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.

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.

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.

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.

A rapid and efficient protocol for in vitro multiplication of genetically uniform Stevia rebaudiana (Bertoni).

Stevia rebaudiana (Bertoni), commonly called candy leaf or sweet leaf, endemic to South America, is an important medicinal plant. As a source of low calorie natural sweetener 'stevoside', it is used in obesity, diabetes, treatment of heartburn and tooth decay, and also serves as a food supplement. Large scale commercial propagation of S. rebaudiana demands a suitable protocol. Here, we propose an improved protocol for in vitro multiplication of S. rebaudiana from nodal explants. In this protocol, the effect of laboratory grade urea on multiple shoot induction from nodal explants was studied. The nodal explants were initially cultured on Murashige and Skoog (MS) basal media for 2 weeks which facilitated the axillary bud break. Further, culturing of these explants on MS medium fortified with 6 benzyl amninopurine (BAP) (2 mg/L) and Naphthalene acetic acid (NAA) (1 mg/L) with and .without urea (5 mg/L) for a period of 40 days revealed maximum shoot production of 44.56 from a single nodal explant in media supplemented with urea as compared to 22.44 without urea. The differences in the number of shoots produced were significant and these shoots readily rooted in MS media with NAA (4 mg/L). Primary and secondary hardening was successful in these plants. There were no visible morphological abnormalities observed in the micropropagated plantlets. Genetic analysis from random samples also revealed that these plants are genetically uniform. The advantage of the present protocol is that the complete process of multiple shoot induction, rooting and hardening could be completed within a period of 6 months as compared to the existing protocols.

Effect of Nitrogen Fertilization and Harvest Time on Steviol Glycosides, Flavonoid Composition, and Antioxidant Properties in Stevia rebaudiana Bertoni.

This work investigated the effect of nitrogen fertilization and harvest time on the flavonoid composition and antioxidant properties of Stevia rebaudiana leaves. At the same time, changes in stevioside (Stev) and rebaudioside A (RebA) contents were recorded. A pot trial under open air conditions was set up, testing five N rates and three harvest times. The results showed that, by using an adequate N rate and choosing an appropriate harvest time, it was possible to significantly increase and optimize the bioactive compound levels. In particular, higher RebA, RebA/Stev ratio, total phenols and flavonoids, luteolin-7-O-glucoside, and apigenin-7-O-glucoside levels and antioxidant capacity were recorded by supplying 150 kg N ha(-1). Reduced or increased N availability in comparison with N150 had no consistent effect on Stevia phytochemicals content. Significant correlations were also found between stevioside and some of the flavonoids, indicating a possible role of flavonoids in the stevioside metabolic pathway, which deserves more investigations.

Crop-ecology and nutritional variability influence growth and secondary metabolites of Stevia rebaudiana Bertoni.

BACKGROUND: Plant nutrition and climatic conditions play important roles on the growth and secondary metabolites of stevia (Stevia rebaudiana Bertoni); however, the nutritional dose is strongly governed by the soil properties and climatic conditions of the growing region. In northern India, the interactive effects of crop ecology and plant nutrition on yield and secondary metabolites of stevia are not yet properly understood. Thus, a field experiment comprising three levels of nitrogen, two levels of phosphorus and three levels of potassium was conducted at three locations to ascertain whether the spatial and nutritional variability would dominate the leaf yield and secondary metabolites profile of stevia. RESULTS: Principal component analysis (PCA) indicates that the applications of 90 kg N, 40 kg P2O5 and 40 kg K2O ha-1 are the best nutritional conditions in terms of dry leaf yield for CSIR-IHBT (Council of Scientific and Industrial Research- Institute Himalayan Bioresource Technology) and RHRS (Regional Horticultural Research Station) conditions. The spatial variability also exerted considerable effect on the leaf yield and stevioside content in leaves. Among the three locations, CSIR-IHBT was found most suitable in case of dry leaf yield and secondary metabolites accumulation in leaves. CONCLUSIONS: The results suggest that dry leaf yield and accumulation of stevioside are controlled by the environmental factors and agronomic management; however, the accumulation of rebaudioside-A (Reb-A) is not much influenced by these two factors. Thus, leaf yield and secondary metabolite profiles of stevia can be improved through the selection of appropriate growing locations and proper nutrient management.

Production of chlorogenic acid and its derivatives in hairy root cultures of Stevia rebaudiana.

Chlorogenic acid and its derivatives (CADs) are valuable bioactive plant secondary metabolites with many health benefits. In the present study, Stevia rebaudiana hairy root cultures were established, and the culture conditions for the production of CADs were optimized. The hairy roots were induced by coculture of S. rebaudiana leaves and Agrobacterium rhizogenes (C58C1) after infection, which were further verified by PCR detection of rolB and rolC genes. HPLC-MS and HPLC analysis showed that chlorogenic acid (3-caffeoylquinic acid, 3-CQA), 3,5-dicaffeoylquinic acid (3,5-CQA), and 4,5-dicaffeoylquinic acid (4,5-CQA) were the major CADs in the hairy roots. Eight single roots with rapid growth rate were selected. Among them, T3 had the highest yield of CADs. B5 medium supplemented with 40 g/L sucrose was more suitable for the production of CADs than others. Under optimal culture conditions, the total content of these three compounds reached 105.58 mg/g and total yield was 234.40 mg/100 mL.

Stevia rebaudiana Bertoni as a source of bioactive compounds: the effect of harvest time, experimental site and crop age on steviol glycoside content and antioxidant properties.

BACKGROUND: This study was aimed at identifying the effect of harvest time, experimental site and crop age on the no-calorie sweetener steviol glycosides (SG) and on the antioxidant properties of stevia leaf extracts. The experiment was conducted over two growing seasons at two sites in the northeastern plain of Italy. RESULTS: The results showed that all analysed factors played an important role in defining the SG profile and the antioxidant properties of stevia extracts. A high level of phenols (78.24 mg GAE g⁻¹ DW by Folin-Ciocalteu method) and high antioxidant activity (812.6 µmol Fe²âº g⁻¹ DW by FRAP assay) were observed. The inhibition of DPPH free radicals was evaluated and an IC50 mean value of 250 µg mL⁻¹ was obtained. Significant relationships among the total antioxidant capacity and the analysed compounds were found. CONCLUSION: The results showed the possibility of obtaining, in the tested environments, very high SG yields thanks to the long-day conditions during the spring/summer season. The harvest time played a key role in determining the stevia quality, influencing the rebaudioside A/stevioside ratio. The strong antioxidant properties make very interesting the possibility of using stevia extracts to improve functional food properties.

Comportamiento del cultivo de Stevia rebaudiana (Bertoni) Bertoni en Cuba / Behavior of the cultivation of Stevia rebaudiana (Bertoni) Bertoni in Cuba

Se presenta la recopilación de los resultados obtenidos en las investigaciones que sobre el cultivo de Stevia rebaudiana (Bertoni) Bertoni, se realizaron en la Estación Experimental de Plantas Medicinales de San Antonio de los Baños, provincia La Habana, Cuba. Bajo estas condiciones se pudo conocer mediante dos fechas de siembra: diciembre 2000 y enero 2001, que las semillas germinan a los 5 días de la siembra, aunque con bajo porcentaje y que se pueden trasplantar dos meses después. Se evaluaron tres cosechas en cada una de las fechas de siembra y se demostró que la de diciembre resultó la mejor pues casi se duplicaron los rendimientos. Como patógenos que la afectaron se observó el hongo Alternaria sp. y los insectos Agromyza sp., además de pequeñas afectaciones por crisomélidos durante la fase vegetativa. Para el control de estos agentes nocivos se probaron extractos acuosos al 30 por ciento y 50 por ciento de follaje de Carica papaya L. contra el hongo y extractos acuosos de Tabaquina® (Nicotiana tabacum L.) al 30 y 50 por ciento para los insectos; se comprobó que las dosis mayores ejercieron mejor control sobre estas plagas. El tamizaje fitoquímico evidenció la presencia de triterpenos, esteroides, carotenos, taninos y flavonoides en el material vegetal recolectado.

The results obtained in the investigations on the cultivation of Stevia rebaudiana (Bertoni) Bertoni conducted at the Experimental Station of Medicinal Plants in San Antonio de los Baños, Havana province, Cuba, are shown. Under these conditions, it was possible to know by 2 sowing dates, December 2000 and January 2000, that the seed germinates at 5 days, although with a low percentage, and that they can be transplanted 2 months later. Three crops were evaluated in every sowing date, and it was proved that the best was obtained in December, when the yields almost doubled. Some of the pathogens that affected the crop were the Alternaria sp. fungus and the Agromyza sp. insects, besides little affections caused by chrysomelids during the vegetative phase. To control these harmful agents, aqueous extracts 30 percent and 50 percent of foliage of Carica papaya L. were used against the fungus, whereas aqueous extracts of Tabaquina® (Nicotiana tabacum L.) 30 percent and 50 percent were utilized against the insects. It was proved that the higher doses exerted a better control on these plagues. The phytochemical screening evidenced the presence of tripertenes, steroids, carotenes, tannins and flavonoids in the vegetal material collected.

Comportamiento del cultivo de Stevia rebaudiana (Bertoni) Bertoni en Cuba / Behavior of the cultivation of Stevia rebaudiana (Bertoni) Bertoni in Cuba

Se presenta la recopilación de los resultados obtenidos en las investigaciones que sobre el cultivo de Stevia rebaudiana (Bertoni) Bertoni, se realizaron en la Estación Experimental de Plantas Medicinales de San Antonio de los Baños, provincia La Habana, Cuba. Bajo estas condiciones se pudo conocer mediante dos fechas de siembra: diciembre 2000 y enero 2001, que las semillas germinan a los 5 días de la siembra, aunque con bajo porcentaje y que se pueden trasplantar dos meses después. Se evaluaron tres cosechas en cada una de las fechas de siembra y se demostró que la de diciembre resultó la mejor pues casi se duplicaron los rendimientos. Como patógenos que la afectaron se observó el hongo Alternaria sp. y los insectos Agromyza sp., además de pequeñas afectaciones por crisomélidos durante la fase vegetativa. Para el control de estos agentes nocivos se probaron extractos acuosos al 30 por ciento y 50 por ciento de follaje de Carica papaya L. contra el hongo y extractos acuosos de Tabaquina® (Nicotiana tabacum L.) al 30 y 50 por ciento para los insectos; se comprobó que las dosis mayores ejercieron mejor control sobre estas plagas. El tamizaje fitoquímico evidenció la presencia de triterpenos, esteroides, carotenos, taninos y flavonoides en el material vegetal recolectado(AU)

The results obtained in the investigations on the cultivation of Stevia rebaudiana (Bertoni) Bertoni conducted at the Experimental Station of Medicinal Plants in San Antonio de los Baños, Havana province, Cuba, are shown. Under these conditions, it was possible to know by 2 sowing dates, December 2000 and January 2000, that the seed germinates at 5 days, although with a low percentage, and that they can be transplanted 2 months later. Three crops were evaluated in every sowing date, and it was proved that the best was obtained in December, when the yields almost doubled. Some of the pathogens that affected the crop were the Alternaria sp. fungus and the Agromyza sp. insects, besides little affections caused by chrysomelids during the vegetative phase. To control these harmful agents, aqueous extracts 30 percent and 50 percent of foliage of Carica papaya L. were used against the fungus, whereas aqueous extracts of Tabaquina® (Nicotiana tabacum L.) 30 percent and 50 percent were utilized against the insects. It was proved that the higher doses exerted a better control on these plagues. The phytochemical screening evidenced the presence of tripertenes, steroids, carotenes, tannins and flavonoids in the vegetal material collected(AU)

Interaction effect between phosphorus and zinc on their availability in soil in relation to their contents in stevia (Stevia rebaudiana).

A greenhouse experiment was conducted at the Indian Institute of Horticultural Research (IIHR), Bangalore to study the interaction effect between phosphorus and zinc on their availability in soil in relation to their contents in stevia (Stevia rebaudiana). The results show that the amount of available P and Zn content in soil has been found to increase initially and, thereafter, the amount of the same decreased with the progress of plant growth up to 60 days irrespective of treatments. The amount of P and Zn in soils showed an increase with their separate applications either as soil or foliar spray while that of the same value significantly decreased both in soils and plants due to their combined applications, suggesting a mutual antagonistic effect between Zn and P affecting each other's availability in soil and content in the stevia plant.

Interaction between phosphorus and zinc on the biomass yield and yield attributes of the medicinal plant stevia (Stevia rebaudiana).

A greenhouse experiment was conducted at the Indian Institute of Horticultural Research (IIHR), Bangalore to study the interaction effect between phosphorus (P) and zinc (Zn) on the yield and yield attributes of the medicinal plant stevia. The results show that the yield and yield attributes have been found to be significantly affected by different treatments. The total yield in terms of biomass production has been increased significantly with the application of Zn and P in different combinations and methods, being highest (23.34 g fresh biomass) in the treatment where Zn was applied as both soil (10 kg ZnSO4/ha) and foliar spray (0.2% ZnSO4). The results also envisaged that the different yield attributes viz. height, total number of branches, and number of leaves per plant have been found to be varied with treatments, being highest in the treatment where Zn was applied as both soil and foliar spray without the application of P. The results further indicated that the yield and yield attributes of stevia have been found to be decreased in the treatment where Zn was applied as both soil and foliar spray along with P suggesting an antagonistic effect between Zn and P.

Features of development of Stevia rebaudiana shoots cultivated in the roller bioreactor and their production of steviol glycosides.

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.