The Effect of Long and Short Day Length upon the Growth of Whole Plants and the Level of Soluble Proteins, Sugars, and Stevioside in Leaves of Stevia rebaudiana Bert.

Long-day conditions increase internode length, leaf area, anddry weight and reduce the interval between the appearance ofsuccessive leaf pairs in S. rebaudiana as compared with shortdays. Total soluble leaf sugars, protein, and stevioside contentare also augmented in both absolute and relative terms and thebiosynthesis of steviol, the aglucone present in stevioside,is increased by 45%. The interaction between dry matter, steviosidecontent, and day length is discussed and a theory for the biologicalrole of stevioside as a possible defence mechanism against insectherbivory is proposed: as such this is the first known caseof a tetracyclic diterpene glycoside assuming this function.     

In Vitro Regeneration of Stevia rebaudiana (Bert) from the Nodal Explant

Procedure for micropropagation of Stevia rebaudiana Bertoni, containing stevioside, a natural noncaloric sweetner, has been developed using nodal segments as explant. Higher proliferation of shoots and multiplication was obtained on Murashige and Skoog basal medium (MS) supplemented with 1.0 mg l-1 indoleacetic acid (IAA) plus 10.0 mg l^-1 kinetin and 30.0 mg l-1 adenine sulphate. Sprouting of 90% of the axillary buds was observed within 4 weeks of inoculation, producing >10.0 shoots per explant within 12 weeks. Profuse roots were induced from 90% of the regenerated shoots within 4 weeks of inoculation on half strength MS solid medium supplemented with 1.0 mg l^-1 IAA. High survival rate, > 60%, was obtained when the plantlets were transferred to field conditions. The survival rate of taller plants was always higher. The in vitro regenerated plants were morphologically indistinguishable from the donor plants and leaves were of intense sweet taste upon chewing. The heterogenic nature of S. rebaudiana necessitates establishment of protocol for every genotype independently.     

Steviol Glycoside Content in Different Organs of Stevia rebaudiana and Its Dynamics during Ontogeny

The contents of three major steviol glycosides (SGs) (stevioside and rebaudiosides A and C) in vegetative and generative organs during ontogeny of Stevia rebaudiana Bertoni were analysed with HPLC. Plant organs contained different amounts of the SGs, which declined in the following order: leaves, flowers, stems, seeds, roots. The highest content of the SGs was found in upper young actively growing shoot sections, whereas lower senescent shoot sections exhibited the lowest amount of such compounds. During ontogeny a gradual increase in the SG content was observed in both mature stevia leaves and stems, and this process lasted up to the budding phase and the onset of flowering. This revised version was published online in July 2006 with corrections to the Cover Date.     

Determination of Microgram Quantities of Stevioside from Leaves of Stevia rebaudiana Bert. by Two-Dimensional Thin Layer Chromatography

A method is described for the determination of stevioside fromleaves of Stevia rebaudiana Bert. Separation upon non-activatedsilica gel impregnated with boric acid in a two-dimensionalchromatographic system followed by clution and colorimetricassay is reported. Recuperation from chromoplates is over 95%.The observed ratio of the optical density of glucose: steviosidematches that expected theoretically so that glucose may be employedas a standard where pure stevioside is not available. Effects of the plant growth retardant, ancymidol, on the growthand morphology of the shoot system of cucumber (Cucumis sativusL. ) were investigated. Ancymidol inhibited stem elongation,reducing both number and length of internodes. Reduction inleaf area, attributable to a reduction in both cell size andnumber, was accompanied by an increase in chlorophyll per unitarea. The retardant decreased apical dominance and delayed anthesis.Gibberellic acid fully reversed ancymidol-induced inhibitionof stem elongation, internode length and production, and leafexpansion. GA_4/7 and ancymidol gave a synergistic promotionof stem elongation by increasing elongation of younger internodesand increasing internode production. Synergistic promotion ofpetiole elongation by this combination was also observed. Ancymidol,applied 7 d previously either to the shoot or root, severelyreduced the level of gibberellin-like activity in bleeding sapcollected from decapitated plants.     

Influence of insect pollinators and harvesting time on the quality of Stevia rebaudiana (Bert.) Bertoni seeds

Stevia rebaudiana (Bert.) Bertoni has sporophytic self-incompatibility and this may lead to poor seed yield and quality, which are obstacles to large-scale crop establishment. This research investigated the influence of insect pollinators and the harvesting time on the qualitative and quantitative characteristics of the ripe seeds. A pot trial under open field conditions was established to evaluate the abundance of insect pollinators and three harvesting times on the production of ripe seeds, germination percentage, mean germination time and thousand seed weight (TSW) in 36 F1 open-pollinated genotypes. Large variability in the number of corymbs of capitula, flowering time, reproductive period and seed yield was observed among the genotypes. Genotypes with promising phenological and reproductive properties were identified. The worst germination percentage and TSW were recorded in the latest harvest. Significant linear regressions were found among the investigated parameters (insect number, germination percentage, TSW, seeds yield per plant and cycle length), confirming complex interactions on the yield and quality of stevia seeds. Results indicate that earlier flowering genotypes produced higher seed quantity, due to favourable weather conditions during the reproductive phase. Moreover, cross-pollination and the insect abundance, as Apidae and Syrphidae, play a crucial role for enhancing the stevia seed yield and quality.     

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

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

Ultrastructural Study of Mesophyll Cells During Their Dediff erentiation in Stevia rebaudiana

Authors deal with the changs of cell ultrastructure ,nucleic acid and soluble protein contents as well as peroxidase activity during mesophyll cell dedifferentiation of leaf explant of stevia rebaudiaha Bertoni. Electron microscopic observations indicated that the mature mesophyll cells had returning to meristematic cells had to go through three main phases: (1)Initiation phase :The main characteristics of cells in this phase were cytoplasmic expansion, stretching out of cytoplasmic filaments to cell centre and appearance of the protein bodies in vacuoles. (2)Medial evolving phase :The main characteristics of cells in this phase were formation of cytoplasmic bridges ;the chloroplasts were dedifferentiated to proplastids and the nuclei started to move toward centre . (3)Finishing and dividing phase :During this phase cell dedifferentiation had finished and cell division was about to start. The main characteristics of cells in this phase were the appearance of meristematic state ; the nuclei, sometimes irregularly shaped, occupied a major proportion of the cell ; the nucleoli were vaculated and the nucleopores increased in number and size. Biochemic analyses showed that the contents of the total nucleic acids , RNA and the soluble proteins as well as the peroxidase activity gradully increased during formation of meristematic cell aggregates and then reduced. However, the change of DNA contents was not obvious.     

Observations of the Leaf Cell Vacuole of Stevia rebaudiana Bertani Under the Electron Microscope

By applying electron microscopic technique, we aimed to observe the morphology of leaf cell vacuole of Stevia rebaudiana Bertani, and the change of inclusion during its different stages of growth. We ascertain that the size of vacuoles is small in younger leaf cell, that the number of vacuoles is more than that in other stages, and that vacuoles appear translucent, either round or elliptical in form. Also those leaf cells that are at the stage of active growth appear to have many morphologically specialized vacuoles containing a lot of inclusions in different forms of granule and vesicle. Furthermore, the inclusion of the vacuols in the elder leaf cells decompose gradually, eventually disappear. Comparing with the substructural observation of chloroplast, by PAS reaction, and with different content of steviaside, we discover that there exists corresponding relationship. Thus, we may infer that steviaside is stored mainly in the vacuoles and it is relevant with the function of production.     

Septoria Leaf Spot of Stevia rebaudiana in Canada and Methods for Screening for Resistance

The herb Stevia rebaudiana is a potential source of low-calorie sweeteners. In 1995, a severe leaf spot and blight was observed in stevia production fields and research plots in the provinces of Ontario and British Columbia, Canada. The disease was characterized by angular, shiny, olive-grey lesions that rapidly coalesced and were often surrounded by a chlorotic halo. Leaves quickly became necrotic and often dropped off the plant. The disease progressed upwards in the foliage during the growing season. A Septoria sp. was isolated from diseased leaves. Ten isolates (five from each of the two provinces) of the Septoria sp. were compared with respect to conidial size. Across isolates, conidia lengths and widths overlapped (grand means for length and width were 71.4 μm and 1.4 μm, respectively). Conidiogenesis was holoblastic. Morphological characteristics and disease symptoms were similar to those of Septoria steviae, previously reported only from Japan. It was concluded that the Canadian isolates belonged to S. steviae. Isolates from Canada did not differ significantly from one another with respect to effects of temperature on colony growth or germination of conidia. Optimum temperatures for these parameters were between 20 and 25°C. In field trials, the pathogen was shown to successfully over-winter in diseased leaf tissue. In order to develop procedures for identification of resistant germplasm and greenhouse screening of candidate fungicides, effects of leaf wetness period, inoculum concentration, and plant age on disease development were determined. Thirty-six hours of leaf wetness were required for consistent development of leaf spots. Inoculum concentrations of 5 × 10⁵ conidia/ml or more were required to produce high disease severities; 6-week-old plants were more susceptible than older plants. In the growth chamber, greenhouse, and field trials, germplasm selections with high levels of resistance to S. steviae were identified. This is the first report of resistance to this disease in S. rebaudiana.     

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.     

一种作用于花青素和甜菊醇的甜菊糖基转移酶的基因克隆和功能分析

本文对一种新的甜菊糖基转移酶进行了基因克隆和功能分析。获得的基因cDNA全长1419bp,编码473个氨基酸,蛋白质分子量约53．2KDa。与常见的糖基转移酶基因比较,相似性达44％以上,工具有糖基转移酶的保守序列。体外异源表达获得的融合蛋白,具有在花青素类和甜菊醇等糖基受体上转糖基的酶活性。在对一系列不同底物的酶活性进行比较后,推测这种糖基转移酶在体内参与了甜菊糖苷的合成。结果表明,具有广泛的底物活性的类黄酮类糖基转移酶,在甜菊体内不仅对类黄酮转糖基,而且在生成水溶性甜菊糖苷的过程中也扮演重要的角色。     

温度对番茄花叶病毒(TOMV)增殖及番茄叶片可溶性蛋白变化的影响

局部枯斑法测定结果表明,番茄GCR—267品系叶片内ToMV的含量仅为番茄GCR-26品系叶片内病毒含量的1/40—1/50;GCR-267品系含有的Tm-2~(?)基因对ToMV增殖的抑制作用不受温度变化的影响;而ToMV在GCR-26体内的增殖却依赖于环境温度,高温对它有部分的抑制作用。用非变性聚丙烯酰胺凝胶电泳和SDS—聚丙烯酰胺凝胶电泳分析上述两个品系在常温和高温下接种ToMV后其叶片可溶性蛋白的变化,结果发现在GCR-26品系中,ToMV的增殖与寄主体内新产生的14.2KD蛋白呈正相关,而在GCR—267品系中未检测到这种蛋白。我们推测14.2KD蛋白可能是一种能参与或促进ToMV增殖过程的温度敏感因子,称之为番茄“S”蛋白。     

Spectral lights trigger biomass accumulation and production of antioxidant secondary metabolites in adventitious root cultures of Stevia rebaudiana (Bert.)

Stevia rebaudiana (S. rebaudiana) is the most important therapeutic plant species and has been accepted as such worldwide. It has a tendency to accumulate steviol glycosides, which are 300 times sweeter than marketable sugar. Recently, diabetic patients commonly use this plant as a sugar substitute for sweet taste. In the present study, the effects of different spectral lights were investigated on biomass accumulation and production of secondary metabolites in adventitious root cultures of S. rebaudiana. For callus development, leaf explants were excised from seed-derived plantlets and inoculated on a Murashige and Skoog (MS) medium containing the combination of 2,4-dichlorophenoxy acetic acid (2, 4-D, 2.0 mg/l) and 6-benzyladenine (BA, 2.0 mg/l), while 0.5 mg/l naphthalene acetic acid (NAA) was used for adventitious root culture. Adventitious root cultures were exposed to different spectral lights (blue, green, violet, red and yellow) for a 30-day period. White light was used as control. The growth kinetics was studied for 30 days with 3-day intervals. In this study, the violet light showed the maximum accumulation of fresh biomass (2.495 g/flask) as compared to control (1.63 g/flask), while red light showed growth inhibition (1.025 g/flask) as compared to control. The blue light enhanced the highest accumulation of phenolic content (TPC; 6.56 mg GAE/g DW), total phenolic production (TPP; 101 mg/flask) as compared to control (5.44 mg GAE/g DW; 82.2 mg GAE/g DW), and exhibited a strong correlation with dry biomass. Blue light also improved the accumulation of total flavonoid content (TFC; 4.33 mg RE/g DW) and total flavonoid production (TFP; 65 mg/flask) as compared to control. The violet light showed the highest DPPH inhibition (79.72%), while the lowest antioxidant activity was observed for control roots (73.81%). Hence, we concluded that the application of spectral lights is an auspicious strategy for the enhancement of the required antioxidant secondary metabolites in adventitious root cultures of S. rebaudiana and of other medicinal plants.     

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

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

Phosphate Deficiency in Maize. VI. Changes in the Two-Dimensional Electrophoretic Patterns of Soluble Proteins from Second Leaf Blades Associated with Induced Senescence

The effects of P deprivation on the two-dimensional electrophoreticpatterns of soluble proteins were evaluated in maize (Zea maysL.) leaves. P deprivation resulted in decreases in the relativeabundance of 11 of more than 450 polypeptides and increasesin that of 18 polypeptides. These changes are discussed in relationto leaf senescence. (Received March 23, 1995; Accepted June 30, 1995)     

Effect of crude extract of Stevia rebaudiana on renal water and electrolytes excretion.

To evaluate the effect of crude extract of Stevia rebaudiana on renal water, Na+ and K+ excretion, male Wistar rats (250-350 g each) under antidiuresis or water diuresis conditions, were evaluated. During intravenous infusion of the extract (0.05 mg/min/100 g) no significant differences were detected in mean arterial pressure or renal hemodynamics parameters. In contrast, fractional water and sodium excretion and solute clearance increased significantly, in both groups of animals. In antidiuresis rats the extract significantly increased reabsorption of water by the collecting duct and in water diuresis animals the extract significantly increased free water clearance. The data suggest preferential action of the extract in the proximal tubular cells involved with salt transport mechanism.     

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

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

The Fertilization and Early Development of Embryo and Endosperm in Stevia rebaudiana Bertani

The mature embryo sac is surrounded by endothelium tapetum. It is composed or an egg apparatus, one central cell with secondary nucleus, and 1–6 antipodal cells. About the 6th hour after pollination, female and male nuclei fuse with each other. The syngamy occurred almost simultaneously with the fusion of an other sperm nucleus and the secondary nucleus, but the velocity of the latter is faster than that of the syngamy. The fertilization of Stevia rebaudiana Bertani belongs to the premitotic type. About the 8th hour after pollination, primary endosperm nucleus is in mitosis, its dividing orientation may parallel or at right angle to the long axis of the embryo sac, and gives rise to two initial endosperm cells. The first five divisions of the endosperm cells are of synchronism. At the stage of heart-shaped embryo, the endosperm cells show the signs of digestion and absorbed. The endosperm development is of the cellular type. About the 10th hour after pollination, zygote divides for the first time. The division of the zygote is always transverse. The embryo development conforms to the Asterad type.