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The genetic stability of Stevia rebaudiana Bertoni plants, propagated through in vitro stored synthetic seeds was assessed. In vitro grown micro shoots of Stevia rebaudiana were encapsulated in 5% sodium alginate and 50 mM CaCl2. The synthetic seeds were stored in vitro for 4 months and re-grown under the tissue culture conditions (16-h photoperiod, 25 oC) on Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ 0.2 mg L-1) [1]. Well rooted plantlets were successfully transferred to a climatic controlled indoor cultivation facility for further cultivation with mother plant. Leaf samples were taken from nine, randomly selected, fully developed, healthy in vitro propagated plants and a mother plant for the molecular analysis of genetic fidelity using eight inter simple sequence repeat (ISSR) markers. Each tested primer produced clear and scorable amplification products ranging in size from about 176 bp (UBC 817) to 1354 bp (UBC 826) with an average of 5.1 products per primer. A total of 41 bands were generated, giving rise to monomorphic patterns across all 10 plantlets analyzed. No ISSR polymorphism was observed in the micropropagated plants. Furthermore, leaf samples from the plants used for molecular analysis were also evaluated for rebaudioside A and stevioside content using high performance liquid chromatography (HPLC) [2]. Our data show no significant differences (p < 0.05) in rebaudioside A and stevioside content among re-grown plants and the mother plant following four months of in vitro storage, thus confirming the clonal fidelity of Stevia rebaudiana plants. Acknowledgement: This research was partially funded by a grant from the USDA, Agricultural Research Service Specific Cooperative Agreement No. 58 – 6408 – 1-603 – 07. References: [1] Hemant Lata, Suman Chandra et al. (2013) American Journal of Plant Sciences, 4: 117 – 128. [2] Yan-Hong Wang, Bharathi Avula et al. (2015) Food Additives & Contaminants: Part A, 32: 674 – 685.

The purpose of the present study was to compare the effect of the oral treatment (gavage) with Stevia rebaudiana (Bert.) Bertoni (SRB) and stevioside (STV) on glycaemia and gluconeogenesis of 15-h fasted rats. For this purpose, the rats received SRB (20 mg/kg x day), STV (5.5 mg/kg x day) or an equal volume of water (controls) during 15 days. To measure hepatic gluconeogenesis, liver perfusion and isolated hepatocytes were used. Glycaemia and gluconeogenesis from L-alanine (5 mM), L-glutamine (5 mM) and L-lactate (2 mM) were decreased (P < 0.05) after pre-treatment with SRB. However, the treatment with STV did not influence glycaemia and gluconeogenesis. Moreover, to get further information about the mechanism by which SRB leaves inhibit gluconeogenesis their potential role as a PPARgamma agonist was investigated. The data showed absence of activation of PPARgamma receptors. In summary, our results showed that the reduction of glycaemia promoted by the treatment with SRB leaves was mediated, at least in part, by an inhibition of hepatic gluconeogenesis. However, this effect did not involve stevioside and the activation of PPARgamma receptors.

We have studied the effects of stevioside on the glucose and insulin metabolism in 2 models of diabetes in rats, STZ-induced diabetic rats and NIDDM diabetic rats induced by feeding with fructose. Stevioside (0.5 mg/kg), lowered the blood glucose levels in STZ-induced diabetic rats, peaking at 90 min. Stevioside administered twice daily also demonstrated dose-dependent effects in lowering the glucose levels in both diabetic rat models. Stevioside reduced the rise in glucose during glucose tolerance testing in normal rats. Stevioside dose-dependently decreased protein levels of phosphoenol pyruvate carboxykinase (PEPCK) and PEPCK mRNA after 15 days of treatment. Stevioside also reduced insulin resistance in the diabetic animals as shown by the glucose lowering effects of tolbutamide. In conclusion, stevioside was able to regulate blood glucose levels by enhancing not only insulin secretion, but also insulin utilization in insulin-deficient rats; the latter was due to decreased PEPCK gene expression in rat liver by stevioside's action of slowing down gluconeogenesis. Further studies of this agent for the treatment of diabetes appear warranted.

Isosteviol is a derivative of stevioside, a constituent of Stevia rebaudiana, and is commonly used as a non-caloric sugar substitute in Japan and Brazil. The present study attempted to elucidate the role of potassium (K (+)) channels in the action of isosteviol on intracellular calcium concentrations ([Ca (2+)]i) in cultured vascular smooth muscle (A7r5) cells using the Ca (2+)-sensitive dye Fura-2 as an indicator. The increase of [Ca (2+)]i in A7r5 cells produced by vasopressin (1 micromol/L) or phenylephrine (1 micromol/L) was attenuated by isosteviol from 0.01 micromol/L to 10 micromol/L. The attenuation by isosteviol of the vasopressin- and phenylephrine-induced increase in [Ca (2+)]i was inhibited by glibenclamide, apamin and 4-aminopyridine but not by charybdotoxin. Furthermore, the inhibitory action of isosteviol on [Ca (2+)]i was blocked when A7r5 cells co-treated with glibenclamide and apamin in conjunction with 4-aminopyridine were present. Therefore, not only did the ATP-sensitive potassium (K (ATP)) channel affect the action of isosteviol on [Ca (2+)]i modulation in A7r5 cells, but also those on the small conductance calcium-activated potassium (SK (Ca)) channels and voltage-gated (Kv) channels. However, the blockers of large-conductance Ca (2+)-activated potassium channels failed to modify the inhibitory action of isosteviol on [Ca (2+)]i. The obtained results indicated that a decrease of [Ca (2+)]i in A7r5 cells by isosteviol is mainly mediated by the selective opening of K (ATP) channel or/and SK (Ca) channel. Alteration in the Kv channel also plays a critical role in the inhibitory action of isosteviol.

Stevioside is a sweet-tasting glycoside occurring abundantly in the leaves of Stevia rebaudiana (Compositae). It has been used popularly in Japan and Brazil as a sugar substitute for decades. Previous study has shown that it lowered blood pressure in spontaneously hypertensive rats (SHRs) when administered intravenously. This study shows that intraperitoneal injection of stevioside 25 mg/kg also has antihypertensive effect in SHRs. In isolated aortic rings from normal rats, stevioside could dose-dependently relax the vasopressin-induced vasoconstriction in both the presence and absence of endothelium. However, stevioside had no effect on phenylephrine- and KCl-induced phasic vasoconstriction. In addition, stevioside lost its influence on vasopressin-induced vasoconstriction in Ca(2+)-free medium. The results indicate that stevioside caused vasorelaxation via an inhibition of Ca(2+) influx into the blood vessel. This phenomenon was further confirmed in cultured aortic smooth muscle cells (A7r5). Using 10(-5) M methylene blue for 15 min, stevioside could still relax 10(-8) M vasopressin-induced vasoconstriction in isolated rat aortic rings, showing that this vasorelaxation effect was not related to nitric oxide. The present data show that the vasorelexation effect of stevioside was mediated mainly through Ca(2+) influx inhibition.

Antihypertensive and antidiabetic effects of stevia, Stevia rebaudiana (Asteraceae), have been demonstrated in several human and animal models. The current study aims to define steviaʼs role in modifying the electrophysiological and mechanical properties of cardiomyocytes, blood vessels, and gastrointestinal smooth muscle. Tissues from thoracic aorta, mesenteric arteries, ileum, and left ventricular papillary muscles were excised from 8-week-old healthy Wistar rats. The effects of stevia (1 × 10 −9 M to 1 × 10 −4 M) were measured on these tissues. Steviaʼs effects in the presence of verapamil, 4-AP, and L-NAME were also assessed. In cardiomyocytes, stevia attenuated the force of contraction, decreased the average peak amplitude, and shortened the repolarisation phase of action potential – repolarisation phase of action potential 20 by 25 %, repolarisation phase of action potential 50 by 34 %, and repolarisation phase of action potential 90 by 36 %. Stevia caused relaxation of aortic tissues which was significantly potentiated in the presence of verapamil. In mesenteric arteries, incubation with L-NAME failed to block stevia-induced relaxation indicating the mechanism of action may not be fully via nitric oxide-dependent pathways. Stevia concentration-dependently reduced electrical field stimulated and carbachol-induced contractions in the isolated ileum. This study is the first to show the effectiveness of stevia in reducing cardiac action potential duration at 20 %, 50 %, and 90 % of repolarisation. Stevia also showed beneficial modulatory effects on cardiovascular and gastrointestinal tissues via calcium channel antagonism, activation of the M 2 muscarinic receptor function, and enhanced nitric oxide release.

In vitro propagation has been extensively used for the rapid multiplication of many medicinal plants. However, its wider use is often restricted by the high percentage of plant loss or damage when transferred to ex vitro conditions. In the present study, a successful attempt has been made to acclimatize the tissue culture raised plants of Stevia rebaudiana Bert. under climate controlled conditions with a 95% survival rate. Well rooted cultures were carefully taken out of the medium, washed thoroughly and pre-incubated in coco natural growth medium (Canna-Continental, Los Angeles, CA, www. canna-hydroponics.com) in small jiffy pots for 10 days at 25oC and 18 hours under cool florescent lights. The pots were covered with polythene bags to maintain humidity. These plants were then transferred to a sterile potting mix, fertilome (Canna-Continental, Los Angeles, CA), in larger pots. All these plantlets were kept under controlled environmental conditions (Temperature, 25±3oC; light, ˜800µmol m-2s-1 and RH, 55±5%) with mother plants in the indoor cultivation facility at Coy-Waller laboratory, University of Mississippi. Well grown tissue culture raised plants were then evaluated for their ecophysiological (photosynthesis, transpiration, stomatal conductance, internal CO2 concentration and water use efficiency) and leaf anatomical (stomatal length, width, frequency and index; leaf transverse section; epidermal cells; palisade cells and spongy parenchyma cells) characteristics and were compared with the mother plant. Our results reveal no statistical differences in gross morphology, ecophysiology and anatomical characteristics of the two types of plants. Acknowledgements: This research was partially funded by the USDA, Agricultural Research Service Specific Cooperative Agreement No. 58–6408–7-012.

Stevia rebaundiana (Bert.) is an important source of natural sweetener. This perennial species belongs to the Asteraceae family and is native to certain regions of South America. Plant, extracts, and glycosides have been used for several years as natural sweeteners. Several studies revealed that Stevia exerts beneficial effects on human health, including antihypergycemic, antihypertensive, noncariogenic, anti-human rotavirus activities [1], while there is little information reported on crop production. It is important to mention that for pharmaceutical use, the plant material must be free of contamination from heavy metals, soil and soil-born organisms, herbicides and pesticides. Thus, hydroponics may provide a suitable growing system for high quality biomass production while allowing the regulation of secondary metabolism by managing the nutrient solution. Two experiments were carried out in the greenhouse at the University of Mississippi's Biological Field Station. The experimental design was a randomized complete block with a split-plot in time arrangement of the treatments, in which the main treatments were the growth systems (floating hydroponic versus soil) and the nitrogen level (200mg L–1 and 300mg L–1 of N in hydroponic; 20 and 70kg ha–1 of N (ammonium nitrate) in soil-filled pots. Seedlings were transplanted in 07/24/09 and the first harvest was done in 09/14/09. Secondary treatments were conducted at harvest times (45 and 90 days after transplanting). Fresh and dried biomass, water consumption and macronutrients analysis were evaluated. In conclusion, the biomass yields were significantly higher using the hydroponic cultivation method as compared to soil and no significant differences were found between levels of nitrogen treatment. Acknowledgments: Partial financial support from the USDA Specific Cooperative Agreement No 58–6408–7-012. Reference: [1] Tadhani MB, Patel VH, et al. (2007)J Food Comp Anal, 20(3/4): 323–329.

Stevia is a generic term for extracts from the herb Stevia rebaudiana (Bertoni), while the sweet components are more precisely known as steviol glycosides. Long-standing questions about the specifications or characterization of the materials, safety, and special population effects have previously prevented steviol glycosides from being considered a mainstream natural sweetener. In order to provide the answers as well as bridge to the safety gaps, a strategic step-wise, research program was undertaken. Essential elements of the program included: complete characterization of the ingredient, general and reproductive toxicology, metabolism and pharmacokinetic analysis, clinical research, intake/exposure assessment, assurance of appropriate GMP to support specifications, and stability in food systems. A holistic approach to the communication of technical and scientific supporting data was used to ensure general recognition of safety by qualified individuals (GRAS). Efforts are ongoing to promote consistent quality standards within the industry, and to provide due diligence with respect to safety from the post-marketing perspective.

Growth and development of Stevia rebaudiana shoots cultivated in the roller bioreactor and their production of steviol glycosides (SGs) were investigated. It was found that, owing to the highly favorable conditions of shoot cultivation created in such an apparatus, the intensity of shoot growth and SG production appeared to be 1.5 - 2.0 times higher than those of the shoots grown in tubes. These results indicate the existence of a positive correlation between these two processes. The data obtained suggest that the enhanced SG production is due to the differentiation of chlorenchyma cells and formation of specific subcellular structures for the glycoside to be accumulated.

A multiple shoot culture was induced from nodal segments on MS medium containing half concentration of macro-elements, 1 % sucrose, and supplemented with NAA (0.01 mg/ I). A bioreactor with hormone-free MS medium (300 ml) was inoculated with 1.5 g of the multiple shoot culture and cultivated for a month. The cultivating process of the multiple shoot culture in the bioreactor and the transfer into ex vitro conditions took about 8-9 weeks and produced approx. 600 new seedlings, that could be transferred from greenhouse to field conditions.

A micropropagation method for STEVIA REBAUDIANA plants was established by culturing young leaves of adult plants on medium containing 6-benzyladenine (BA) (2.0 mg/l) in the light or BA (2.0 mg/l) plus 1-naphthaleneacetic acid (NAA) (2.0 mg/l) in the dark. An open-ended system was obtained by transfer of the regenerating material to medium containing BA (0.1 mg/l), with continuous subculture and isolation of suitable shoots. Auxin addition to the rooting medium (especially at 0.1 mg/l 3-indolebutyric acid - IBA) favored root formation and improved EXTRA VITRUM plant survival. Regenerated plants transferred to the soil were uniform and cytological observation in root tip cells showed normal chromosome number (2n = 22). Cultures treated with colchicine originated altered plants.

Shoot primordia, which were able to propagate vegetatively with a very high rate and to redifferentiate easily to new plants, were induced from shoot tips of Stevia rebaudiana Bertoni on Gamborg B5 medium containing 6-benzylaminopurine (BAP) and alpha-naphthaleneacetic acid (NAA) under light. The propagation of the shoot primordia of Stevia rebaudiana is rapid, and they are highly stable in chromosome number and karyotype. The shoot primordia can propagate at a high rate for a long time without differentiation. At any time, the shoot primordia readily developed into plantlets with shoots and roots within 2 or 3 weeks in static culture on B5 medium containing 0.02 mg/l BAP and 2% sucrose. The plantlets were transplanted to sterilized soil to grow to normal adult plants.