Glória Pinto

Factors influencing somatic embryogenesis induction in Eucalyptus globulus Labill.: basal medium and anti-browning agents

The low induction rates of somatic embryogenesis (SE) in Eucalyptusglobulus hamper scaling up the process for commercialization. We analyzed the effectiveness of several media (MS, 1/2MS, B5, WPM, DKW and JADS) during SE induction and expression. MS and B5 were the best media for SE induction and embling regeneration. In general, MS was the best medium for expression, independently of the medium previously used during induction. Several anti-browning compounds (ascorbic acid, charcoal, DTE, DTT, PVP, PVPP and silver nitrate) were added to the expression medium (MS), but all decreased SE potential and only DTE, charcoal and silver nitrate reduced explant browning. When added only during the induction period, anti-browning agents reduced accumulation of phenolics but also severely reduced SE potential. Continuous exposure completely inhibited the SE response. The negative impact of anti-browning agents on SE potential raises a question about the role of production/accumulation of phenolics in the SE process.

Micropropagation of Juniperus phoenicea from adult plant explants and analysis of ploidy stability using flow cytometry

We report here the successful micropropagation of adult Juniperus phoenicea L. with respective ploidy stability studies. Microcuttings with axillary buds were grown on five media supplemented with different growth regulator combinations. Best elongation rates were achieved on Driver and Kuniyuki (DKW) medium supplemented with kinetin alone or with naphthaleneacetic acid (NAA), while Rugini olive (OM) medium stimulated the development of new branches. Shoots growing on Murashige and Skoog (MS) medium browned and showed necrotic zones. Shoots of second to fourth subcultures usually had higher elongation rates than those of the first culture. For rooting assays, half strength DKW and OM media, different concentrations of growth regulators, auxin continuous exposure vs. dipping and the type of solid matrix were assessed. During rooting assays, two morphotypes were observed with one type having well developed internodes and the other showing hyperhydratation and no internode development. High rooting rates (40 %) were only obtained in the first morphotype shoots exposed for 5 min to 2.4 µM IBA and then transferred to OM medium without growth regulators. Plants were acclimatized in pots containing a mixture of peat and Perlite (3:2) in greenhouse with progressive reduction of relative humidity. A flow cytometric screening for major ploidy changes revealed no differences among the morphotypes and between them and the mother plant. Also the nuclear DNA content of this species was estimated for the first time using flow cytometry (2C = 24.71 pg).

Nutrient responses and glutamate and proline metabolism in sunflower plants and calli under Na2SO4 stress

The growth of Helianthus annuus L. calli and plants was reduced in the presence of Na2SO4 (10, 25, 50, and 100 mM). SO42— and Na concentrations increased in stressed calli and plants while NO3—, Cl—, P, K, and Mg decreased in stressed plants and Ca in shoots. Stressed calli showed decreases of NO3—, Ca, K, and Mg concentrations. Calli adapted to 50 mM Na2SO4 accumulated more K and Ca and less ammonium than stressed non-adapted calli. Proline exhibited increases in stressed calli and plants that were accompanied by decreases of proline oxidase activities while pyrroline-5-carboxylate reductase (P5CR) and ornithine aminotransferase (OAT) activities increased. Adapted calli accumulated more proline and had higher P5CR and OAT activities than stressed non-adapted calli. Glutamate concentration decreased with stress, together with a stimulation of cytosolic glutamine synthetase (GS1) and a decrease of plastidal GS (GS2) activity. These data strongly suggest that the increase of P5CR and GS1 activities are responsible for the decrease of glutamate concentration leading, together with the stimulation of OAT and the inhibition of the proline oxidation metabolism, to an increase of proline levels in Na2SO4-stressed sunflower cells. These data also show that salt stress increases the release of endogenous ammonium and suggests that the increase of GS1 activity plays an important role in its elimination. Nahrstoffgehalte und Glutamat- und Prolinstoffwechsel in Sonnenblumenpflanzen und Calli unter Na2SO4-Stress Das Wachstum von Helianthus annuus L. Pflanzen und deren Calli wird in Gegenwart steigender Na2SO4-Belastung (10, 25, 50 bzw. 100 mM) gehemmt. Sowohl in gestressten Pflanzen wie in den Calli waren die SO4- und Na-Konzentrationen erhoht. In den gestressten Pflanzen waren die Konzentrationen von NO3., Cl, P, K und Mg in Wurzel und Spross, die von Ca lediglich im Spross gesenkt, wahrend in gestressten Calli NO3, Ca, K und Mg erniedrigt waren. Calli, die uber 8 Monate an 50 mM Na2SO4 adaptiert waren, akkumulierten unter Salzstress im Vergleich zu nicht-adaptierten mehr K und Ca, aber weniger Ammonium. Am starksten stieg in den Pflanzen sowie Calli unter Salzstress die Prolin-Konzentration, bedingt durch einen Abfall der Prolinoxidase-Aktivitat sowie einen Anstieg der Pyrrolin-5-carboxylat-Reductase (P5CR) und der Ornithin-Aminotransferase (OAT). Die an Salinitat adaptierten Calli akkumulierten mehr Prolin und zeigten hohere Aktivitaten an P5CR und OAT. Bei Salzstress nahm die Konzentration an Glutamat ab. Dies war begleitet von einer Stimulierung der cytosolischen Glutaminsynthetase (GS1), jedoch einem Abfall der plastidaren GS2. Auf Grund der Ergebnisse kann der Anstieg der Prolingehalte nach Salzstress durch die Anstiege in der Aktivitat von P5CR und GS1, die die Absenkung der Glutamatkonzentration bewirken, sowie von OAT und durch die Hemmung der Prolinoxidase erklart werden. Der durch Salzstress ebenfalls geforderten Freisetzung endogenen Ammoniums wirkt die gesteigerte GS1-Aktivitat entgegen.

Somatic embryogenesis in leaf callus from a mature Quercus suber L. Tree

SummarySomatic embryos were obtained from a 60-yr-old Quercus suber L. tree. Leaf explants were cultivated on Murashige and Skoog medium with 30 gl−1 sucrose, 3 gl−1 gelrite, pH adjusted to 5.8, and different growth regulator combinations. Callus induction took place at 24±1°C in the dark during the first 3 wk. After 3 mo, calluses that showed embryogenic structures were transferred to the same medium without growth regulators. Somatic embryogenesis was only observed in calluses induced on E3 medium (supplemented with 4.5 μM 2,4-dichlorophenoxyacetic acid and 9.0 μM zeatin). On average, 7.5% of the initial explants formed embryogenic calluses in this medium. Somatic embryo proliferation was high due to secondary embryogenesis. On average, 10% of the somatic embryos germinated and 40% of these germinated embryos converted into plants. Plants were elongated on the same medium without growth regulators and acclimated to greenhouse conditions.

Is the interplay between epigenetic markers related to the acclimation of cork oak plants to high temperatures

Trees necessarily experience changes in temperature, requiring efficient short-term strategies that become crucial in environmental change adaptability. DNA methylation and histone posttranslational modifications have been shown to play a key role in both epigenetic control and plant functional status under stress by controlling the functional state of chromatin and gene expression. Cork oak (Quercus suber L.) is a key stone of the Mediterranean region, growing at temperatures of 45°C. This species was subjected to a cumulative temperature increase from 25°C to 55°C under laboratory conditions in order to test the hypothesis that epigenetic code is related to heat stress tolerance. Electrolyte leakage increased after 35°C, but all plants survived to 55°C. DNA methylation and acetylated histone H3 (AcH3) levels were monitored by HPCE (high performance capillary electrophoresis), MS-RAPD (methylation-sensitive random-amplified polymorphic DNA) and Protein Gel Blot analysis and the spatial distribution of the modifications was assessed using a confocal microscope. DNA methylation analysed by HPCE revealed an increase at 55°C, while MS-RAPD results pointed to dynamic methylation-demethylation patterns over stress. Protein Gel Blot showed the abundance index of AcH3 decreasing from 25°C to 45°C. The immunohistochemical detection of 5-mC (5-methyl-2′-deoxycytidine) and AcH3 came upon the previous results. These results indicate that epigenetic mechanisms such as DNA methylation and histone H3 acetylation have opposite and particular dynamics that can be crucial for the stepwise establishment of this species into such high stress (55°C), allowing its acclimation and survival. This is the first report that assesses epigenetic regulation in order to investigate heat tolerance in forest trees.

Photosynthetic parameters of Ulmus minor plantlets affected by irradiance during acclimatization

In order to set up large-scale acclimatization protocols of micropropagated plants, an in-depth knowledge of their physiological responses during in vitro to ex vitro transfer is required. This work describes the photosynthetic performance of Ulmus minor micropropagated plants during acclimatization at high irradiance (HI; 200 ± 20 μmol m−2 s−1 or low irradiance (LI; 100 ± 20 μmol m−2 s−1). During this experiment, leaf pigment content, chlorophyll a fluorescence, gas exchange, stomata morphology, the activity of the Calvin cycle enzymes and saccharides were measured in persistent and new leaves. The results indicated that HI induces a higher photosynthetic performance compared to LI. Therefore, plants acclimatized under HI are likely to survive better after field transfer.

Acclimatization of micropropagated plantlets induces an antioxidative burst: a case study with Ulmus minor Mill.

In this article, the effects of increased light intensities on antioxidant metabolism during ex vitro establishment of Ulmus minor micropropagated plants are investigated. Three month old in vitro plants were acclimatized to ex vitro conditions in a climate chamber with two different light intensities, 200 μmol m−2 s−1 (high light, HL) and 100 μmol m−2 s−1 (low light, LL) during 40 days. Immediately after ex vitro transfer, the increase of both malondialdehyde (MDA) and electrolyte leakage in persistent leaves is indicative of oxidative stress. As the acclimatization continues, an upregulation of the superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GR) enzyme activities were also observed. Simultaneously, MDA content and membrane permeability stabilized, suggesting that the antioxidant enzymes decrease the deleterious effects of reactive oxygen species (ROS) generation. Unexpectedly, newly formed leaves presented a different pattern of antioxidative profile, with high levels of MDA and membrane leakage and low antioxidant enzyme activity. Despite these differences, both leaf types looked healthy (e.g. greenish, with no necrotic spots) during the whole acclimatization period. The results indicate that micropropagated U. minor plantlets develop an antioxidant enzyme system after ex vitro transfer and that, in general, LL treatment leads to lower oxidative stress. Moreover, new leaves tolerate higher levels of ROS without the need to activate the antioxidative pathway, which suggests that the environment at which leaves are exposed during its formation determinate their ability to tolerate ROS.

Integrated physiological and hormonal profile of heat-induced thermotolerance in Pinus radiata.

Despite great interest, not only from the economic point of view but also in terms of basic science, research on heat stress tolerance in conifers remains scarce. To fill this gap, a time-course experiment using expected temperature increase was performed aiming to identify physiological and biochemical traits that allow the characterization of heat-induced thermotolerance and recovery in Pinus radiata D. Don plants. Several physiological parameters were assessed during heat exposure and after recovery, and multiple phytohormones-abscisic acid (ABA), indole-3-acetic acid (IAA), cytokinins (CKs), gibberellins, jasmonic acid, salicylic acid (SA) and brassinosteroids-were quantified by ultra-performance liquid chromatography-mass spectrometry from unique sample. Furthermore, tissue specific stress-signaling was monitored by IAA and ABA immunolocalization. Multivariate statistical analysis of the data enabled clustering of the shorter- and longer-term effects of heat stress exposure. Two sequential physiological responses were identified: an immediate and a delayed response, essentially determined by specific phytohormones, proline, malondialdehyde and total soluble sugar patterns. Results showed that ABA and SA play a crucial role in the first stage of response to heat stress, probably due to the plants urgent need to regulate stomatal closure and counteract the increase in oxidative membrane damage demonstrated in shorter-term exposures. However, in longer exposures and recovery, proline, total sugars, IAA and CKs seem to be more relevant. This integrated approach pinpointed some basic mechanisms of P. radiata physiological responses underlying thermotolerance processes and after recovery.

Genetic control of somatic embryogenesis induction in Eucalyptus globulus Labill.

A reproducible protocol for somatic embryogenesis (SE) induction in Eucalyptus globulus from mature zygotic embryos is available since 2002. However, for the use of SE in tree breeding programs, the frequency of SE initiation needs to be improved and controlled, and this was investigated in 13 open-pollinated (OP) families over three consecutive years. A diallel mating design with five parent trees was used to study genetic control of SE induction. Results showed that SE induction varies across E. globulus families and over the years of seed production tested. Somatic embryogenesis was initiated on explants from 84% of the OP families tested in 2002 and 100% of the families tested in 2003 and 2004. The year 2003 gave best results for percentage of induction and total number of somatic embryos produced. Results concerning genetic control showed that SE induction is under the control of additive genetic effects, as 22.0% of variation in SE initiation was due to general combining ability (GCA) effect, whereas 6.4% was due to maternal effects. Neither specific combining ability (SCA) nor reciprocal effects were significant.

Acclimatization of secondary somatic embryos derived plants of Eucalyptus globulus Labill.: an ultrastructural approach

This paper reports the complete process from secondary emblings (SE-derived plants) regeneration to acclimatization of Eucalyptus globulus and describes histocytological changes that occur in leaves from in vitro to ex vitro acclimatization for a 3-month period. After elongation, plants were transferred to pots with sterilized peat:perlite and acclimatized in a phytotron, with progressive reduction of RH and increase of light intensity. Histocytological analyses were performed in fixed material using light microscopy and ultrastructural changes followed by electron microscopy (SEM and TEM). The protocol used allowed the successful acclimatization of the emblings. Plants looked morphologically normal and FCM screening revealed no ploidy or DNA content abnormalities. Histocytological analyses showed significant changes along time, mostly in stomata shape and aperture, starch reserves, chloroplast morphology and mesophyll differentiation. This is the first report concerning emblings acclimatization to ex vitro conditions in Eucalyptus. It was clearly demonstrated that during acclimatization emblings suffered profound changes in leaf morphology in order to successfully adapt to ex vitro conditions.