yield.     

盐胁迫对甜菊细胞内游离Ca^2＋浓度的影响（简报）

Fluo-3/AM, a calcium indicator, was introduced by low temprature loading method into callus protoplasts of Stevia rebaudiana Bertoni. The microscope observation results showed that NaCl in different concentrations (30-200mmol/L) can elevate the intracellular calcium concentration in protoplasts. The elevation was related to the amount of salinity. The effect of salinity stress was inhibited by LiCl pretreatment but restored by inositol pretreatment. This suggested that salinity stress promoted the cytoplasmic calcium activity, perhaps by activating the phosphoinoditide system. As a result of salinity stress signals, elevated calcium activity may trigger corresponding metabolic changes, such as synthesis of enzyme via activating other members of calcium signal system, fit the cells for the change of the environment.     

Melatonin alleviates NaCl-induced damage by regulating ionic homeostasis,antioxidant system,redox homeostasis,and expression of steviol glycosides-related biosynthetic genes in in vitro cultured Stevia rebaudiana Bertoni

In Vitro Cellular & Developmental Biology - Plant - Melatonin has been shown to play a variety of roles in regulating plant growth and response to various abiotic stresses, but its main...