0000000000146853
AUTHOR
Juan Segura
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.
Plant hormones and Agrobacterium tumefaciens strain 82.139 induce efficient plant regeneration in the cardenolide-producing plant Digitalis minor
Summary Shoot formation in explants of Digitalis minor was achieved, through axillary bud proliferation and adventitious bud differentiation, by varying the amount and source of plant hormones. Shoot regeneration was also obtained after infection of D. minor with the wild-type Agrobacterium tumefaciens strain 82.139, which induced shooty tumours. These shoots were not transgenic, as revealed by nopaline assays and the use of a C58pMP90/T139GUS-INT strain harbouring the intron inactivated gusA gene. Plants were easily rooted and transplanted into the greenhouse. Shoot cultures of D. minor accumulated up to 226 μg cardenolides per g dry mass when cultured on agar-solidified medium. Cardenolid…
The phosphorylated pathway of serine biosynthesis is essential both for male gametophyte and embryo development and for root growth in Arabidopsis.
This study characterizes the phosphorylated pathway of Ser biosynthesis (PPSB) in Arabidopsis thaliana by targeting phosphoserine phosphatase (PSP1), the last enzyme of the pathway. Lack of PSP1 activity delayed embryo development, leading to aborted embryos that could be classified as early curled cotyledons. The embryo-lethal phenotype of psp1 mutants could be complemented with PSP1 cDNA under the control of Pro35S (Pro35S:PSP1). However, this construct, which was poorly expressed in the anther tapetum, did not complement mutant fertility. Microspore development in psp1.1/psp1.1 Pro35S:PSP1 arrested at the polarized stage. The tapetum from these lines displayed delayed and irregular devel…
RAPD variation within and among natural populations of outcrossing willow-leaved foxglove (Digitalis obscura L.)
Random amplified polymorphic DNA (RAPD) markers were used to assess levels and patterns of genetic diversity in Digitalis obscura L. (Scrophulariaceae), an outcrossing cardenolide-producing medicinal plant species. A total of 50 plants from six natural populations on the Iberian Peninsula were analysed by six arbitrarily chosen decamer primers resulting in 96 highly reproducible RAPD bands. To avoid bias in parameter estimation, analyses of population genetic structure were restricted to bands (35 of 96) whose observed frequencies were less than 1–3/n in each population. The analysis of molecular variance (AMOVA) with distances among individuals corrected for the dominant nature of RAPDs (g…
Cardenolide variation within and among natural populations of Digitalis obscura
Summary Cardenolide content in 49 wild-growing Digitalis obscura plants from six natural populations on the Iberian Peninsula was determined by HPLC. Series A and B glycosides were the predominant cardenolides in all samples, but absolute values varied among and within populations. Hierarchic analysis of variance showed that the proportion of variation attributable to individuals was significantly higher than that attributable to population differences. Furthermore, analyses from greenhouse-grown plants, generated from seeds collected from selected parental genotypes, attributed nearly all the variability in cardenolide content to single plants. Some individual plants showed a leaf content …
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…
Maritime Pine Pinus Pinaster Aiton
Maritime pine (Pinus pinaster Aiton) is the most abundant conifer in the Mediterranean basin. Currently, maritime pine is considered to be a model conifer species for study of the adaption responses to drought stress from a genomics approach. In this context, the availability of protocols that allow not only mass vegetative propagation of selected families or genotypes, but also facilitate the functional analyses needed to verify and further to study the effects of candidate genes are necessary. Here we describe an improved protocol to generate maritime pine plants through somatic embryogenesis from immature megagametophytes. Accurate procedures for explant preparation, somatic embryo induc…
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.
Axillary shoot proliferation in cultures of explants from mature Juniperus oxycedrus trees.
We developed procedures for the micropropagation of Juniperus oxycedrus L. using shoot apices or nodal segments from mature plants. Of the media and explants examined, best culture establishment was obtained with shoot apices cultured on modified Schenk and Hildebrandt medium (SH medium) without growth regulators; however, shoot multiplication was only achieved when shoot apices isolated from shoots grown on SH medium without growth regulators were subcultured on SH medium containing 0.5 micro M benzyladenine. None of the auxins and methods tested for root induction provided satisfactory results.
Somatic embryogenesis from hypocotyl callus cultures of Digitalis obscura L.
Hypocotyl-derived calli obtained in agar solidified medium with several growth regulator combinations gave rise to proembryonal masses and globular embryos when transferred to liquid media with lower growth regulator and higher NH4HO3 levels. By transferring cultures from liquid media to different solidified media, new embryo formation took place, but further development of these embryos or those previously induced depended on the characteristics of these media. Normal development was only achieved on 8 g/l agar solidified medium without growth regulators. Typical cotyledonary embryos developed into whole plants when transferred to this same medium.
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.
Biotechnological Approaches to Increase Essential Oil Yield and Quality in Aromatic Plants: The Lavandula latifolia (Spike Lavender) Example. Past and Recommendations for the Future
Increasing knowledge about isoprenoid biosynthesis pathways has provided new tools for aromatic plant breeding using biotechnological approaches. Notably, there are possibilities to modify essential oil profiles and enhance production of valuable monoterpenes. This attains a particular significance in Lavandula latifolia Medik. (spike lavender), one of the most important essential oil crops in Spain. This chapter summarizes work done to improve essential oil yield and quality by engineering: (1) the enzymes controlling regulatory steps of methyl-D-erythritol 4-phosphate (MEP) and mevalonic acid (MVA) pathways to increase C5 units employed for monoterpene biosynthesis and (2) the monoterpene…
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.
The specific role of plastidial glycolysis in photosynthetic and heterotrophic cells under scrutiny through the study of glyceraldehyde-3-phosphate dehydrogenase
The cellular compartmentalization of metabolic processes is an important feature in plants where the same pathways could be simultaneously active in different compartments. Plant glycolysis occurs in the cytosol and plastids of green and non-green cells in which the requirements of energy and precursors may be completely different. Because of this, the relevance of plastidial glycolysis could be very different depending on the cell type. In the associated study, we investigated the function of plastidial glycolysis in photosynthetic and heterotrophic cells by specifically driving the expression of plastidial glyceraldehyde-3-phosphate dehydrogenase (GAPCp) in a glyceraldehyde-3-phosphate de…
Isolation, culture and plant regeneration from mesophyll protoplasts of Digitalis obscura
High yields of protoplasts were obtained from mesophyll tissue of Digitalis obscura L. Osmotic potential of the isolation medium and Ca2+ were important in obtaining a high viability of the preparations. In different culture techniques employed, liquid-over-agar-solidified medium was superior to liquid medium alone. Agar plating technique was ineffective. On Murashige and Skoog modified medium with casein hydrolysate and several indoleacetic acid and benzyladenine combinations, isolated protoplasts underwent sustained mitotic division and produced calli. The calli formed shoots when transferred to regeneration media. Regenerated shoots could be easily rooted and developed into whole plants …
Digitalis spp.: In Vitro Production of Haploids
Digitalis, a member of the Scrophulariaceae, is composed of biennial or perennial herbs and occasionally of small shrubs. Several Digitalis species are used therapeutically, as they are a source of cardiac glycosides. The importance and distribution of these species are summarized below, with special attention to D. purpurea, D. lanata and D. obscura. For a detailed review see Thtin et al. (1972), Morton (1977), and Font Quer (1978).
Isolation, culture and plant regeneration from protoplasts of Sideritis angustifolia
Summary Factors promoting high yields of viable protoplasts from leaves of Sideritis angustifolia are reported. Isolated protoplasts divided and gave rise to callus when plated in modified liquid MS medium with growth regulators. Best plating efficiency was obtained when protoplast were cultured in the presence of 6 μM BA and 2 μM NAA. Calli were then transferred onto solidified regeneration media. Adventitious bud differentiation was only observed in calli grown on medium containing 8 μM BA and 2 μM NAA. These buds proliferated quickly when placed on hormone-free medium and subsequendy regenerated new buds and shoots. Once developed shoots were excised, the remaining tissues maintained thi…
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.
Genetic diversity and structure of natural and managed populations of Cedrus atlantica (Pinaceae) assessed using random amplified polymorphic DNA
Cedrus atlantica (Pinaceae) is a large and exceptionally long-lived conifer native to the Rif and Atlas Mountains of North Africa. To assess levels and patterns of genetic diversity of this species, samples were obtained throughout the natural range in Morocco and from a forest plantation in Arbúcies, Girona (Spain) and analyzed using RAPD markers. Within-population genetic diversity was high and comparable to that revealed by isozymes. Managed populations harbored levels of genetic variation similar to those found in their natural counterparts. Genotypic analyses of molecular variance (AMOVA) found that most variation was within populations, but significant differentiation was also found b…
Effect of methyl jasmonate in gene expression, and in hormonal and phenolic profiles of holm oak embryogenic lines before and after infection with Phytophthora Cinnamomi
The dieback syndrome affecting Quercus ilex and other oak species impels the search for tolerant plant genotypes, as well as methods of plant immunization against such infections. Elicitation treatments can be an effective strategy to activate plant defense response and embryogenic lines represent a promising tool to generate new tolerant genotypes and also to study early markers involved in defense response. The aim of the presented work was to investigate changes in gene expression, and in hormonal and phenolic profiles induced in three holm oak embryogenic lines (ELs) elicited with methyl jasmonate (MeJA) before and after infection with the oomycete Phytophthora cinnamomi, which is the m…
Population genetic study in the Balearic endemic plant speciesDigitalis minor(Scrophulariaceae) using RAPD markers
Digitalis minor (Scrophulariaceae) is a cardenolide-producing plant endemic to the eastern Balearic Islands (Mallorca, Menorca, and Cabrera) that occurs in two morphologically distinct varieties: D. minor var. minor (pubescent) and D. minor var. palaui (glabrous). Levels and patterns of genetic diversity in 162 individuals from 17 D. minor populations across the entire geographic range were assessed using random amplified polymorphic DNA (RAPD) markers. Comigrating RAPD fragments tested were found to be homologous by Southern hybridization in both var. minor and var. palaui. To avoid bias in parameter estimation, analyses of population genetic structure were restricted to those RAPD bands t…
An assessment of genetic relationships within the genus Digitalis based on PCR-generated RAPD markers
RAPD markers were used to study inter-specific variation among six species of the genus Digitalis: D. obscura, D. lanata, D. grandiflora, D. purpurea, D. thapsi and D. dubia, and the hybrid D. excelsior (D. purpurea×D. grandiflora). A total of 91 highly reproducible bands amplified with four arbitrarily chosen decamer primers were obtained. Homology of the co-emigrating RAPD markers was tested by blot hybridisation and sequencing of selected bands. The application of a range of statistical approaches for RAPD data analysis, including distance and parsimony methods, family clustering and the analysis of molecular variance (AMOVA), indicated that these molecular markers were taxonomically inf…
Agrobacterium tumefaciens-mediated genetic transformation of the cardenolide-producing plant Digitalis minor L.
A repeatable transformation system has been established for Digitalis minor using Agrobacterium tumefaciens. Leaf explants from 30-day-old seedlings were inoculated with either EHA105 (carrying the nptII and gusA genes) or AGL1 (with the bar and gusA genes) strains. Among the tested factors influencing T-DNA transfer to plants, the EHA105 strain and the addition of acetosyringone to the co-culture medium increased transformation. The highest transformation efficiency (8.4%) was obtained when freshly isolated explants, soaked in a bacterial suspension with an OD 550 of 0.9, were subcultured on selection medium after a 4-day co-culture with the bacteria. Evidence of stable transgene integrati…
Essential oil variation within and among natural populations of Lavandula latifolia and its relation to their ecological areas
Essential oil yield and composition in seven natural populations of Lavandula latifolia from the eastern Iberian Peninsula were determined by GC/MS. Twenty-eight constituents were identified, accounting for 92.0e95.4% of the total oils. These oils were dominated by the monoterpene fraction and three of them (linalool, cineole and camphor) constituted 79.5e86.9% of the oil from flowers. Essential oil yield in leaves and flowers varied among and within populations, but hierarchic analyses of variance showed that the proportion of variation attributable to individuals was significantly higher than that attributable to population differences. Principal component and cluster analyses allowed thr…
Regeneration of Sideritis angustifolia (Labiatae) Plants from Single Cell Cultures
Summary Mechanically isolated cells from hypocotyl-derived calli of Sideritis angustifolia Lag. were inoculated on agar-solidified Murashige and Skoog (MS) nutrient medium containing CH and different concentrations and combinations of NAA and BA. Isolated cells underwent continued divisions giving rise to calli after 30 days of culture. Best results were obtained when medium was supplemented with 0.5 μM NAA and 4.4 or 8.8 μM BA or with 5 μM NAA and 4.4μM BA. Calli obtained in the presence of 0.5 μM NAA and 8.8 μM BA regenerated shoot-buds when transferred to MS medium without CH and supplemented or not with these growth regulators. Once shoots were excised, the calli maintained their morpho…
Serine biosynthesis by photorespiratory and nonphotorespiratory pathways: and interesting interplay with unknown regulatory networks
[EN] Photorespiration is a primary metabolic pathway, which, given its energy costs, has often been viewed as a wasteful process. Despite having reached the consensus that one important function of photorespiration is the removal of toxic metabolite intermediates, other possible functions have emerged, and others could well emerge in the future. As a primary metabolic pathway, photorespiration interacts with other routes; however the nature of these interactions is not well known. One of these interacting pathways could be the biosynthesis of serine, since this amino acid is synthesised through photorespiratory and non-photorespiratory routes. At present, the exact contribution of each rout…
Factors controlling adventitious bud induction and plant regeneration in matureJuniperus oxycedrus leaves cultured in vitro
The morphogenetic capacity of matureJuniperus oxycedrus L. leaves cultured in vitro has been studied, noting nutritive, hormonal, and environmental factors inducing differentiation and development of adventitious shoots. Bud primordia formed directly from the leaves. Highest bud differentiation rates were obtained when the explants were cultured for at least 21 days on a modified Schenk and Hildebrandt solidified medium containing 0.5 μM benzyladenine under a 16-h photoperiod. Maximum bud development and elongation was achieved on cytokinin-free medium containing 4% (wt/vol) sucrose and 0.05% (wt/vol) activated charcoal. Regenerated shoots were excised and induced to root on media with auxi…
Random amplified polymorphic DNA assessment of diversity in western Mediterranean populations of the seagrass Posidonia oceanica.
Posidonia oceanica is an endemic Mediterranean seagrass species that has often been assumed to contain low levels of genetic diversity. Random amplified polymorfic DNA (RAPD) markers were used to assess genetic diversity among five populations from three geographical regions (north, central, and south) of the western Mediterranean Sea. Stranded germinating seeds from one of the central populations were also included in the analysis. Forty-one putative genets were identified among 76 ramets based on 28 RAPD markers. Genotypic diversity strongly depended on the spatial structure, age, and maturity of the meadows. The lowest clonal diversity was found in the less structured and youngest prairi…
Sowing date, transplanting, plant density and nitrogen fertilization affect indigo production from Isatis species in a Mediterranean region of Spain
Abstract The increasing interest in natural products from a renewable source has encouraged growers to reintroduce indigo-producing crops into the European agriculture. We studied agronomic conditions (sowing date, plant density, nitrogen fertilization, irrigation rate, seedling transplanting) influencing production of the blue pigment indigo, from Isatis tinctoria and I. indigotica crops in a Mediterranean region of Spain (Valencia). I. tinctoria was more suitable for cultivation in our climate conditions than I. indigotica . Indigo yield from Spanish I. tinctoria trials was greater than in Northern and Central Europe. Furthermore, indigo production was maintained when water and nitrogen s…
Phosphoglycerate Kinases Are Co-Regulated to Adjust Metabolism and to Optimize Growth
[EN] In plants, phosphoglycerate kinase (PGK) converts 1,3-bisphosphoglycerate into 3-phosphoglycerate in glycolysis but also participates in the reverse reaction in gluconeogenesis and the Calvin-Benson cycle. In the databases, we found three genes that encode putative PGKs. Arabidopsis (Arabidopsis thaliana) PGK1 was localized exclusively in the chloroplasts of photosynthetic tissues, while PGK2 was expressed in the chloroplast/plastid of photosynthetic and nonphotosynthetic cells. PGK3 was expressed ubiquitously in the cytosol of all studied cell types. Measurements of carbohydrate content and photosynthetic activities in PGK mutants and silenced lines corroborated that PGK1 was the phot…
In Vitro Propagation of Sideritis angustifolia Lag.
Summary Hypocotyl, root and cotyledon explants from 30 day old seedlings of Sideritis angustifolia Lag. were cultured on Murashige and Skoog nutrient medium. Organogenesis of roots and shoots was induced with treatments containing BA or KIN in combination with NAA, IAA or 2,4-D, however, those combinations including NAA and BA were optimal. Most regenerated shoots developed roots when transferred to medium without growth regulators. Rooted plantlets were successfully transplanted into soil.
Arabidopsis plants deficient in plastidial glyceraldehyde-3-phosphate dehydrogenase show alterations in abscisic acid (ABA) signal transduction: interaction between ABA and primary metabolism
Abscisic acid (ABA) controls plant development and regulates plant responses to environmental stresses. A role for ABA in sugar regulation of plant development has also been well documented although the molecular mechanisms connecting the hormone with sugar signal transduction pathways are not well understood. In this work it is shown that Arabidopsis thaliana mutants deficient in plastidial glycolytic glyceraldehyde-3-phosphate dehydrogenase (gapcp1gapcp2) are ABA insensitive in growth, stomatal closure, and germination assays. The ABA levels of gapcp1gapcp2 were normal, suggesting that the ABA signal transduction pathway is impaired in the mutants. ABA modified gapcp1gapcp2 gene expressio…
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.
Deficiency in the Phosphorylated Pathway of Serine Biosynthesis Perturbs Sulfur Assimilation.
Although the plant Phosphorylated Pathway of l-Ser Biosynthesis (PPSB) is essential for embryo and pollen development, and for root growth, its metabolic implications have not been fully investigated. A transcriptomics analysis of Arabidopsis (Arabidopsis thaliana) PPSB-deficient mutants at night, when PPSB activity is thought to be more important, suggested interaction with the sulfate assimilation process. Because sulfate assimilation occurs mainly in the light, we also investigated it in PPSB-deficient lines in the day. Key genes in the sulfate starvation response, such as the adenosine 5′phosphosulfate reductase genes, along with sulfate transporters, especially those involved in sulfat…
Cryopreservation of Digitalis obscura selected genotypes by encapsulation-dehydration
Shoot-tips from several genotypes of the cardenolide-producing perennial shrub Digitalis obscura L. were successfully cryopreserved using the encapsulation-dehydration technique. Precultivation on MS medium containing 0.5 M sucrose, followed by 2.5 h dehydration (final weight 30 %) induced shoot regrowth in 42 % of cryopreserved shoot-tips. Cold-hardening of the in vitro cultures before sucrose treatment dramatically increased shoot recovery up to 86 %. The optimized cryopreservation protocol was then employed using different shoot cultures from five D. obscura genotypes. Responses to cryopreservation depended mainly on the genotype, best results being obtained when shoot tips from HU3 and …
New Approaches to Optimize Somatic Embryogenesis in Maritime Pine
Maritime pine (Pinus pinaster Aiton) is a coniferous native of the Mediterranean basin. Because of its adaptability to a wide range of environmental conditions, the species have become a model for studies in coniferous forest management and functional genomics. Somatic embryogenesis (SE) has been so far, the preferred biotechnological strategy for maritime pine breeding programs initiated at the middle-end of the 20th century. To overcome the limitations of the induction and maturation phases in maritime pine SE, we analyzed the possible maternal influence on the embryogenic capability of megagametophytes from controlled crosses, as well as the effect of the temperature and water availabili…
Morphogenesis in callus and single-cell cultures ofLavandula latifoliaMedicus
Procedures and factors promoting plant regeneration from callus and isolated cells were investigated in Lavandula latifolia Medicus. Callus was obtained from cotyledon explants cultured on MS medium supplemented with 0.34 (xM 2,4-D. These calli regenerated adventitious buds when transferred to MS medium containing either BA or several auxin (IAA or NAA)/BA combinations, best results being obtained in the presence of 8.80 (mi BA alone or in combination with 0.06 and 0.60 (xM IAA or NAA. Single cells from cotyledon-derived calli were able to proliferate as callus when plated in MS medium supplemented with IAA or several IAA/BA combinations. Best plating efficiency was achieved when cells were…
Plant regeneration from isolated cells ofLavandula latifolia medicus
Single cells were obtained from hypocotyl-derived callus ofLavandula latifolia Medicus. Cells were plated in Murashige and Skoog medium supplemented with indoleacetic acid (IAA), benzyladenine (BA), and several IAA-BA combinations. Cell division required the simultaneous presence of IAA and BA in the culture medium, but callus formation was only achieved with 0.1 or 1 mg/liter IAA and 2 mg/liter BA. To induce organogenesis, calli were transferred to various regeneration media. Shoot-bud differentiation efficiency depended on the composition of both the callus induction and the shoot regeneration media, best results being obtained when calli grown in 1 mg/liter IAA and 2 mg/liter BA were sub…
In vitro control of adventitious bud differentiation by inorganic media components in leaves of matureJuniperus oxycedrus
The effects of changes in the concentration of macronutrients on BA-induced caulogenesis from leaves of matureJuniperus oxycedrus cultured on modified Murashige and Skoog or Schenk and Hildebrandt media are reported. The bud-forming capacity of the explants depended mainly on the ratios among the levels of ammonium, nitrate, and potassium. The most favorable media formulations for differentiation of adventitious buds were those with nitrate:potassium, ammonium:potassium, and nitrate:ammonium ratios near 1, around 0.1, and between 9 and 15, respectively. The total ionic strength of the media limited bud induction, but only when a disequilibrium of these ratios was produced.
Somatic Embryogenesis and Plant Regeneration from Hypocotyl Cultures of Digitalis obscura L.
Summary Hypocotyl sections from 20-day-old seedlings of Digitalis obscura L. were used to examine the effect of IAA on somatic embryogenesis. Although embryogenesis was obtained with all IAA concentrations (0.1, 0.5 and 1 ppm) and under both illumination conditions tested (16 h photoperiod and darkness), incubation in photoperiod increased yield of embryos and shortened the embryo induction time. Optimum results were obtained with 1 ppm IAA. About 30 % of the embryos obtained developed into complete plantlets when transferred to basal medium without growth regulators.
Priming maritime pine megagametophytes during somatic embryogenesis improved plant adaptation to heat stress
In the context of global climate change, forest tree research should be addressed to provide genotypes with increased resilience to high temperature events. These improved plants can be obtained by heat priming during somatic embryogenesis (SE), which would produce an epigenetic-mediated transgenerational memory. Thereby, we applied 37 °C or 50 °C to maritime pine (Pinus pinaster) megagametophytes and the obtained embryogenic masses went through the subsequent SE phases to produce plants that were further subjected to heat stress conditions. A putative transcription factor WRKY11 was upregulated in priming-derived embryonal masses, and also in the regenerated P37 and P50 plants, suggesting …
Dynamics of Monoterpene Formation in Spike Lavender Plants
The metabolic cross-talk between the mevalonate (MVA) and the methylerythritol phosphate (MEP) pathways was analyzed in spike lavender (Lavandula latifolia Med) on the basis of 13CO2-labelling experiments using wildtype and transgenic plants overexpressing the 3-hydroxy-3-methylglutaryl CoA reductase (HMGR), the first and key enzyme of the MVA pathway. The plants were labelled in the presence of 13CO2 in a gas chamber for controlled pulse and chase periods of time. GC/MS and NMR analysis of 1,8-cineole and camphor, the major monoterpenes present in their essential oil, indicated that the C5-precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) of both monoterpenes …
Plastidial Glyceraldehyde-3-Phosphate Dehydrogenase Deficiency Leads to Altered Root Development and Affects the Sugar and Amino Acid Balance in Arabidopsis
[EN] Glycolysis is a central metabolic pathway that, in plants, occurs in both the cytosol and the plastids. The glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate with concomitant reduction of NAD(+) to NADH. Both cytosolic (GAPCs) and plastidial (GAPCps) GAPDH activities have been described. However, the in vivo functions of the plastidial isoforms remain unresolved. In this work, we have identified two Arabidopsis (Arabidopsis thaliana) chloroplast/plastid-localized GAPDH isoforms (GAPCp1 and GAPCp2). gapcp double mutants display a drastic phenotype of arrested root development, dwarfism, and steri…
Spermidine and morphogenesis in single cell cultures of Sideritis angustifolia lag.
Abstract Single cells from hypocotyl-derived callus of Sideritis angustifolia were evaluated for morphogenesis when cultured in either solidifier or liquid Murashige and Skoog medium supplemented with various concentrations of spermidine, naphthaeneacetic acid (NAA), benzyladenine (BA) and dicyclohexylamine (DCHA). Spermidine (Spd) did not replace the hormonal requirements for cell division and callus formation, but affected growth induced by NAA or BA. This polyamine increased the plating efficiency of cells cultured in the presence of NAA (0.05, 0.54 and 5.40 μM) or BA (0.40, 4.40 and 8.80 μM). In contrast, Spd reduced growth induced by 10.80 μM NAA, optimal auxin concentration for callus…
Adventitious shoot regeneration from hypocotyl cultures of service tree(Sorbus domesticaL.)
Effects of growth regulators and N03:NH4 ratio were investigated to determine the mor- phogenic capacity of hypocotyl explants of service tree (Sorbus domestica L.) Organogenic responses were preceded by an initial callus phase and depended mainly on the auxin type used. NAA induced root differentiation whereas IAA added to media containing BA promoted adventitious shoot regeneration. Best results were obtained with 2.5 μM each of BA and IAA (19% of caulogenic explants). This reponse was increased (up to 44%) by varying nitrate to ammonium ratio from 2:1 to 4:1.
Enhanced levels of S-linalool by metabolic engineering of the terpenoid pathway in spike lavender leaves
Transgenic Lavandula latifolia plants overexpressing the linalool synthase (LIS) gene from Clarkia breweri, encoding the LIS enzyme that catalyzes the synthesis of linalool were generated. Most of these plants increased significantly their linalool content as compared to controls, especially in the youngest leaves, where a linalool increase up to a 1000% was observed. The phenotype of increased linalool content observed in young leaves was maintained in those T1 progenies that inherit the LIS transgene, although this phenotype was less evident in the flower essential oil. Cross-pollination of transgenic spike lavender plants allowed the generation of double transgenic plants containing the …
Morphogenic potential of mechanically isolated single cells from Digitalis obscura L. callus
Calli from hypocotyl and root explants of Digitalis obscura L. showed regeneration of adventitious shoots, roots and embryos when transferred to Murashige & Skoog medium supplemented with cytokinins alone or in combination with auxins. Optimum shoot-bud formation was achieved in the presence of IAA and BA, while roots mainly appeared either in absence of growth regulators or with IAA and Kn. Embryo formation took place only in those combinations that included Kn. Embryo development was influenced by the type of auxin, and precocious germination occurred in media with NAA. Mechanically isolated cells from hypocotyl- and root-derived calli were plated in MS medium supplemented with several IA…
Up-regulation of an N-terminal truncated 3-hydroxy-3-methylglutaryl CoA reductase enhances production of essential oils and sterols in transgenic Lavandula latifolia
Spike lavender (Lavandula latifolia) essential oil is widely used in the perfume, cosmetic, flavouring and pharmaceutical industries. Thus, modifications of yield and composition of this essential oil by genetic engineering should have important scientific and commercial applications. We generated transgenic spike lavender plants expressing the Arabidopsis thaliana HMG1 cDNA, encoding the catalytic domain of 3-hydroxy-3-methylglutaryl CoA reductase (HMGR1S), a key enzyme of the mevalonic acid (MVA) pathway. Transgenic T0 plants accumulated significantly more essential oil constituents as compared to controls (up to 2.1- and 1.8-fold in leaves and flowers, respectively). Enhanced expression …
Up-regulation of 1-deoxy-D-xylulose-5-phosphate synthase enhances production of essential oils in transgenic spike lavender.
Abstract Spike lavender (Lavandula latifolia) is an aromatic shrub cultivated worldwide for the production of essential oils. The major constituents of these oils are monoterpenes, which are obtained from isopentenyl diphosphate and dimethylallyl diphosphate precursors through the plastidial methylerythritol phosphate (MEP) pathway and/or the cytosolic mevalonate pathway. 1-Deoxy-d-xylulose-5-P synthase (DXS) catalyzes the first step of the MEP pathway. A cDNA coding for the Arabidopsis (Arabidopsis thaliana) DXS was constitutively expressed in spike lavender. Gas chromatography/mass spectrometry analyses revealed that transgenic plants accumulated significantly more essential oils compared…
Plastidial Glycolytic Glyceraldehyde-3-Phosphate Dehydrogenase Is an Important Determinant in the Carbon and Nitrogen Metabolism of Heterotrophic Cells in Arabidopsis
This study functionally characterizes the Arabidopsis (Arabidopsis thaliana) plastidial glycolytic isoforms of glyceraldehyde-3-phosphate dehydrogenase (GAPCp) in photosynthetic and heterotrophic cells. We expressed the enzyme in gapcp double mutants (gapcp1gapcp2) under the control of photosynthetic (Rubisco small subunit RBCS2B [RBCS]) or heterotrophic (phosphate transporter PHT1.2 [PHT]) cell-specific promoters. Expression of GAPCp1 under the control of RBCS in gapcp1gapcp2 had no significant effect on the metabolite profile or growth in the aerial part (AP). GAPCp1 expression under the control of the PHT promoter clearly affected Arabidopsis development by increasing the number of later…
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…
Micropropagation of juvenile and adult Sorbus domestica L.
Successful propagation of seedlings and mature trees of Sorbus domestica L. has been achieved by in vitro methods. Multiple shoot formation was obtained by placing shoot apices or nodal segments on a modified Schenck and Hildebrandt medium containing benzyladenine. Regenerated shoots were excised and induced to root on media with auxin. In the best treatments 75–85% of shoots from juvenile material rooted. Rooting capacity of shoots from mature explants was lower (30%) and was not improved by dipping the base of shoots in concentration solutions of indolebutyric or naphthaleneacetic acids. Plantlets were ultimately established in soil.
The essential role of the phosphorylated pathway of serine biosynthesis inArabidopsis
[EN] In plants, 3 different pathways of serine biosynthesis have been described: the Glycolate pathway, which is associated with photorespiration, and 2 non-photorespiratory pathways, the Glycerate and the Phosphorylated pathways. The Phosphorylated Pathway of Serine Biosynthesis (PPSB) has been known since the 1950s, but has been studied relatively little, probably because it was considered of minor significance as compared with the Glycolate pathway. In the associated study1 , we described for the first time in plants the in vivo functional characterization of the PPSB, by targeting the phosphoserine phosphatase (PSP1), the last enzyme of the pathway. Following a gain—and loss-of-function…
Deoxyxylulose 5-phosphate reductoisomerase is not a rate-determining enzyme for essential oil production in spike lavender
[EN] Spike lavender (Lavandula latifolia) is an economically important aromatic plant producing essential oils, whose components (mostly monoterpenes) are mainly synthesized through the plastidial methylerythritol 4-phosphate (MEP) pathway. 1-Deoxy-d-xylulose-5-phosphate (DXP) synthase (DXS), that catalyzes the first step of the MEP pathway, plays a crucial role in monoterpene precursors biosynthesis in spike lavender. To date, however, it is not known whether the DXP reductoisomerase (DXR), that catalyzes the conversion of DXP into MEP, is also a rate-limiting enzyme for the biosynthesis of monoterpenes in spike lavender. To investigate it, we generated transgenic spike lavender plants con…
Lavandula spp. (Lavender): In Vitro Culture, Regeneration of Plants, and the Formation of Essential Oils and Pigments
Lavandula species are of great interest due to their content of essential oils, which are important to the perfume, cosmetic, flavoring and pharmaceutical industries. They also have numerous other applications, including their uses as ornamental and melliferous plants.
In vivo and in vitro effects of nickel and cadmium on the plasmalemma ATPase from rice (Oryza sativa L.) shoots and roots
Abstract Plasmalemma vesicles isolated from rice (Oryza sativa L.) shoots and roots were used to study the effect of different nickel and cadmium concentrations on the Mg2+-ATPase activity. In vivo application of the metals for 5 or 10 days resulted in a stimulation of the enzyme activity in shoots, while either increases or decreases were observed in roots depending on the concentration and the type of ion tested. In contrast, an inhibitory effect was always observed on shoot and root plasmalemma ATPase activity when these ions were applied in vitro. At the organ level, the content of Cd in plants treated with 0.5 mM metal was higher than that of Ni, especially in roots. No correlation bet…
Metabolic cross-talk between pathways of terpenoid backbone biosynthesis in spike lavender
Abstract The metabolic cross-talk between the mevalonate (MVA) and the methylerythritol phosphate (MEP) pathways in developing spike lavender ( Lavandula latifolia Med) was analyzed using specific inhibitors and on the basis of 13 C-labeling experiments. The presence of mevinolin (MEV), an inhibitor of the MVA pathway, at concentrations higher than 0.5 μM significantly reduced plant development, but not the synthesis of chlorophylls and carotenoids. On the other hand, fosmidomycin (FSM), an inhibitor of the MEP pathway, at concentrations higher than 20 μM blocked the synthesis of chlorophyll, carotenoids and essential oils, and significantly reduced stem development. Notably, 1.2 mM MVA cou…
Expression of spearmint limonene synthase in transgenic spike lavender results in an altered monoterpene composition in developing leaves.
We generated transgenic spike lavender (Lavandula latifolia) plants constitutively expressing the limonene synthase (LS) gene from spearmint (Mentha spicata), encoding the LS enzyme that catalyzes the synthesis of limonene from geranyl diphosphate. Overexpression of the LS transgene did not consistently affect monoterpene profile in pooled leaves or flowers from transgenic T(0) plants. Analyses from cohorts of leaves sampled at different developmental stages showed that essential oil accumulation in transgenic and control plants was higher in developing than in mature leaves. Furthermore, developing leaves of transgenic plants contained increased limonene contents (more than 450% increase c…
Factors influencing axillary shoot proliferation and adventitious budding in cedar.
We developed procedures for in vitro cloning of Cedrus atlantica Manetti and C. libani A. Rich explants from juvenile and mature plants. Explant size was one determinant of the frequency of axillary bud break in both species. Shoot tips and nodal explants mainly developed calli, whereas bud sprouting occurred in defoliated microcuttings cultured on a modified Murashige and Skoog medium without growth regulators. Isolation and continuous subculture of sprouted buds on the same medium allowed cloning of microcuttings from C. atlantica and C. libani seedlings and bicentennial C. libani trees, thus providing a desirable alternative for multiplying mature trees that have demonstrated superior ch…
Plant regeneration from cultured leaves of Lavandula latifolia Medicus: Influence of growth regulators and illumination conditions
Leaves were obtained from 4-week-old seedlings of Lavandula latifolia Medicus grown in vitro. Leaf explants were then cultured on MS medium supplemented with different concentrations and combinations of the auxins IAA or NAA with the cytokinin BA and maintained under three illumination conditions, 16h photoperiod, darkness or darkness followed by a photoperiod, to assess morphogenic responses. Irrespective of illumination conditions, bud regeneration was achieved only in media containing BA or BA/auxin combinations, with the best results being obtained in the presence of BA and 0.06 or 0.6 μM IAA or NAA. A photoperiod of 16h appeared to yield the best response in terms of bud regeneration p…
Morphogenesis in leaf and single-cell cultures of mature Juniperus oxycedrus
Single cells were mechanically isolated from leaf-derived callus of mature Juniperus oxycedrus L. These cells divided and gave rise to callus when plated on medium containing growth regulators. Best plating efficiency was obtained on a modified Schenk and Hildebrandt medium supplemented with 0.6 micro M 2,4-dichlorophenoxyacetic acid and 100 mg l(-1) casein hydrolyzate. Although single-cell-derived callus showed poor morphogenic potential, both adventitious shoots and embryogenic tissues differentiated from the callus. We also achieved induction of somatic embryogenesis in leaf explants of mature J. oxycedrus trees cultured in the presence of 6.0 or 10.0 micro M 2,4-dichlorophenoxyacetic ac…
Trends and patterns in the evolution of vascular plants: macroevolutionary implications of a multilevel taxonomic analysis
Cascales-Minana, B., Munoz-Bertomeu, J., Ros, R., Segura, J. 2010: Trends and patterns in the evolution of vascular plants: macroevolutionary implications of a multilevel taxonomic analysis. Lethaia, 10.1111/j.1502-3931.2009.00212.x Studying the macroevolutionary patterns of vascular plants from the Silurian to the present-day provides a global record of plant life history. Evolutionary rates (origination, extinction and diversification) for families, orders, classes and divisions were analysed, as was abundance and richness for 21 time intervals. An accumulative analysis, based on the total plant fossil record, the accumulated extinctions and relative diversity, was also carried out. The d…
Micropropagation of adultLavandula dentataplants
SummaryA protocol for in vitro propagation of adult Lavandula dentata plants has been achieved. Cultures were established by placing nodal segments on Murashige and Skoog medium containing BA, KIN, and NAA. Highest shoot multiplication rates were obtained when explants grown in the presence of 5.0 μM BA or 20 (JLM KIN were transferred to medium with 8.8 μM BA and 15% coconut milk. Multiplication efficiency through subcultures was significantly affected by the cytokinin concentration in the initial culture medium. Subculture reduced drastically the final number of shoots produced on nodal segments isolated from shoots grown in the presence of 2.0 μM BA or 40.0 μM KIN. Shoots were easily root…
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.
The Plastidial Glyceraldehyde-3-Phosphate Dehydrogenase Is Critical for Viable Pollen Development in Arabidopsis
Plant metabolism is highly coordinated with development. However, an understanding of the whole picture of metabolism and its interactions with plant development is scarce. In this work, we show that the deficiency in the plastidial glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPCp) leads to male sterility in Arabidopsis (Arabidopsis thaliana). Pollen from homozygous gapcp double mutant plants (gapcp1gapcp2) displayed shrunken and collapsed forms and were unable to germinate when cultured in vitro. The pollen alterations observed in gapcp1gapcp2 were attributed to a disorganized tapetum layer. Accordingly, the expression of several of the genes involved in tapetum development was d…
A critical role of plastidial glycolytic Glyceraldehyde-3-Phosphate Dehydrogenase in the control of plant metabolism and development
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