Rebaudiana Plants Research Articles

An Assessment of Major Nutritional Components and Some Secondary Metabolites of In-Vitro Propagated Stevia Rebaudiana (Cultured in Bangladesh) Plant Leaves Dry Powder

Stevia rebaudiana, belongs to the family of Asteraceae, is a perennial and medicinal shrub. It’s leaves dry powder widely used as a natural sweetener which has no calories but reported to be 50– 300 times sweetener as sweet as sugar, that are said to be having insulin balancing properties without any side effects. This research was first time conducted in Bangladesh where a quantitative analysis were performed for determining the major constituents of in vitro propagated Stevia rebaudiana plant leaves dry powder that was prepared through oven dry after sun dry. The analysis revealed the powder as mild acidic (PH 5.345). Total chlorophyll contents were found to be 0.845mg% where chlorophyll–a and -b were 0.088 and 0.761 mg% respectively. The major macro nutrients such as ash, crude fiber, total carbohydrate, total protein and fat were determined at the amount of 7.05, 10.5, 53.52, 13.13 and 3.55 gm% respectively where the micro nutrients like Iron (Fe), Sodium (Na), Potassium (K), Calcium (Ca), Magnesium (Mg), Phosphorus (P) and Chloride (Cl) were found at the amount of 34.2, 184.3, 2500, 534.43, 465.34, 304.7 and 49.6 mg% accordingly. Screening for secondary metabolites showed the presence of alkaloids, flavonoids, saponins and tannins as the amount of 2.83, 17.20, 6.69 and 5.84 mg% respectively in dry powder. These findings also have been compared with the other reported values on Stevia from different countries of the world. Int J Appl Sci Biotechnol, Vol 3(4): 721-726

Functionalization of the double bond in the glycoside of the Stevia rebaudiana plant steviolbioside, as a way to macrocyclic glycosides

The C16=C17 bond in the glycoside steviolbioside from the plant Stevia rebaudiana was oxidized for the first time. Ketone, thiosemicarbazone, and oxime of steviolbioside with a heptaacetylated sophorosyl fragment, as well as binuclear and tetranuclear macrocyclic derivatives of this rebaudioside were synthesized.

Steviol glycosides targeted analysis in leaves of Stevia rebaudiana (Bertoni) from plants cultivated under chilling stress conditions

Stevia rebaudiana is an important agricultural crop for the production of a high-potency natural sweetener, sensitive to low temperature during the developmental stage. Stimulation of chilling stress with a pre-treatment with endogenous signalling components and in particular with salicylic acid (SA), hydrogen peroxide (H2O2), 6-benzylaminopurine (BAP) and calcium chloride (CaCl2) could induce tolerance to chilling and could constitute a suitable way to maintain quality and quantity of steviol glycosides under controlled artificial environment. In the present work the effects of different putative signalling molecules on the morpho-physiological parameters were evaluated, and a specific method for the quali-quantitative analysis of steviol glycosides in S. rebaudiana plants cultivated under controlled conditions was developed, by using LC–ESI-FT (Orbitrap) MS, LC–ESI–QqQ-MS/MS and multivariate data analysis. This approach underlined that the pre-treatment has influence on the production of secondary metabolites. In particular Stevia plants characterised by higher contents of rebaudioside A and stevioside, were identified.

Design and synthesis of isosteviol triazole conjugates for cancer therapy.

One of the keys for successfully developing drugs against the broad spectrum of cancer cell types is structural diversity. In the current study, we focused on a family of isosteviol derivatives as potential novel antitumor agents. Isosteviol is a tetracyclic diterpenoid obtained by acid hydrolysis of steviol glycoside extracts isolated from abundant Stevia rebaudiana plants. In this work, we have designed and synthesized a panel of isosteviol triazole conjugates using “click” chemistry methodology. Evaluation of these compounds against a series of cancer cell lines derived from primary and metastatic tumors demonstrated that these conjugates exhibit cytotoxic activities with IC50 in the low μM range. In addition, their anti-proliferative activities are cancer cell type specific. Taken together, our studies underscore the importance of structural diversity in achieving cancer cell type specific drug development.

Assessment of Heavy Metals Tolerance in Leaves, Stems and Flowers of Stevia Rebaudiana Plant

Stevia has become rather widespread over a wide range of climatic locations around the world and can apparently be successfully grown under different cultivation conditions. Heavy metal accumulation of Stevia extract is dependent on obtaining heavy metals from the soil and water. Heavy metals from plant sources may also vary from place to place because soil heavy metals content varies geographically, thus, they have become the subject of many research projects. Stevia plant is easily contaminated during growth, development and processing and for this, an extensive research is needed to explore the characteristics of the heavy metal produced by the plant. The heavy metals produced from the herb and its toxicity of Stevia plant is not well documented and scientific evidence is limited to establishing Stevia plant as a medicinal plant. The samples were collected from Malacca, Malaysia. The fresh leaves, stems and flowers of the Stevia rebaudiana plant were dried using oven equipment and were grinded until fine to make powder and then of each extracted using Microwave digester. The analysis of samples was carried out by using an Inductively Coupled Plasma Mass-Spectrophotometer (ICP-MS) with different mode equipment to compare results of heavy metals in Stevia rebaudiana plant. Heavy metal accumulation in Stevia rebaudiana from leaves, stems, and flowers extraction is reported. Heavy metals in leaves, stems and flowers of Stevia rebaudiana presented variety of elements such as As, Cd, Cr, Cu, Fe, Mg, Pb, Se, Zn, Al, Ag, Co, Ca, Mn and Ni. The high tolerance to heavy metals in leaves, stems, and flowers of Stevia rebaudiana were presented at fifteen parameters below the permissible limit in plant and can be used as food product or therapeutic agent in traditional medicine.

Purple phototrophic bacterium enhances stevioside yield by Stevia rebaudiana Bertoni via foliar spray and rhizosphere irrigation.

This study was conducted to compare the effects of foliar spray and rhizosphere irrigation with purple phototrophic bacteria (PPB) on growth and stevioside (ST) yield of Stevia. rebaudiana. The S. rebaudiana plants were treated by foliar spray, rhizosphere irrigation, and spray plus irrigation with PPB for 10 days, respectively. All treatments enhanced growth of S. rebaudiana, and the foliar method was more efficient than irrigation. Spraying combined with irrigation increased the ST yield plant -1 by 69.2% as compared to the control. The soil dehydrogenase activity, S. rebaudiana shoot biomass, chlorophyll content in new leaves, and soluble sugar in old leaves were affected significantly by S+I treatment, too. The PPB probably works in the rhizosphere by activating the metabolic activity of soil bacteria, and on leaves by excreting phytohormones or enhancing the activity of phyllosphere microorganisms.

Gene transcription of steviol glycoside biosynthesis in Stevia rebaudiana Bertoni under polyethylene glycol, paclobutrazol and gibberellic acid treatments in vitro

This experiment was carried out to measure the transcript levels of some genes involved in biosynthesis of steviol glycosides. Stevia rebaudiana plants were treated with polyethylene glycol (PEG), paclobutrazol (PBZ) and gibberellic acid (GA). Using quantitative real-time poly- merase chain reaction, the transcript levels of kaurene synthase (KS), kaurene oxidase (KO), kaurenoic acid hydroxylase (KAH) and three UDP-dependent glycosyl- transferases, UGT85C2, UGT74G1 and UGT76G1, were studied. The transcription of ent-KS1-1, ent-KAH and UGT74G1 were fairly stable under different treatments. The transcription of ent-KO, UGT85C2 and UGT76G1 significantly decreased by PBZ and PEG treatments. The results indicate that both PBZ and PEG treatments resulted in the same negative effect on genes transcription, which could not be reversed by GA treatment.

TDZ-Induced High Frequency Plant Regeneration through Direct Shoot Organogenesis in <i>Stevia rebaudiana</i> Bertoni: An Important Medicinal Plant and a Natural Sweetener

An efficient high frequency plant regeneration protocol through direct organogenesis was developed for Sevia rebaudiana Bert. Nodal segments containing axillary buds were used as an explant and inoculated on Murashige and Skoog’s (MS) medium containing 3% (w/v) sucrose, 0.8% (w/v) agar supplemented with various concentrations of benzyladenine (BA), kinetin (Kn) and thidiazuron (TDZ) ranging from 1.00 to 9.00 μM. Maximum multiple shoots (96%) were obtained in MS medium supplemented with 1.0 μM TDZ with an average of 60 shoots per culture, having an average shoot length of 6.0 cm. The best in vitro root induction (89%) was achieved on half strength MS medium without any growth regulator with an average of 24 roots per culture and root length of7 cm. The rooted plantlets were successfully established in soil and grown to maturity at the survival rate of 95% in the indoor grow room. High-performance liquid chromatography was used to assess the stability in chemical profile and quantification of stevioside and rebaudioside A content of in vitro propagated S. rebaudiana plants and compared with their mother plant at the peak vegetative stage. Our results show no significant differences (p in vitro propagated plants. Furthermore, fully developed in vitro propagated S. rebaudiana plants were also compared with mother plant for their gas and water vapour exchange characteristics and leaf anatomy. The results show that in vitro propagated and hardened plants of S. rebaudiana are morphologically as well as functionally comparable to each other and to their mother plant.

The use of esterase polymorphism for analysis of the genetic diversity and structure of stevia (Stevia rebaudiana Bert. Bertoni) populations

Native polyacrylamide gel electrophoresis was used in the current study to identify polymorphisms in α- and β-esterase loci in the leaves of Stevia rebaudiana Bert. Bertoni. The esterases produced from the Est-2 and Est-4 loci were observed as strongly stained bands, and four alleles were detected at each of these loci from the 428 analysed S. rebaudiana plants. The observed and expected mean heterozygosity were higher in populations maintained by genetically breeding plants for higher glycoside production than in plant populations propagated by seeds or maintained by vegetative propagation. The esterase analysis employed in this study showed that the artificial selection of plant samples with specific uniform characteristics, such increased height and precocious flowering, may lead to the fixation of alleles and α/β-esterase phenotype patterns in S. rebaudiana populations. The uniform α/β-esterase phenotype may be applied in the monitoring of genetic stability in selected populations for specific traits of interest.

UDP-dependent glycosyltransferases involved in the biosynthesis of steviol glycosides

A short-term experiment was designed to measure the transcript levels of downstream genes contributing to the biosynthesis of steviol glycosides. Stevia rebaudiana plants were subjected to long- and short-day conditions for different time intervals. Samples from both lower and upper leaves were collected. Using quantitative real-time polymerase chain reaction, the transcript levels of three UDP-dependent glycosyltransferases, UGT85C2, UGT74G1 and UGT76G1, were studied. The results were compared with the steviol glycoside contents measured in the leaves, which were quantified by reversed phase HPLC. In the same daylength condition, steviol glycoside concentration and the transcript levels of the three UGT genes were higher in upper leaves than in lower leaves. Steviol glycosides accumulated more in plants under short-day conditions. Under these conditions, a highly significant correlation was found between UGT85C2 transcription and total steviol glycoside accumulation in the upper leaves. This suggests that the glycosylation of steviol to form steviolmonoside is the rate-limiting step in the glycosylation pathway of steviol glycosides. In these upper leaves, a relatively high accumulation of rebaudioside A compared to stevioside was also observed, however, without correlation with the transcription of UGT76G1.

IMPROVED RP-HPLC METHOD FOR QUANTITATIVE ESTIMATION OF STEVIOSIDE IN STEVIA REBAUDIANA BERTONI BURM

An RP-HPLC method with UV array detection was established for the determination of stevioside, an extract of herbal S. rebaudiana plant . The stevioside was separated using isocratic solvent system consisting of methanol and 0.1% orthophosphoric acid (v/v) in water (70:30) at flow rate of 1.0 ml/min and the detection wavelength of 219 nm. The method was validated for linearity, precision, accuracy, limit of detection (LOD), and limit of quantitation (LOQ). The linearity of the proposed method was obtained in the range of 5.0-75 μg/ml with regression coefficient of 0.9999. Intraday and interday precision studies showed the relative standard deviation less than 2.5%. The accuracy of the proposed method was determined by a recovery study conducted at 3 different levels. The average recovery was 97-99%. The LOD and LOQ were 0.02 and 0.05 µg/ml, respectively. The content of stevioside obtained in the dried leaves powder was within the ranges of 6.83 – 7.91% and 1.7 – 2.9 % w/w, respectively. The proposed method is simple, sensitive, yet reproducible. It is therefore suitable for routine analysis of stevioside in S. rebaudiana Bertoni.

Stimulatory effect of phosphate-solubilizing bacteria on plant growth, stevioside and rebaudioside-A contents of Stevia rebaudiana Bertoni

The effect of four phosphate-solubilizing bacteria (PSB), ( Burkholderia gladioli 10216, Burkholderia gladioli 10217, Enterobacter aerogenes 10208 and Serratia marcescens 10238) as identified on the basis of 16S rRNA gene sequencing was evaluated on plant growth and commercially important glycosides, stevioside (ST) and rebaudioside-A (R-A) of Stevia rebaudiana in pots containing tricalcium phosphate (TCP) supplemented soil. The PSB were isolated from the rhizosphere of S. rebaudiana plants and tested for P-solubilization ability, biocompatibility, indole acetic acid (IAA) and siderophore production. In greenhouse study, treatment of either individual PSB or a consortium (of PSB) resulted in increased plant growth, ST and R-A contents. The stimulatory effect was observed with consortium treatment in plant growth parameters (shoot length, 22.5%; root length, 14.7%; leaf dry weight, 89.0%; stem dry weight, 76.3% and shoot biomass, 82.5%) and glycoside contents (ST, 150% plant −1 and R-A, 555% plant −1) as compared to the un-inoculated plants. Among individual PSB treatments, B. gladioli 10216 showed most promising response in majority of the parameters studied. The root colonization potential of PSB, assayed by RAPD technique, showed the colonization of all PSB isolates, though their extent of colonization varied.

Genotoxicity studies on a high-purity rebaudioside A preparation

Rebaudioside A (Reb A) is a steviol glycoside isolated from the leaves of the Stevia rebaudiana plant. This non-nutritive, natural sweetener is reported to be 250–450 times sweeter than sucrose and has potential for wide use in the US diet, and is used in Japan and South America today. The safety of Reb A has been investigated in several recently published studies and information on genotoxicity is described herein. Reb A was investigated for its potential to induce genotoxicity in three in vitro and two in vivo assays (conducted according to OECD guidelines). Reb A was non-mutagenic in an Ames test using Salmonella typhimurium and Escherichia coli, in a chromosomal aberration test using Chinese Hamster V79 cells and in a mouse lymphoma assay using L5178Y+/− cells, all studies were conducted at concentrations up to 5000 μg/ml, with and without metabolic activation. Also, Reb A was non-genotoxic in a bone marrow micronucleus test in mice at doses up 750 mg/kg bw and in an unscheduled DNA synthesis test in rats at 2000 mg/kg bw. These studies provide additional evidence that Reb A is not genotoxic at the doses tested and further support the generally recognized as safe determination of Reb A.

First Report of Gray Mold Caused by Botrytis cinerea on Stevia rebaudiana in Italy.

Stevia rebaudiana (sweetleaf) is a perennial shrub belonging to the Asteraceae family and is widely grown for its sweet leaves. With its extracts having as much as 300 times the sweetness of sugar, this species is used in many countries for the production of sugar substitutes. However, in Italy, as well as in other countries, this species cannot be grown for the use of its leaf extracts. This plant is grown in a few nurseries in the Albenga Region (northern Italy) as potted plants. In February of 2008, 3-month-old plants grown in plastic pots (14-cm diameter) under glasshouse on heated benches started showing symptoms of a previously unknown blight. The temperature in the glasshouse ranged between 16 and 20°C and plants were watered by sprinkle irrigation. Leaves, starting from the basal ones, showed small, brown spots that spread across the entire leaf surface. Subsequently, the crown and stem were infected, and the pathogen developed abundant, soft, gray mycelium on leaves and stems and in the middle of the heads of S. rebaudiana. Flowers were not present when the symptoms appeared. Severely infected leaves dried out and became necrotic. The disease was observed in one nursery in which 5% of the plants were affected. The margins of the lesions were excised from leaves, immersed in a solution containing 1% sodium hypochlorite, and then cultured on potato dextrose agar (PDA) medium. A fungus produced abundant mycelium when incubated under constant fluorescent light at 22 ± 1°C after 10 days. The conidia were smooth, hyaline, ovoid, measuring 15.5 to 8.3 × 11.1 to 7.3 (average 11.6 × 8.6) μm, and were similar to those described for Botrytis cinerea. Conidiophores were slender and branched with enlarged apical cells bearing conidia on short sterigmata. The identity of the fungus was also confirmed by the production of numerous, small, black sclerotia on PDA plates incubated for 20 days at 8 ± 1°C. Sclerotia were dark and irregular with a diameter ranging from 1 to 2 mm. These morphological characters identified the fungus as B. cinerea (2). The internal transcribed spacer (ITS) region of rDNA was amplified using primers ITS4/ITS6 and sequenced. BLAST analysis (1) of the 780-bp segment showed a 100% homology with the sequence of Botryotinia fuckeliana (perfect stage of B. cinerea). The nucleotide sequence has been assigned GenBank Accession No. FJ486270. Pathogenicity tests were performed by spraying leaves of six healthy 6-month-old potted S. rebaudiana plants with a 105 conidia/ml suspension. Six plants sprayed with water only served as controls. Plants were covered with plastic bags for 3 days after inoculation to maintain high relative humidity and were placed in a growth chamber at 20 ± 1°C. The first foliar lesions developed on leaves 4 days after inoculation, whereas control plants remained healthy. B. cinerea was consistently reisolated from these lesions. The pathogenicity test was completed twice. To our knowledge, this is the first report of the presence of B. cinerea on S. rebaudiana in Italy. The disease has been reported in Ukraine (3) and more recently in Japan (4). The economic importance of this disease is at the moment limited.

Plant growth regulators affecting in vitro cultivation of Stevia rebaudiana

In order to maximize efficiency of plant propagation via direct organogenesis, the influence of plant growth regulators on the growth and development of Stevia rebaudiana grown in vitro was studied. Results indicated that benzyl adenine increased multiplication rate, vitrification and somaclonal variation. However, the best results were recorded with MS nutrient medium without plant growth regulators during in vitro growth and development of Stevia rebaudiana. MS basal medium supplemented with 2 mg/l BA recorded the highest number of shoots, but these shoots were very thin and vitrified and not suitable for multiplication through several subcultures. The nutrient medium (MS basal medium) supplemented with 10 mg/l kinetin recorded 45 shoots / explant as compared to MS nutrient medium which recorded 6.63 shoots / explant but growth parameters for Stevia rebaudiana plantlets grown in MS medium without kinetic is better than MS medium containing kinetin at high concentrations. Nutrient medium (MS basal medium) supplemented with IBA at low concentrations (0.01mg/l) or without auxins achieved the best in vitro growth of Stevia rebaudiana plantlets (100 % root formation). IBA was better than NAA and IAA for shoot and root formation. Increasing NAA concentrations decreased gradually number of shoots. Regarding the effect of NAA on root formation, data indicated that the per cent of shoots formed roots was 87% on MS basal medium without plant growth regulators as compared to MS basal medium supplemented with NAA at low concentrations 0.001, 0.01 and 0.1 mg/l NAA where root per cent was 80%, 73 % and 53 % respectively. On the other hand, NAA at 1.0 and 1.5 mg/l did not help shoots to form roots. Statistical analysis of variance showed no significant differences amongst IAA treatments for in vitro growth of plantlets. Stevia rebaudiana plants were adapted and grown well in planting media containing peat moss, sand and vermiculite at equal volume.

Propagación in vitro de Stevia rebaudiana Bert. (Asteraceae-Eupatorieae) a través de organogénesis

Secciones de entrenudos tomados de plantas de Stevia rebaudiana establecidas en condiciones in vitro fueron inducidos a formar callos en presencia de diferentes concentraciones de Bencilaminopurina (BAP) combinadas con varias cantidades de ácido naftalenacético (ANA) evaluándose su efecto sobre el porcentaje de inducción de callo, friabilidad y formación de órganos. Posteriormente, los callos inducidos fueron multiplicados y el efecto de cuatro combinaciones de ANA y BAP sobre el incremento de masa fresca evaluado. Finalmente, los tallos proliferados fueron transferidos sobre medio de regeneración suplementado con diferentes cantidades de BAP solo o en combinación con ANA con el fin de evaluar su efecto sobre la regeneración de brotes. Todos los tratamientos fueron distribuidos utilizando un diseño completamente al azar con un mínimo de 15 repeticiones por tratamiento. Los resultados demostraron que la presencia de ANA es necesaria y suficiente para la formación de callo. La regeneración de brotes vía organogénesis ocurre de forma independiente a la presencia de RCV en el medio. Los tratamientos aplicados no afectaron el incremento de masa fresca de callo como tampoco tuvieron ningún efecto sobre la regeneración de plantas a partir del callo proliferado. Los datos colectados permiten recomendar un suplemento combinado de 1.0 mg L-1 ANA + 4.0 mg L-1 BAP para obtener plantas de Stevia rebaudiana a través de organogénesis.

Occurrence of Tomato spotted wilt virus in Stevia rebaudiana and Solanum tuberosum in Northern Greece.

Tomato spotted wilt virus (TSWV) (genus Tospovirus, family Bunyaviridae) was first reported in Greece during 1972 (3) and currently is widespread in the central and northern part of the country infecting several cultivated and wild plant species (1,2). In June 2006, virus-like symptoms similar to those caused by TSWV were observed on sweet honey leaf (Stevia rebaudiana (Bertoni) Bertoni, [synonym Eupatorium rebaudianum Bertoni], family Asteraceae) plants in an experimental field in the prefecture of Kilkis and on potato (Solanum tuberosum, family Solanaceae) plants growing close to tobacco in the prefecture of Kozani in northern Greece. Diseased S. rebaudiana plants expressed chlorotic and necrotic rings and line patterns on systemically infected leaves and occasionally a general chlorosis or dwarfing of the plant. Potato plants expressed only necrotic rings or lesions and a mild plant dwarfing. Seven percent of the S. rebaudiana plants, but only 0.1% of the potato plants, were showing symptoms. TSWV was detected by ELISA using polyclonal antisera in all symptomatic plants of both species, but those plants tested negative for Potato Y virus (PVY), Cucumber mosaic virus (CMV), Alfalfa mosaic virus (AMV), and Potato leafroll virus (PLRV); all viruses known to occur in these areas. Sap from symptomatic plants was used to mechanically inoculate Petunia × hybrida cv. Blue magic, Nicotiana tabacum cvs. Samsun NN and Basmas, N. benthamiana, N. glutinosa, N. rustica, Datura stramonium, and Physalis floridana. Sap from both S. rebaudiana and potato produced typical symptoms of TSWV on the diagnostic test plants; all Nicotiana species and cultivars, as well as D. stramonium and P. floridana, reacted with chlorotic and necrotic lesions on the inoculated leaves followed by systemic mosaic and mottling of the leaves. None of the isolates produced systemic infection on P. × hybrida. Adult thrips, identified as Thrips tabaci, were collected from infected plants of both hosts in the field and determined to be infected with TSWV by the petunia leaf disc method (4). Symptoms observed in the field on both hosts where reproduced in the laboratory after infestation of healthy plants with TSWV-infected individuals from a tobacco population of T. tabaci. To our knowledge, this is the first official report of a TSWV infection in S. rebaudiana. Potato is a known host for TSWV, however, to our knowledge, this is the first natural infection detected in Greece.

First report of Alternaria alternata causing leaf spot on Stevia rebaudiana

Stevia rebaudiana is a herbaceous perennial plant of the Asteraceae and native to Paraguay. It has been recognized world wide for its excellent sweetening property. In India the plant has been introduced in the states of Maharastra, Tamil Nadu, Karanataka, Rajasthan and West Bengal. For the last three years, during February when temperature ranges from 20–25°C, severe foliar infections were observed in the region of South Bengal. Symptoms initially appeared as small circular spots, light brown in colour. Later, many became irregular and dark brown to grey, while others remained circular with concentric rings or zones. On severely affected leaves several spots coalesced to form large necrotic areas. On older leaves concentric spots were more common at the tips. Leaf spots varied from 2–18 mm in diameter. Conidial dimensions varied from 10–40 × 6–12 mm, mid to dark brown or olive-brown in colour, short beaked, borne in long chains, oval and bean shaped with 3–5 transverse septa. A fungus was isolated on potato dextrose agar media and produced abundant branched septate, brownish mycelia. Conidiophores were simple, olive-brown, septate, variable in length with terminal conidia, which were solitary or in short chains. Conidial characteristics from culture were similar to the conidia isolated from infected plants. Based on the morphological characters, the organism was identified as Alternaria alternata and the identification was confirmed by the Agharkar Research Institute, Pune, India. A pathogenicity test was performed by spraying leaves of 10 healthy, 3-month-old potted S. rebaudiana plants with a spore suspension of 105 conidia per mL. Control plants were sprayed with sterile water. Plants were covered with plastic bags for 10 days and kept in the laboratory garden at 30 ± 20°C. The pathogenicity tests were repeated three times. The first lesions appeared after a period of 12 ± 2 days. The pathogen was consistently reisolated from the lesions. Fungal diseases reported on S. rebaudiana include Erysiphe cichoracearum, Rhizoctonia solani, Sclerotium dephinii, Sclerotium rolfsii, Septoria steviae, Sclerotinia sclerotiorum and A. steviae (Thomas, 2000; Lovering & Reeleeder, 1996; Kamalakannan et al., 2007; Ishiba et al., 1982). This is the first report of A. alternata on stevia in India. Authors are thankful to the Ramakrishna Mission Ashrama, Narendrapur, India for providing facilities to perform field experiments in their Medicinal Plant Garden, as well as thankful to the Directorate of Food Processing Industries and Horticulture, Govt. of West Bengal, India for financial assistance.

Monitoring the sweetener content in leaves of Stevia Rebaudiana plant using Raman and SERS spectroscopy

Monitoring the sweetener content in leaves of Stevia Rebaudiana plant using Raman and SERS spectroscopy

Micropropagation ofStevia rebaudianaThrough Leaf Explants from Adult Plants

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.