Background: In Quercus suber, cork oak, a Mediterranean forest tree of economic and social interest, rapid production of isogenic lines and clonal propagation of elite genotypes have been achieved by developing in vitro embryogenesis from microspores and zygotic embryos respectively. Despite its high potential in tree breeding strategies, due to their recalcitrancy, the efficiency of embryogenesis in vitro systems in many woody species is still very low since factors responsible for embryogenesis initiation and embryo development are still largely unknown. The search for molecular and cellular markers during early stages of in vitro embryogenesis constitutes an important goal to distinguish, after induction, responsive from non-responsive cells, and to elucidate the mechanisms involved in embryogenesis initiation for their efficient manipulation. In this work, we have performed a comparative analysis of two embryogenesis pathways derived from microspores and immature zygotic embryos in cork oak in order to characterize early markers of reprogrammed cells in both pathways. Rearrangements of the cell structural organization, changes in epigenetic marks, cell wall polymers modifications and endogenous auxin changes were analyzed at early embryogenesis stages of the two in vitro systems by a multidisciplinary approach.

Somatic embryogenesis is a reliable system for in vitro plant regeneration, with biotechnological applications in trees, but the regulating mechanisms are largely unknown. Changes in cell wall mechanics controlled by methylesterification of pectins, mediated by pectin methylesterases (PMEs) and pectin methyl esterase inhibitors (PMEIs) underlie many developmental processes. Arabinogalactan proteins (AGPs) are highly glycosylated proteins located at the surface of plasma membranes, in cell walls, and in extracellular secretions, with key roles in a range of different processes. In this study, we have investigated changes in two cell wall components, pectins and AGPs, during somatic embryogenesis in Quercus suber, a forest tree of high economic and ecologic value. At early embryogenesis stages, cells of proembryogenic masses showed high levels of esterified pectins and expression of QsPME and QsPMEI genes encoding a PME and a putative PMEI, respectively. At advanced stages, differentiating cells of heart, torpedo and cotyledonary embryos exhibited walls rich in de-esterified pectins, while QsPME gene expression and PME activity progressively increased. AGPs were detected in cell walls of proembryogenic masses and somatic embryos. QsLys-rich-AGP18, QsLys-rich-AGP17, and QsAGP16L1 gene expression increased with embryogenesis progression, as did the level of total AGPs, detected by dot blot with β-glucosyl Yariv reagent. Immuno dot blot, immunofluorescence assays and confocal analysis using monoclonal antibodies to high- (JIM7, LM20) and low- (JIM5, LM19) methylesterified pectins, and to certain AGP epitopes (LM6, LM2) showed changes in the amount and distribution pattern of esterified/de-esterified pectins and AGP epitopes, that were associated with proliferation and differentiation and correlated with expression of the PME and AGP genes analyzed. Pharmacological treatments with catechin, an inhibitor of PME activity, and Yariv reagent, which blocks AGPs, impaired the progression of embryogenesis, with pectin de-esterification and an increase in AGP levels being necessary for embryo development. Findings indicate a role for pectins and AGPs during somatic embryogenesis of cork oak, promoting the cell wall remodeling during the process. They also provide new insights into the regulating mechanisms of somatic embryogenesis in woody species, for which information is still scarce, opening up new possibilities to improve in vitro embryo production in tree breeding.

Quercus suber L. is a forest tree with remarkable ecological, social and economic value in the southern Europe ecosystems. To circumvent the difficulties of breeding such long-lived species like Q. suber in a conventional fashion, clonal propagation of Q. suber elite trees can be carried out, although this process is sometimes unsuccessful. To help decipher the complex program underlying the development of Q. suber somatic embryos from the first early stage until maturity, in deep studies become necessary. This article is part of a Special Issue entitled: Translational Plant Proteomics.

The present study analyzes some effects of nano-CeO2 particles on the growth of in vitro plantlets of Medicago arborea when the nanoceria was added to the culture medium. Various concentrations of nano-CeO2 and bulk ceric oxide particles in suspension form were introduced to the agar culture medium to compare the effects of nanoceria versus ceric oxide bulk material. Germination rate and shoot dry weight were not affected by the addition of ceric oxide to the culture media. Furthermore, no effects were observed on chlorophyll content (single-photon avalanche diode (SPAD) measurements) due to the presence of either nano- or micro-CeO2 in the culture medium. When low concentrations of nanoceria were added to the medium, the number of trifoliate leaves and the root length increased but the root dry weight decreased. Also the values of maximum photochemical efficiency of PSII (F(v)/F m) showed a significant decrease. Dark-adapted minimum fluorescence (F 0) significantly increased in the presence of 200 mg L(-1) nanoceria and 400 mg L(-1) bulk material. Root tissues were more sensitive to nanoceria than were the shoots at lower concentrations of nanoceria. A stress effect was observed on M. arborea plantlets due to cerium uptake.

Quercus suber L., cork oak, is a forest tree of high social and economic value. The cork is traditionally used in the wine industry to produce bottle stoppers, but it is also a very good material for both thermal and acoustic insulation in construction. Since its harvest does not harm the tree, the use of cork in the industry has a positive impact on the environment.Somatic embryogenesis is considered a feasible system for in vitro regeneration procedures, with many advantages in woody species. Classical genetic breeding programs have important limitations in forest trees, like cork oak, due to their long life span and difficulties of seed conservation and vegetative reproduction. Therefore, somatic embryogenesis has a great potential for large-scale propagation and cryopreservation of elite genotypes, as well as for transformation strategies. In the case of Q. suber, several in vitro propagation systems through somatic embryogenesis have been reported, with different efficiency rates.In the present chapter, updated information is reported about an efficient protocol for induction of somatic embryogenesis of Q. suber from immature zygotic embryos, as well as methods for proliferation and maturation of somatic embryos, germination, plantlet regeneration, and acclimatization.

Grapevine (Vitis vinifera L.) was genetically modified with a construct containing a cDNA insert encoding the stilbene synthase gene (Vst1) from grapevine, under the control of the cauliflower mosaic virus 35S promoter in order to test the potential of over-production of resveratrol to protect plants from fungal attack. Southern blot hybridization and quantitative real-time PCR analysis demonstrated the presence and integration of one copy of exogenous DNA sequences in two grapevine-modified lines. Relative expression of the Vst1 gene in different modified lines was confirmed by using gene-specific quantitative real-time PCR. Compared to the control, the concentration of trans-resveratrol quantified by HPLC was up to 7.5 fold higher in the modified plants. The necrotic lesion size of leaves of intact modified plants inoculated by Botrytis cinerea B05.10 strain was consistently smaller and significantly different (p B 0.05) than in control plants, showing that modified grapevine plants were more resistant to the pathogen than the control plants.

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In a climate change scenario, Mediterranean forest species such as pines may be endangered by rising temperatures and reduced precipitation, thus calling for studies on the transpiration and water balance in pines. In this paper, the response of young plants of Pinus sylvestris L., Pinus pinea L. and Pinus halepensis Mill. to different irrigation treatments has been studied. Significant differences were found in water potential, sap flow, leaf-level gas exchange and spectral variables. P. sylvestris had higher pre-dawn and midday water potentials, sap flow rates and leaf-level gas exchange rates compared to the other two species in well-watered conditions. Vapor pressure gradient correlated with stomatal conductance, net assimilation and transpiration, but the association between stomatal conductance and sap flow was weak. The environmental variables more strongly associated with sap flow were solar radiation and reference evapo-transpiration, especially in the well-watered plants, but those associations were weaker in the stressed plants. All three pine species showed the isohydric, drought-avoiding strategy common in the genus Pinus, maintaining relatively high water potentials in dry conditions. Nevertheless, P. halepensis showed a water-saving strategy, with a stomatal closure behavior under drought. Stomatal regulation was less strict in P. sylvestris, closer to a water-spending pattern, while P. pinea showed an intermediate behavior. Significant differences were recorded among species in spectral reflectance in the visible and infra-red regions. Photochemical Reflectance Index, Normalized Difference Vegetation Index and combinations of other ratios permitted the discrimination among the three pine species. These spectral variables showed association with sap flow rate, water potential and leaflevel gas exchange variables. Both cluster analysis and k-means classification discriminated Scots pine and Aleppo pine in two different groups. On the other hand, Stone pine showed differences in spectral behavior depending on the hydric status of the plants. Well-watered Stone pine plants had the same spectral behavior as Scots pine, while the plants subjected to drought stress were closer to Aleppo pine plants in spectral response. These findings may help to quantify the impacts of early and mid-summer water deficit on Mediterranean pines in future climate regimes.