Isabel Gavidia

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.

Digitalis purpurea P5βR2, encoding steroid 5β‐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, whereas P5 beta R2 is highly responsive. P5 beta R2 expression is regulated by ethylene and hydrogen peroxide. The correlation between P5 beta R2 expression and cardenolide formation demonstrates the key role of this gene in cardenolide biosynthesis, and therefore in the chemical defense of foxglove plants.

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 manganese ions in the nutrient media did not significantly affect the biosynthetic capacity of shoot-tip cultures, which was not correlated with increases or decreases observed in growth and development of the regenerated shoots or in their contents in photosynthetic pigments.

Micropropagation of juvenile and adultDigitalis obscura and cardenolide content of clonally propagated plants

SummaryCultures 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 performance liquid chromatography and radioimmunoassay, respectively. Several cardenolides of series A and C were identified in the regenerants; no significant differences were found in the cardenolide patterns. Digoxigenin derivatives were found in all clonally propagated plants, but the amount of these glycosides was much higher in those obtained from axillary buds. This is the first report on micropropagation ofD. obscura from mature plants.

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.

Variants of Digitalis obscura from irradiated shoot tips

Shoot tips excised from genotype T4 of Digitalis obscura were exposed to gamma rays (20–100 Gy). Radiosensitivity was assessed and LD50 determined was about 60 Gy. The effect of the herbicide amitrole on shoot-tip culture was investigated, efficient bleaching was obtained with 10 mg/l amitrole. Shoot tips mutagenized (20–40 Gy) were cultured on several selective amitrole-containing media, but none of them permitted prolonged survival of the green shoots formed. Ploidy level of the plantlets developed was analysed by flow cytometry 8 and 18 months after culture establishment. Variations detected corresponded to aneuploid changes and increased in parallel with the gamma radiation dose. Plantlets developed from irradiated shoot tips presented a high variability in their cardenolide production (878 to 3291 μg/g d.w.), including variants with similar or even higher productivities than the native T4 plant.

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.

Cover crops and pruning in Bobal and Tempranillo vineyards have little influence on grapevine nutrition

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 and total organic carbon (TOC) were determined in samples taken from the Ap1 horizon in January prior to vine pruning. Foliar N, P, K contents were measured in leaves sampled upon grape veraison. The differences between vineyards with cover cropping and bare soils suggest that legumes positively affected soil N (1.55 vs. 1.68 g kg−1 and 1.49 vs. 1.76 g kg−1 in Bobal and Tempranillo vineyards, respectively) and soil organic matter (SOM) (12.5 vs. 15.5 g kg−1 and 12.9 vs. 17.2 g kg−1 in Bobal and Tempranillo vineyards, respectively). The use of cover crops did not affect grapevine yields nor quality of Bobal and Tempranillo berry . Cover crops, or light pruning, did not alter the foliar N, P, K contents of both cultivars since their concentrations were similar to those found in the leaves from vineyards with soil tillage or severe pruning.