Pedro Pérez-Bermúdez

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.

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 from 22.0 μM cytokinin, but these shoots showed less and slower development than those induced with low dosages of cytokinin. The presence of naphthaleneacetic acid in combination with cytokinin in the induction medium decreased the frequency of bud formation.

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.

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.

Pollen plant formation from anther cultures of Digitalis obscura L.

Factors favouring pollen callus proliferation, induction of embryogenesis and plant regeneration from cultured anthers of Digitalis obscura L. were determined. The presence of auxins was essential for cell proliferation and morphogenesis, and incubation in darkness singificantlyincreased these responses. Callus proliferation usually preceded embryo development, although sometimes direct embryogenesis was observed. On the other hand, bud differentiation was achieved only when callus was transferred to media containing cytokinin or several auxin/cytokinin combinations. Different ploidy levels] were observed in the regenerated plants, with approximately 50% being haploid.

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.

Morphogenesis in hypocotyl cultures of Digitalis obscura: Influence of carbohydrate levels and sources

Abstract Hypocotyl explants of Digitalis obscura L. were grown on Murashige and Skoog medium supplemented with 0.57 μM IAA and 4.40 μM BA. The effects of sucrose, maltose, glucose, galactose or mannitol on their growth and bud formation were investigated. None of the carbohydrate sources tested was superior to sucrose, and best results were obtained with 2.0% (w/v) of this disaccharide. Although mannitol did not support morphogenesis, it had a promotive effect on bud formation when added to 1.0 or 1.5% sucrose-supplied media to give the molar sucrose equivalent to 2.0%. The inhibitory effect of high sucrose concentrations could be duplicated by substituting mannitol for sucrose on a molar basis. Our data suggest a dual role of sucrose as osmotic and energy source in D. obscura hypocotyl cultures.

Seasonal Variation in Nutrient Status of Foxglove Leaves

ABSTRACT 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 seasonal variations, significant fluctuations of N/P and Ca/Mg ratios were observed in young leaves. Strong positive correlations existed among N, P and K, while negative correlations were found between Ca and N, P, or K.

Soil characteristics, mineral nutrients, biomass, and cardenolide production in Digitalis obscura wild populations

ABSTRACT 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 of P in plants and those of Cu in soils, suggesting that these elements may affect cardenolide biosynthesis.