Elicitors directed in vitro growth and production of stevioside and other secondary metabolites in Stevia rebaudiana (Bertoni) Bertoni.

Stevia rebaudiana (stevia) is a plant in the Asteraceae that contains several biologically active compounds including the antidiabetic diterpene glycosides (e.g. stevioside, rebaudioside and dulcoside) that can serve as zero-calorie sugar alternatives. In this study, an elicitation strategy was applied using 5% polyethylene glycol (PEG), sodium chloride (NaCl; 50 and 100 mM) and gibberellic acid (2.0 and 4.0 mg/L GA3) to investigate their effect on shoot morphogenesis, and the production of phenolics, flavonoids, total soluble sugars, proline and stevioside, as well as antioxidant activity, in shoot cultures of S. rebaudiana. Herewith, the media supplemented with 2 mg/L and 4 mg/L GA3 exhibited the highest shooting response (87% and 80%). The augmentation of lower concentrations of GA3 (2 mg/L) in combination with 6-benzylaminopurine (BAP) resulted in the maximum mean shoot length (11.1 cm). The addition of 100 mM NaCl salts to the media led to the highest observed total phenolics content (TPC; 4.11 mg/g-DW compared to the control 0.52 mg/g-DW), total flavonoids content (TFC; 1.26 mg/g-DW) and polyphenolics concentration (5.39 mg/g-DW) in shoots cultured. However, the maximum antioxidant activity (81.8%) was observed in shoots raised in media treated with 50 mM NaCl. The application of 2 mg/L of GA3 resulted in the highest accumulation of proline (0.99 µg/mL) as compared to controls (0.37 µg/mL). Maximum stevioside content (71 µL/mL) was observed in cultures supplemented with 100 mM NaCl and 5% PEG, followed by the 4 mg/L GA3 treatment (70 µL/mL) as compared to control (60 µL/mL). Positive correlation was observed between GA3 and stevioside content. Notably, these two compounds are derived from a shared biochemical pathway. These results suggest that elicitation is an effective option to enhance the accumulation of steviosides and other metabolites and provides the groundwork for future industrial scale production using bioreactors.

Influence of the Nitrogen Fertilization on the Yield, Biometric Characteristics and Chemical Composition of Stevia rebaudiana Bertoni Grown in Poland.

Stevia rebaudiana Bertoni is a plant native to South America that has gathered much interest in recent decades thanks to diterpene glycosides, called steviosides, which it produces. These compounds are characterised by their sweetness, which is 250-300 times higher than saccharose, and they contain almost no caloric value. Stevia is currently also grown outside the South American continent, in various countries characterised by warm weather. This research aimed to determine whether it is viable to grow Stevia rebaudiana plants in Poland, a country characterised by a cooler climate than the native regions for stevia plants. Additionally, the impact of adding various dosages and forms of nitrogen fertiliser was analysed. It was determined that Stevia rebaudiana grown in Poland is characterised by a rather low concentration of steviosides, although proper nitrogen fertilisation can improve various characteristics of the grown plants. The addition of 100 kg or 150 kg of nitrogen per hectare of the field in the form of urea or ammonium nitrate increased the yield of the stevia plants. The stevioside content can be increased by applying fertilisation using 100 kg or 150 kg of nitrogen per hectare in the form of ammonium sulfate. The total yield of the stevia plants grown in Poland was lower than the yield typically recorded in warmer countries, and the low concentration of steviosides in the plant suggests that more research about growing Stevia rebaudiana in Poland would be needed to develop profitable methods of stevia cultivation.

Beyond the PAR spectra: impact of light quality on the germination, flowering, and metabolite content of Stevia rebaudiana (Bertoni).

BACKGROUND: Stevia rebaudiana is a high value crop due to the strong commercial demand for its metabolites (steviol glycosides) but has limited geographical cultivation range. In non-native environments with different daylength and light quality, Stevia has low germination rates and early flowering resulting in lower biomass and poor yield of the desired metabolites. In this study, artificial lighting with light-emitting diodes (LEDs) was used to determine if different light quality within and outside of the photosynthetically active radiation (PAR) range can be used to improve germination rates and yields for production of steviol glycosides for the herbal supplement and food industry. RESULTS: Plants treated with red and blue light at an intensity of 130 µmol m-2  s-1 supplemented with 5% of UV-A light under a 16-h photoperiod produced the most desirable overall results with a high rate of germination, low percentage of early flowering, and high yields of dry leaf, stevioside and rebaudioside A, 175 days after planting. CONCLUSION: While red and blue light combinations are effective for plant growth, the use of supplemental non-PAR irradiation of UV-A wavelength significantly and desirably delayed flowering, enhanced germination, biomass, rebaudioside A and stevioside yields, while supplemental green light improved yield of biomass and rebaudioside A, but not stevioside. Overall, the combination of red, blue and UV-A light resulted in the best overall productivity for Stevia rebaudiana. © 2021 Society of Chemical Industry.

Salt Stress Mitigation via the Foliar Application of Chitosan-Functionalized Selenium and Anatase Titanium Dioxide Nanoparticles in Stevia (Stevia rebaudiana Bertoni).

High salt levels are one of the significant and major limiting factors on crop yield and productivity. Out of the available attempts made against high salt levels, engineered nanoparticles (NPs) have been widely employed and considered as effective strategies in this regard. Of these NPs, titanium dioxide nanoparticles (TiO2 NPs) and selenium functionalized using chitosan nanoparticles (Cs-Se NPs) were applied for a quite number of plants, but their potential roles for alleviating the adverse effects of salinity on stevia remains unclear. Stevia (Stevia rebaudiana Bertoni) is one of the reputed medicinal plants due to their diterpenoid steviol glycosides (stevioside and rebaudioside A). For this reason, the current study was designed to investigate the potential of TiO2 NPs (0, 100 and 200 mg L-1) and Cs-Se NPs (0, 10 and 20 mg L-1) to alleviate salt stress (0, 50 and 100 mM NaCl) in stevia. The findings of the study revealed that salinity decreased the growth and photosynthetic traits but resulted in substantial cell damage through increasing H2O2 and MDA content, as well as electrolyte leakage (EL). However, the application of TiO2 NPs (100 mg L-1) and Cs-Se NPs (20 mg L-1) increased the growth, photosynthetic performance and activity of antioxidant enzymes, and decreased the contents of H2O2, MDA and EL under the saline conditions. In addition to the enhanced growth and physiological performance of the plant, the essential oil content was also increased with the treatments of TiO2 (100 mg L-1) and Cs-Se NPs (20 mg L-1). In addition, the tested NPs treatments increased the concentration of stevioside (in the non-saline condition and under salinity stress) and rebaudioside A (under the salinity conditions) in stevia plants. Overall, the current findings suggest that especially 100 mg L-1 TiO2 NPs and 20 mg L-1 Cs-Se could be considered as promising agents in combating high levels of salinity in the case of stevia.

Comparative transcriptome analysis provides insights into steviol glycoside synthesis in stevia (Stevia rebaudiana Bertoni) leaves under nitrogen deficiency.

KEY MESSAGE: Transcriptome analysis revealed the potential mechanism of nitrogen regulating steviol glycosides synthesis via shifting of leaf carbon metabolic flux or inducing certain transcription factors. Nitrogen (N) plays key regulatory roles in both stevia (Stevia rebaudiana) growth and the synthesis of its functional metabolite steviol glycosides (SGs), but the mechanism by which this nutrient regulates SGs synthesis remains to be elucidated. To address this question, a pot experiment was performed in a greenhouse where stevia plants fertilized with N (the control as CK plants) and compared with plants without the supply of N. Physiological and biochemical analyses were conducted to test the growth and metabolic responses of plants to N regimes. Our results showed that N deficiency significantly inhibited plant growth and leaf photosynthesis, while increased leaf SGs contents in stevia (49.97, 46.64 and 84.80% respectively for rebaudioside A, stevioside, and rebaudioside C), which may be partly due to "concentration effect". Then, transcriptome analysis was conducted to understand the underlying mechanisms. A total of 535 differentially expressed genes were identified, and carbon metabolism-related events were highlighted by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. Many of these genes were significantly upregulated by N-deficiency, including those involved in "phenylpropanoid biosynthesis", "flavonoid biosynthesis" and "starch and sucrose metabolism". Our study also analyzed the expression patterns of SGs synthesis-related genes under two N regimes and the potential transcription factors linking N nutrition and SG metabolism. N-deficiency may promote SGs synthesis by changing the carbon metabolism flux or inducing certain transcription factors. Our results provide deeper insight into the relationship between N nutrition and SGs synthesis in stevia plants.

Optimization of regeneration and Agrobacterium-mediated transformation of Stevia (Stevia rebaudiana Bertoni): a commercially important natural sweetener plant.

Stevia rebaudiana Bertoni is a commercially important zero calorie natural-sweetener herb which produce sweet compounds known as steviol glycosides. Rising demands of steviol glycosides by food and beverage industries has led to an increase in its cultivation in various countries. Unfortunately, stevia cultivation faces 2-25% yield penalty due to weeds which further adds to its cultivation cost. To resolve this major challenge, Agrobacterium-mediated genetic transformation of in vitro derived stevia-nodal explants using herbicide resistance gene (bar) has been optimized, for the production of stable transgenic stevia plants. Several parameters including explant type, pre-incubation duration, acetosyringone (As) concentration, Agrobacterium cell density, Agro-inoculation duration, co-cultivation duration, selection regime and plant growth regulators (PGRs) combination and concentration, have been successfully optimized. Among the two types of explants used, nodal explants showed a higher regeneration response of 82.85%, with an average of 25 shoots/explant. The best PGRs combination and concentration for shoot-induction, shoot-elongation and root-induction was found to be 6-benzyladenine (1.0 mg l-1) + naphthalene acetic acid (0.5 mg l-1), gibberellic acid (1.0 mg l-1), and half-strength MS medium, respectively. The two-step selection (phosphinothricin) regime resulted in an average transformation efficiency of 40.48% with nodal explants. Molecular characterization of putative transformants through PCR, RT-PCR, qRT-PCR and Southern-blot hybridization confirmed the presence, stability, expression as well as copy number of bar gene respectively. Compared to the non-transgenic plants, the T0 transgenic plants successfully tolerated 8 mg l-1 glufosinate ammonium sprays. Thus, the optimized protocol can be useful for the introduction of other genes (inter-kingdom transfer) into stevia genome.

Steviol glycosides profile in Stevia rebaudiana Bertoni hairy roots cultured under oxidative stress-inducing conditions.

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.

Development of Bacillus safensis-based liquid bioformulation to augment growth, stevioside content, and nutrient uptake in Stevia rebaudiana.

The application of chemical fertilizers to enhance crop production is a major concern due to associated environmental pollution and health hazards. Hence, there is an urgent need to develop an eco-friendly solution to improve crop production and promote sustainable agriculture simultaneously. Stevia rebaudiana is an important medicinal crop being substitute for sugar, superior flavor outline, extensive medicinal properties, and also of agronomic interest. In the present study, bacterium STJP isolated from the rhizospheric soil of S. rebaudiana and identified as Bacillus safensis on the basis of 16S rRNA gene sequencing, showed good amount of zinc (4.4 mg/L) and potassium (5.4 mg/L) solubilization. Paneer-whey (a dairy waste) based bioformulation (P-WBF) was developed utilizing isolate B. safensis STJP (accession number NAIMCC TB-2833) and inspected for the quality and ability to enhance the growth, nutrients uptake, and stevioside content in S. rebaudiana. The application of P-WBF displayed a significantly higher concentration (153.12%) of stevioside in S. rebaudiana as compared to control. P-WBF treated Stevia plants showed significantly higher fresh and dry weight as well (as compared to control). Further, enhancement of phosphorous, nitrogen, potassium, and zinc uptake in plant tissue was also recorded by application of P-WBF. This study suggests the use of P-WBF based biofertilizer using B. safensis STJP to increase stevioside content in Stevia plant by a nutrient(s) linked mechanism. This novel approach can also be beneficial for utilization of a dairy waste in preparation of bioformulation and, for enhancement of crop yield by an ecofriendly manner leading to sustainable agriculture.

Nitrogen drives plant growth to the detriment of leaf sugar and steviol glycosides metabolisms in Stevia (Stevia rebaudiana Bertoni).

Steviol glycosides (SGs) in Stevia (Stevia rebaudiana Bertoni) leaves are important due to their high sweetness and low calorific value. The yield of SGs is dependent on fertilization regimes, but the relationship between nitrogen (N) administration and SGs synthesis is still unclear. In this study, we investigate the effects of N rates on SGs production through hydroponic and plot experiments. The SGs yield was not significantly changed by N fertilization, but leaf SGs concentrations were significantly reduced due to the "dilution effect". Additionally, N addition decreased leaf carbon (C)/N ratio and soluble sugar concentration, accompanied with the inhibited phosphoenolpyruvate carboxylase and L-phenylalanine ammonia_lyase activities. A significant positive correlation between leaf SGs concentrations, C/N ratio and soluble sugar concentration was observed. Overall, we suggest that N-driven Stevia growth negatively affects SGs concentrations. The leaf C/N ratio and soluble sugar changes indicated the occurrence of metabolic reprogramming.

Plant densities and harvesting times on productive and physiological aspects of Stevia rebaudiana Bertoni grown in southern Brazil.

Stevia (stevia rebaudiana Bertoni) is a species characterized by producing non-caloric substances with high sweetening potential. Among these substances, rebaudioside A and stevioside are produced in greater quantity. Plant density and harvesting time are factors that affects biomass and sweetening compounds yield in this species. The objective of this research was to evaluate the effect of plant densities and harvesting times on the productive and physiological characteristics of stevia in southern Brazil. The experimental design was in randomized blocks, in a split-plot scheme, with 9 treatments comparing the effect of three planting densities (166 667, 83 333 and 33 333 plants ha-1) in the plots and three harvesting periods (before, in the beginning and in full flowering) in the subplots. Harvesting at the beginning of flowering promoted higher dry leaf biomass yield and, when associated with the lowest planting density, promoted higher levels of rebaudioside A and stevioside. The lowest planting density resulted in greater leaves biomass accumulation, whereas the highest density promoted higher yields per area. Interaction between density of 166 667 plants ha-1 and the harvest at the onset of flowering promoted higher yields of rebaudioside A (43.22 kg ha-1) as well as higher rebaudioside A/stevioside ratio (0.60).

Comparative metabolic and ionomic profiling of two cultivars of Stevia rebaudiana Bert. (Bertoni) grown under salinity stress.

This study provides a comprehensive investigation on the impact of increasing NaCl concentrations on hydroponically grown Stevia rebaudiana cultivars (Shoutian-2 and Fengtian). Growth parameters including plant height, biomass and physiological responses including osmotic potential were measured. In addition, the levels of steviol glycosides, elements and primary metabolites were measured and statistically evaluated. The cultivar Fengtian grew faster, accumulated less Na+ and compatible organic solutes, and more K+ in the leaves, as compared to the cv. Shoutian-2. Metabolite analysis identified 81 differentially accumulated metabolites, indicating an alteration in the metabolite phenotype of both cultivars upon exposure to salinity A general increase in many amino acids, amines, sugars and sugar phosphates with a concurrent decrease in most organic acids; including tricarboxylic acid (TCA) cycle intermediates, was observed. In the more salt tolerant cv. Fengtian, the levels of hexose phosphates and metabolites involved in cellular protection increased in response to salinity. These metabolites remained unchanged in the sensitive cv. Shoutian-2. Interestingly, salt treatment notably increased the rebaudioside A concentration by 53% while at the same time stevioside decreased by 38% in Fengtian which has important implications for controlling the relative amounts of reboudioside A and stevioside. The findings of this study leads to the conclusion that mild salinity stress can increase the yield of sweetener compounds, which is dependent on the cultivar and the level of salinity stress.

Effects of various glutamine concentrations on gene expression and steviol glycosides accumulation in Stevia rebaudiana Bertoni.

Stevia rebaudiana Bertoni is one of the most important biologically sourced and low-calorie sweeteners that contains a lots of Steviol glycosides. Tissue culture is the best method for propagation of stevia and micro nutrients can affect both morphological traits and steviol glycosides production. In the present study, we investigated the effect of different concentrations of glutamine (10, 20, 30 and 40 g/l) on expression of UGT74G1 and UGT76G1 genes and stevioside and rebaudioside A accumulation in the leaves of stevia under in vitro conditions. The highest level of expression for UGT74G1 (1.000 Total lab unit) was seen at plants grown in MS media without glutamine and the highest gene expression level for UGT76G1 (1.321 Total lab unit) was observed at plants grown in 2% glutamine. Based on HPLC results, the highest amount of stevioside (22.74) was accumulated in plants which were under 3% glutamine treatment and the lowest production level of stevioside (16.19) was resulted under MS (0 glutamine) medium. The highest rebaudioside A (12.19) accumulation was observed under 2% glutamine treatment and the lowest accumulation of rebaudioside A (8.41) was seen at plants grown in MS medium.

Effects of life cycle and leaves location on gene expression and glycoside biosynthesis pathway in Stevia rebaudiana Bertoni.

Stevia rebaudiana Bertoni is One of the most important biologically sourced and low-calorie sweeteners that known as "Sweet Weed". It contains steviol glycosides that they are about 200-300 times sweeter than sucrose. Tissue culture is the best method with high efficiency that can overcome to problems of traditional methods, and it is the most useful tools for studying stress tolerance mechanisms under in vitro conditions to obtain drought tolerance. In the present research, we investigated the impact of life cycle, leaves location and the harvesting time on expression of UGT74G1 and UGT76G1 as well as steviol glycosides accumulation. The highest gene expression of both UGT74G1 and UGT76G1 (207.677 and 208.396 Total Lab unit, respectively) was observed in young leaves in the second vegetative year. Also, the highest amount of stevioside accumulation (13.04) was due to the old leaves in vegetative stage which had significant differences with other effects whereas the lowest accumulation (7.47) was seen at young leaves at vegetative stage. Interestingly, the highest level of rebaudioside a production (15.74) was occurred at the young leaves at vegetative stage. There was significant differences between life cycle and leaves location on steviol glycoside production in stevia.

Investigation of different concentrations of MS media effects on gene expression and steviol glycosides accumulation in Stevia rebaudiana Bertoni.

Stevia rebaudiana Bertoni is one of two species that contains steviol glycosides. Among steviol glycosides that extracted from leaves, stevioside and rebaudioside A are the two major and the sweetest glycosides that are about 200-300 times sweeter than sucrose with zero calories. The best method for stevia propagation is tissue culture. So, for investigation of nutrients in medium, we studied the effect of different concentrations of MS media (MS, 0.5 MS, 0.25 MS, 0 MS) on morphological traits, UGT74G1 and UGT76G1 genes expression and accumulation of steviol glycosides in stevia leaves. The best growth rate (0.472 mm/d) has occurred in plants grown in MS media. Also, the highest gene expression of UGT74G1 gene (1.000 Total lab unit) was seen under MS treatment. However, the highest expression level of UGT76G1 gene (1.701 Total lab unit) was observed at plants grown in 0 MS. The highest amount of both Stevioside and Rebaudioside A (14.23 and 8.12, respectively) were accumulated in plants under MS treatment. Obviously, dilution of MS media associated with decreasing in both expression of the intended genes and accumulation of steviol glycosides.

Effect of sucrose concentrations on Stevia rebaudiana Bertoni tissue culture and gene expression.

Stevia rebaudiana (Bert.) Bertoni is known as sweet plant which it contains a high level of steviol glycosides in the leaves.  This plant has been used from centuries ago as a sweetener for tea. One of the most important steviol glycosides is stevioside that is attractive for diabetic persons. Tissue culture is the only rapid process for the mass propagation of stevia. One of the most important factors in the medium is sucrose that is a necessary for plant growth. In the present study, we use nodal segments of the stem as explants in mediums with different sucrose concentration (50 mM, 100mM and 150mM). Several morphological traits were measured in a 28 day period. Results analysis showed a significant variation between treatments. The highest growth rate, rooting and leaf production was obtained in medium with 100mM sucrose. The correlation between measured traits was significant at the 0.01 level. To investigation of UGT74G1, UGT76G1, UGT85C2 and KS genes expression that are involved in the synthesis of SGs, RT- PCR was done with the housekeeping gene of as internal control. There were significant differences between all media. The results showed thatsucrose 100 mM containing media was more desirable than others for expression of UGT76G1 and UGT85C2 genes. Whereas, the best medium for expression of UGT74G1 was sucrose 150 mM and sucrose 50 mM for KS gene. Totally, it seems that sucrose at a concentration of 100 mMprovides the best condition for stevia growth and steviol glycosides production.

Molecular dissection of transcriptional reprogramming of steviol glycosides synthesis in leaf tissue during developmental phase transitions in Stevia rebaudiana Bert.

Stevia is a natural source of commercially important steviol glycosides (SGs), which share biosynthesis route with gibberellic acids (GAs) through plastidal MEP and cytosolic MVA pathways. Ontogeny-dependent deviation in SGs biosynthesis is one of the key factor for global cultivation of Stevia, has not been studied at transcriptional level. To dissect underlying molecular mechanism, we followed a global transcriptome sequencing approach and generated more than 100 million reads. Annotation of 41,262 de novo assembled transcripts identified all the genes required for SGs and GAs biosynthesis. Differential gene expression and quantitative analysis of important pathway genes (DXS, HMGR, KA13H) and gene regulators (WRKY, MYB, NAC TFs) indicated developmental phase dependent utilization of metabolic flux between SGs and GAs synthesis. Further, identification of 124 CYPs and 45 UGTs enrich the genomic resources, and their PPI network analysis with SGs/GAs biosynthesis proteins identifies putative candidates involved in metabolic changes, as supported by their developmental phase-dependent expression. These putative targets can expedite molecular breeding and genetic engineering efforts to enhance SGs content, biomass and yield. Futuristically, the generated dataset will be a useful resource for development of functional molecular markers for diversity characterization, genome mapping and evolutionary studies in Stevia.

Effect of nitrogen sources on some morphological characteristics of in vitro stevia rebaudiana Bertoni.

Stevia rebaudiana Bertoni belongs to Asteraceae family that leaves 200-300 times sweeter than sugar. Low seed fertility is one of the most important problems in Stevia production. So, Plant tissue culture is an efficient method for mass propagation of Stevia. In this research, we studied the effect of various concentrations of nitrogen on some morphological traits of stevia under in vitro conditions. We used axillary nodes as explants and they were cultured on Murashige and Skoog (MS) medium containing inorganic nitrogen sources i.e. NH4NO3(0, 825 and 1650 mg/l), KNO3(0, 950 and 1900 mg/l) were observed. The cultures were kept for 4 weeks at a temperature of 25±2°C with a photoperiod of 16/8 hour low light/dark each day. Maximum shoot length (89.33 mm), dry weight of plants (0.10 mg) and leaf fresh weight (0.42 mg) was observed on MS medium with 1650 mg/l NH4NO3 and 950 mg/l KNO3. Minimum shoot length (6.13 mm), root length (6.60 mm), leaf number (4.26), leaf dry weight (0.01 mg), leaf fresh weight (0.05 mg), total dry and fresh weight (0.02 and 0.15 mg) and growth rate was observed on a MS medium without nitrogen sources. Moreover, presence of nitrogen sources increases both shooting and rooting in Stevia rebaudiana Bertoni.

Micropropagation and Biomass Production of True-to-Type Stevia rebaudiana Bertoni.

Here we describe an efficient micropropagation protocol for Stevia rebaudiana Bertoni. We present experiments carried out to optimize the suitable media for in vitro shoot multiplication and root induction and to study the effect of culture vessel on shoot multiplication. Among all different media tested for in vitro shoot multiplication, hormone-free liquid medium is most suitable. The highest number of nodes per shoot (5.4) and length of shoot (4.76 cm) at 4 weeks after subculturing are observed when single node explants are placed on modified MS medium supplemented with 1 % sucrose and 0.7 % agar. The highest response of multiplication rate (9.56) is observed on half strength of macroelement of MS with full strength of microelement of MS and 170 mg/l KH2PO4, and 185 mg/l MgSO4 in plastic growth container. Further, RAPD marker analysis of in vitro-raised plants maintained their clonal fidelity and true-to-type without showing any somaclonal variation.

Transcript Quantification of Genes Involved in Steviol Glycoside Biosynthesis in Stevia rebaudiana Bertoni by Real-Time Polymerase Chain Reaction (RT-PCR).

Stevia (Stevia rebaudiana Bertoni) is a medicinal plant having sweet, diterpenoid glycosides known as steviol glycosides which are 200-300 times sweeter than sucrose (0.4 % solution). They are synthesized mainly in the leaves via plastid localized 2-C-methyl-D-erythrose-4-phosphate pathway (MEP pathway). Fifteen genes are involved in the formation of these glycosides. In the present protocol, a method for the quantification of transcripts of these genes is shown. The work involves RNA extraction and cDNA preparation, and therefore, procedures for the confirmation of DNA-free cDNA preparation have also been illustrated. Moreover, details of plant treatments are not mentioned as this protocol may apply to relative gene expression profile in any medicinal plant with any treatment. The treatments are numbered as T0 (Control), T1, T2, T3, and T4.

Light-induced biochemical variations in secondary metabolite production and antioxidant activity in callus cultures of Stevia rebaudiana (Bert).

Stevia rebaudiana (S. rebaudiana) is a very important species with worldwide medicinal and commercial uses. Light is one of the major elicitors that fluctuate morphogenic potential and biochemical responses. In the present study, we investigated the effect of various spectral lights on biomass accumulation and secondary metabolite production in callus cultures of S. rebaudiana. Leaf explants were placed on Murashige and Skoog (MS) medium and exposed to various spectral lights. 6-Benzyle adenine (BA) and 2, 4-dichlorophenoxy acetic acid (2, 4-D; 2.0 mgl(-1)) were used for callus induction. The control light (16/8h) produced optimum callogenic response (92.73%) than other colored lights. Compared to other colored lights, control grown cultures displayed maximum biomass accumulation (5.78 gl(-1)) during a prolonged log phase at the 18th day of growth kinetics. Cultures grown under blue light enhanced total phenolic content (TPC; 102.32 µg/g DW), total flavonoid content (TFC; 22.07 µg/g DW) and total antioxidant capacity (TAC; 11.63 µg/g DW). On the contrary, green and red lights improved reducing power assay (RPA; 0.71Fe(II)g(-1) DW) and DPPH-radical scavenging activity (DRSA; 80%). Herein, we concluded that the utilization of colored lights is a promising strategy for enhanced production of antioxidant secondary metabolites in callus cultures of S. rebaudiana.