0000000000724224
AUTHOR
Pedro Pérez-bermúdez
Morphogenesis in Root Tip Meristem Cultures of Digitalis obscura L.
Summary The morphogenic capacity of Digitalis obscura L. root tip meristems cultured in vitro has been studied, noting hormonal factors inducing differentiation and development of shoots as well as those promoting somatic embryogenesis. Caulogenesis was obtained with different auxin/cytokinin combinations but a high rate of shoot regeneration was induced by 0.1 or 0.5 ppm IAA and 1.0 ppm BA. Different stages of somatic embryo development were observed in media supplemented with auxins or high auxin/cytokinin ratios, although no complete plant regeneration was achieved. This is the first report known of shoot regeneration from root tips of Digitalis obscura L.
Cover crops and pruning in Bobal and Tempranillo vineyards have little influence on grapevine nutrition
ABSTRACT Cover crops may improve vineyard soil properties, grapevine nutrient status and berry composition, however, factors such as cover crop type, annual rainfall, climate and irrigation may change their effects on vineyards. From 2008 to 2011, the effects of a non-permanent cover crop and two pruning techniques on soil as well as vine nutrients and grapevine performance of two vineyards (cv. Tempranillo and cv. Bobal) were evaluated. For that purpose, two legumes were sown in inter-rows of hand-pruned vines in February and were tilled at flowering. Soil tillage, or cover cropping, was combined with either light pruning or severe pruning to study foliar nutrient variations. Soil N, P, K …
Characterisation of Bobal and Crujidera grape cultivars, in comparison with Tempranillo and Cabernet Sauvignon: Evolution of leaf macronutrients and berry composition during grape ripening
Abstract We present characterisation data for two Spanish autochthonous grapevines, Bobal and Crujidera, in comparison with the well-known cultivars Tempranillo and Cabernet Sauvignon. Microsatellite markers were used for the molecular characterisation of Crujidera grapevines. Leaf macronutrient contents of the four cultivars were evaluated, as well as their changes at different vine developmental stages, and veraison was seen as the most suitable time to evaluate the nutritional status. Quantitative changes in some physiological parameters and the phenolic composition of the four grape varieties were measured during the last month of ripening. Polyphenols, tannins and anthocyanins increase…
Factors affecting shoot proliferation and vitrification inDigitalis obscura cultures
Variations of composition and consistency of the culture medium and time of exposure to growth regulators were assayed to optimize normal caulogenic response ofDigitalis obscura hypocotyls cultured in vitro. The effects of the culture conditions on physiologic changes related to vitrification of the regenerated plants were also investigated. Liquid medium increased the bud-forming capacity of the explants but induced buds failed to develop into shoots and showed symptoms of vitrification. On agar-solidified media, maximum multiplication rates were achieved with 0.7% agar. Increasing agar concentration reduced vitrification but lowered the propagation rate. Changes in the strength of the mac…
Influence of Microspore Development on Pollen Protoplast Isolation in Digitalis obscura
Summary Protocols for the enzymatic isolation of Digitalis obscura pollen protoplasts are described. The developmental stage of the microspores was a critical factor for successful isolation since protoplasts were obtained only from tetrads. The highest yield of viable protoplasts was achieved with an enzymatic mixture containing helicase, cellulase and pectolyase in 0.5 M sucrose. In different culture techniques employed, cell wall regeneration took place only when protoplasts were plated in agarose drops.
Plant regeneration from juvenile and adult Anthyllis cytisoides, a multipurpose leguminous shrub
Summary Anthyllis cytisoides , a legume shrub used for afforestation and reclamation of degraded Mediterranean areas, was successfully micropropagated from expiants of juvenile (cotyledonary nodes and apical buds) and adult origin (axillary buds). Multiple shoot formation was dependent on the presence of benzyladenine in the induction medium. Of the salt formulation and expiants examined, the higher proliferation rates were obtained when axillary buds from adult plants were cultured on a modified Schenk and Hildebrandt medium. Following the preferred protocol, shoot yield reached values greater than 100 shoots per expiant. Plants were easily rooted and transplanted into greenhouse.
Effects of Gibberellic Acid on Morphogenesis and Cardenolide Accumulation in Juvenile and Adult Digitalis obscura Cultures
Summary Effects of gibberellic acid on morphogenesis and cardenolide accumulation in in vitro cultures of Digitalis obscura are reported. Cultures were established from hypocotyls or leaves of axenic seedlings and shoot tips of mature plants. Irrespective of the kind of explant, gibberellic acid by itself did not induce morphogenesis but modified those morphogenic responses promoted by auxins and/or cytokinins, always inhibited organogenesis and favoured both embryo formation and conversion into plants. Gibberellic acid did not significantly affect cardenolide content of plants regenerated from juvenile or mature D. obscura explants.
SOIL CHARACTERISTICS, MINERAL NUTRIENTS, BIOMASS, AND CARDENOLIDE PRODUCTION INDIGITALIS OBSCURAWILD POPULATIONS
Mature leaves of wild-growing Digitalis obscura plants and soil samples were collected in four different regions. Soil and leaf mineral nutrient contents as well as biomass and cardenolide productions were determined. The soils in the four collection sites, basic and highly calcareous, showed significant interpopulation differences in phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), manganese (Mn), copper (Cu), and zinc (Zn). Foliar mineral nutrients and cardenolides also varied among populations. Highest cardenolide yield was found in those Digitalis populations presenting a better development. The cardenolide content exhibited significant negative correlations with the levels …
Water-depth effects in photosynthetic pigment content of the benthic algae Dictyota dichotoma and Udotea petiolata
Abstract The effect of water depth on the light-harvesting pigments contents in Dictyota dichotoma (Hudson) Lamouroux and Udotea petiolata (Turra) Borgesen was studied. Both species showed higher photosynthetic-pigments contents with increased depth or shade. The ratios of chlorophyll c /chlorophyll a and chlorophyll b / chlrophyll a in D. dichotoma and U. petiolata were lower in light-exposed samples. In D. dichotoma , the ratios of fucoxanthin to chlorophyll a increased with depth or shade; however, it is worth noting that the 0 m (sun) samples showed the highest values. On the other hand, total carotenoids to chlorophyll a ratios in U. petiolata increased with depth.
Effects of Rosmarinus officinalis and Salvia officinalis essential oils on Tetranychus urticae Koch (Acari: Tetranychidae)
Laboratory trials were carried out to characterize Rosmarinus officinal and Salvia officinalis essential oils and to evaluate their pesticidal activities against Tetranychus urticae. Slide-dip and leaf-disk bioassays were employed to study the mortality caused by these plant oils on two-spotted spiders. Different dilutions of both essential oils (0.10-0.25%, v/v) caused acute contact toxicity, although the sage extract showed greater acaricidal activity than rosemary oil. Mortality rates of 95-100% were observed at all the sage oil dosages and when rosemary emulsions contained at least 0.20% of essential oil. In the residual contact experiments (leaf-disk assays), 0.15-0.25% of sage oil or …
Factors affecting adventitious bud induction in Pinus elliottii (Engelm.) embryos cultured in vitro
Embryos of slash pine (Pinus elliottii Engelm.) were induced to form adventitious buds when placed in culture on nutrient media supplemented with cytokinin. Buds were induced on media containing Risser & White major salts. The high content in nitrogen of Murashige & Skoog formulation seems to be deleterious for this in vitro system, since morphogenic responses were only promoted when nitrogen concentration was drastically reduced in the macronutrient formulation. Factors such as concentration of cytokinin (6-benzyladenine) and time and method of exposure (liquid or solid induction medium) strongly influenced bud formation and development. The greatest number of buds and shoots were obtained…
Production of soluble eukaryotic recombinant proteins in E. coli is favoured in early log-phase cultures induced at low temperature
Abstract Background Producing recombinant plant proteins expressed in Escherichia coli produce in high yields and in a soluble and functional form can be difficult. Under overexpression conditions, proteins frequently accumulate as insoluble aggregates (inclusion bodies) within the producing bacteria. We evaluated how the initial culture density, temperature and duration of the expression stage affect the production of some eukaryotic enzymes in E. coli. Findings A high yield of active soluble proteins was obtained by combining early-log phase cultures and low temperatures for protein induction. When IPTG was added at OD600 = 0.1 and cultures were maintained at 4°C for 48-72 h, the soluble …
Plant progesterone 5β-reductase is not homologous to the animal enzyme. Molecular evolutionary characterization of P5βR from Digitalis purpurea
Plants of the genus Digitalis produce cardiac glycosides, i.e. digoxin, which are widely used for congestive heart failure. Progesterone 5beta-reductase (P5betaR) is a key enzyme in the biosynthesis of these natural products. Here, we have carried out the purification and partial amino acid sequencing of the native P5betaR from foxglove (Digitalis purpurea), and isolated a cDNA encoding this enzyme. Similarly to other steroid 5beta-reductases, the recombinant P5betaR catalyzes the stereospecific reduction of the Delta(4)-double bond of several steroids with a 3-oxo,Delta(4,5) structure. The gene encoding P5betaR is expressed in all plant organs, and maximally transcribed in leaves and matur…
Cloning and expression of two novel aldo-keto reductases fromDigitalis purpurealeaves
The aldo-keto reductase (AKR) superfamily comprises proteins that catalyse mainly the reduction of carbonyl groups or carbon–carbon double bonds of a wide variety of substrates including steroids. Such types of reactions have been proposed to occur in the biosynthetic pathway of the cardiac glycosides produced by Digitalis plants. Two cDNAs encoding leaf-specific AKR proteins (DpAR1 and DpAR2) were isolated from a D. purpurea cDNA library using the rat Δ4-3-ketosteroid 5β-reductase clone. Both cDNAs encode 315 amino acid proteins showing 98.4% identity. DpAR proteins present high identities (68–80%) with four Arabidopsis clones and a 67% identity with the aldose/aldehyde reductase from Medi…
Soil‐plant relationships, micronutrient contents, and cardenolide production in natural populations of Digitalis obscura
The production of secondary metabolites by plants growing in natural populations is conditioned by environmental factors. In the present study, we have investigated the relationships among soil properties, micronutrients in soils and plants, and cardenolide production from wild Digitalis obscura (Scrophulariaceae) populations. Young and mature leaves and soil samples were collected in ten different populations, corresponding to three Mediterranean bioclimatic belts (Thermo-, Meso-, and Supramediterranean belts). Soil (total and EDTA-extractable) and leaf micronutrients (Fe, Mn, Zn, and Cu), and leaf cardenolide accumulation have been determined. Significant negative correlations were observ…
Relationships among soil characteristics, plant macronutrients, and cardenolide accumulation in natural populations of Digitalis obscura
In the present study, we have investigated relationships among several soil parameters (pH, organic matter, total carbonate, macronutrients, electrical conductivity, cation-exchange capacity) and macronutrient and cardenolide contents in leaves of wild Digitalis obscura plants. Young and mature leaves and soil samples were collected in ten different areas, corresponding to three Mediterranean bioclimatic belts (thermo-, meso-, and supramediterranean belts). Soil and leaf macronutrient (N, P, K, Ca, and Mg) contents and leaf cardenolide contents were determined. Bioclimatic conditions influenced the development of D. obscura, biomass being lowest in plant populations of the supramediterranea…
A morphogenetic role for ethylene in hypocotyl cultures of Digitalis obscura L.
The effect of exogenously applied ethylene on organogenesis in Digitalis obscura L. hypocotyls cultured in vitro was studied. Interactions of this gas with other growth regulators was also tested. Ethylene by itself only promoted root formation. Shoot regeneration was obtained in presence of indoleacetic acid and kinetin. The addition of ethylene (10 ppm) increased the caulogenetic action of this medium; higher concentrations than 10 ppm reduced this response. Kinetin alone did not promote organogenesis and nullified the promotive effect of ethylene on rhizogenesis.
Estudio de la actividad biocida de aceites procedentes de plantas aromáticas sobre Tetranychus urticae y Ceratitis capitata
Se ha estudiado la actividad biocida de aceites esenciales extraídos de plantas aromáticas como el romero (Rosmarinus officinalis L.) y la salvia (Salvia officinalis L.) sobre la araña roja (Tetranychus urticae Koch) y la mosca de la fruta (Ceratitis capitata Wiedemann). El aceite esencial de salvia a la concentración del 1% se mostró capaz de atraer adultos de C. capitata. La mayor actividad frente a la araña roja se observó por parte del aceite esencial de salvia, provocando, con gran rapidez, una alta tasa de mortalidad. El aceite de salvia al 0,25% consiguió reducir el nivel de puesta de T. urticae, llegando incluso a tasas de reducción de un 50%.
Selection and long-term cultures of high-yielding Digitalis obscura plants: RAPD markers for analysis of genetic stability
Fifteen wild-growing plants of Digitalis obscura collected in three differents regions were characterized according to their capacities to biosynthesize cardenolides and to proliferate in vitro. Great genotype-dependent variabilities were found in both parameters. Selected genotypes were differentiated using randomly amplified polymorphic DNA (RAPD) markers. Once an elite genotype with high-yielding properties was isolated and micropropagated during 2 years, identity of the donor plant and the regenerants was confirmed by RAPD analysis.
Seasonal Variation in Nutrient Status of Foxglove Leaves
The seasonal variation of mineral elements and the relationships among them were studied in natural populations of foxglove (Digitalis obscura). Young and mature leaves were collected in 10 different populations and on four sample dates (May, July, October, and February). Leaf mineral elements [nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), iron (Fe), manganese (Mn), zinc (Zn), and copper (Cu)] were determined. The highest concentrations of N, P, and K in young leaf were recorded in May, followed by a decrease in the other months, while in contrast Ca and Fe showed the lowest concentration in May. Mature leaves showed differential seasonal behavior. Besides seaso…
Extracellular vesicles in food: Experimental evidence of their secretion in grape fruits
In the last decade, the number of studies related with extracellular vesicles (EVs) has dramatically grown since their role as key part of intercellular communication has been confirmed. EVs, as transporter of distinct bioactive molecules, can take part in different physiological mechanisms and have been gaining attention as potential tools with a wide range of therapeutic effects. Whereas a high number of studies have been published related to mammalian derived EVs, including products as food source, the existence of EVs in plants still is controversial. Recent descriptions of vesicles derived from edible plants show that they might contain pharmacological active molecules. In this context…
Micropropagation of juvenile and adultDigitalis obscura and cardenolide content of clonally propagated plants
Cultures ofDigitalis obscura L. were established from axillary buds of mature plants or leaves of seedlings obtained under aseptic conditions. Explants were cultured on Murashige and Skoog medium containing benzyladenine and/or naphthaleneacetic acid. Shoot proliferation from axillary buds was not affected by seasonal fluctuations in the stock plants and increased relative to the cytokinin concentration, but auxin reduced the multiplication rate. Differentiation of somatic embryos and adventitious buds from cultured leaves required naphthaleneacetic acid alone or combined with benzyladenine, respectively. Cardenolide pattern and content of the regenerated plants were determined by high perf…
Morphogenesis and Cardenolide Formation in Digitalis obscura Cultures
Summary Cardenolide formation was investigated in hypocotyl cultures of Digitalis obscura L. established on MS medium with growth regulators selected to promote either callus formation, caulogenesis, embryogenesis or rhizogenesis. Examination of the cardenolide composition of leaves from mature plants was also performed. Several compounds of series A and C were identified by HPLC in in vitro and in vivo grown tissues, no marked differences being found between their cardenolide patterns. RIA determinations showed that shoot- or embryo-forming calli accumulated significantly higher amounts of digoxigenin derivatives than undifferentiated or rhizogenic cultures. In any case, field plants prese…
Effects of traditional and light pruning on viticultural and oenological performance of bobal and tempranillo vineyards
<p style="text-align: justify;"><strong>Aim</strong>: Light pruning may improve vine yield and quality although vineyard responses are variable. We assessed the effects of traditional manual pruning and mechanical light pruning on the viticultural and oenological performance of <em>Vitis vinifera </em>cv. Bobal and cv. Tempranillo.</p><p style="text-align: justify;"><strong>Methods and results</strong>: During 2008-2011, Bobal and Tempranillo vineyards underwent traditional pruning or light pruning. The effects of both pruning techniques were determined in vine vigour and yield, and grape and wine characteristics. Both cultivars responde…
LC-NMR applied to the characterisation of cardiac glycosides from three micropropagatedIsoplexisspecies
Species of the genus Isoplexis are of particular interest with respect to the biochemical pathway leading to the cardenolides. It is important to determine whether or not 5β-configured compounds, typically produced by Digitalis species and used in medicine, are present together with their respective α-isomers in Isoplexis spp. Structure elucidation by LC-NMR of the products isolated from in vitro regenerated Isoplexis canariensis, I. chalcantha and I. isabelliana was carried out, and similarities were observed among the products of the three species, including the presence of digitoxigenin-type cardenolides in I. canariensis and xysmalogenin and canarigenin derivatives in I. chalcantha neve…
Cardenolides of Digitalis obscura: The effect of phosphate and manganese on growth and productivity of shoot-tip cultures
Abstract Cardenolide composition of leaves from wild and micropropagated elite plants of Digitalis obscura (genotype T4) has been investigated and no qualitative differences were found among their major cardenolides (series A). All of the detected glycosides belong to the digitoxose-type cardenolides. Genins represented less than 2% of the overall content, while lanatoside A was the predominant cardenolide ( ca. 65%) in all samples. The cardenolide yield of micropropagated D. obscura plants depended on the age and development of the cultures, but productivity of long-term cultures (2 years) was quite similar to that of the parent plant. Changes in the concentrations of phosphate or manganes…
Digitalis purpurea P5 beta R2, encoding steroid 5 beta-reductase, is a novel defense-related gene involved in cardenolide biosynthesis.
The stereospecific 5 beta-reduction of progesterone is a required step for cardiac glycoside biosynthesis in foxglove plants. Recently, we have isolated the gene P5 beta R, and here we investigate the function and regulation of P5 beta R2, a new progesterone 5 beta-reductase gene from Digitalis purpurea. P5 beta R2 cDNA was isolated from a D. purpurea cDNA library and further characterized at the biochemical, structural and physiological levels. Like P5 beta R, P5 beta R2 catalyzes the 5 beta-reduction of the Delta(4) double bond of several steroids and is present in all plant organs. Under stress conditions or on treatment with chemical elicitors, P5 beta R expression does not vary, wherea…
Seasonal cardenolide production and Dop5βr gene expression in natural populations of Digitalis obscura
Productivity variations and seasonal fluctuations of cardenolides have been studied in 10 natural populations of Digitalis obscura distributed in three bioclimatic belts. Main cardenolides in D. obscura plants are those of the series A and such predominance (ca. 80-85%) over the series B metabolites is independent of the population studied or the degree of maturity of the leaves. Primary glycosides represent ca. 50-60% of total cardenolides; this percentage did not vary among populations or with the leaf age but increased in summer and decreased in winter. A correlation analysis between plant biomass and cardenolide content showed a positive relationship of these parameters, which, accordin…
Digitalis obscura cardenolides. Effect of macronutrient concentration and N source on growth and productivity of shoot-tip cultures
Abstract Changes in nutrient medium formulation in terms of major salt concentration or N source significantly modified cardenolide production by Digitalis obscura (genotype T4) shoot-tip cultures. A positive correlation between morphogenetic responses, culture growth, and proliferative capacity on one side and metabolite biosynthesis on the other cannot be established. The final stages of microprogation, rooting, and acclimatization of D. obscura were improved by using paclobutrazol.
In vitro morphogenesis from excised leaf explants of Digitalis obscura L.
The morphogenic capacity of Digitalis obscura leaf explants cultured in vitro has been studied, noting factors promoting the differentiation of roots, buds and shoots as well as those promoting callus proliferation. Complete plant regeneration was obtained only by first culturing the leaf explants in a medium with NAA and BA to induce formation of buds, and subsequently transferring them to a medium without growth regulators to achieve the further development of shoots.