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12 p.-4 fig., Long-term in vitro maintenance of embryogenic lines of Pinus species has been associated with lower maturation capacity, because of this, cryopreservation protocols for the embryogenic lines are needed to maintain valuable genotypes. Since cryopreservation may induce epigenetic variations, we evaluate changes in DNA methylation levels through the course of the cryopreservation of maritime pine embryogenic lines, as compared to those lines maintained by repeated subcultures. Six maritime pine embryogenic lines were cryopreserved following a protocol that includes pre-treatments inducing osmotic stress in liquid media. The percentage of methylated cytosines (%5-mC) in total DNA was determined by using a colorimetric assay. Reactive oxygen species (ROS) accumulation in cell lines was also determined by quantifying dihydroethidium (DHE) fluorescence under a confocal laser-scanning microscope. In the first experiment, we found that global DNA methylation was significantly reduced during the cryopreservation protocol. Subsequently, we evaluated the methylation status of both cryopreserved and no cryopreserved lines (maintained by subcultures) and found differences among embryogenic lines but overall, cryopreservation did not alter %5-mC of the recovered lines while periodical subcultures increased methylation rates. In addition, maltose pretreatment did not increase significantly ROS production in embryogenic lines. Our results demonstrate that the genetic stability during cryopreservation highly depends on the embryogenic line studied, but the protocol allows maintaining methylation DNA rates in most of the recovered lines., This work was supported by the research project co-financed by MICINN and FEDER from the EU (AGL2013-47400-C4-04-R and AGL2016-76143-C4-01-R).

International audience; The advent of next-generation genome sequencing technologies has allowed approaching the sequencing and analysis of large and complex conifer genomes. Maritime pine (Pinus pinaster Ait.) is an economically and ecologically important conifer species widely distributed in South-West Europe, which shows a significant genetic and adaptive variability. This chapter takes on the task of reviewing the insights into the maritime pine genome sequencing breakthrough and its impact on downstream analysis. Maritime pine genome sequencing and assembly approaches are described along with the impact of related tools. A section of the state-of-the-art research on comparative, functional, structural, and translational genomics aimed at dissecting the genetic basis and the specific regulation of biological processes underlying the expression of traits of interest in maritime pine and other conifers is also described. Perspectives about the impact of these tools as well as additional research approaches are discussed.

Libro sobre buenas prácticas en universidades FORTHEM en 5 áreas clave: internacionalización, ciencia abierta, co-creación con el ecosistema social y empresarial, y comunicación de la ciencia. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 101017248. Book on existing good practices developed in FORTHEM universities regarding the key research areas identified - on the topics: internationalisation, open science, co-creation with the social and business ecosystem, and communication of science

Resumen: Objetivo: Identificar el perfil sociodemográfico del usuario de la homeopatía en España. Diseño: Estudio cuantitativo. Emplazamiento: España. Corpus: Base de datos del Centro de Investigaciones Sociológicas (Estudio 3205, febrero de 2018), con un total de 2.486 entrevistas, y se analiza una submuestra (n = 124), que agrupa a todos los ciudadanos que afirman que han usado la homeopatía en España en los 12 meses anteriores. Mediciones principales: Se utilizaron los porcentajes, las medias y/o la desviación estándar de las medias, así como la significación de los cambios en las distintas variables analizadas entre la población en general y los usuarios específicos de la homeopatía en España. Esto se determinó mediante el análisis de la varianza o prueba χ2 de Pearson, dependiendo de la naturaleza de la variable en el estudio. Resultados: El perfil tipo del usuario de la homeopatía en España es una mujer, de clase media/alta, con estudios superiores universitarios y con una ideología política progresista. Conclusiones: El perfil trazado es similar al de otros estudios internacionales. Abstract: Objective: To identify the sociodemographic profile of the homeopathy user in Spain. Design: Quantitative study. Location: Spain. Corpus: The Centre for Sociological Research database (Study 3205, February 2018), with a total of 2,486 interviews, and the sub-sample that includes all patients who have claimed to use homeopathy in Spain in the last 12 months (n = 124). Main measurements: The percentages, means and/or standard deviation of the factors were calculated, as well as the significance of the changes in the different variables analysed in the general population, and the specific users of homeopathy in Spain. This was determined by the variance analysis or Pearson's χ2 test, depending on the nature of the variable in the study. Results: The homeopathy user-type profile in Spain is a woman, of middle/high social status, with higher university studies, and with a progressive political ideology. Conclusions: The Spanish profile is similar to that in other international studies. Palabras clave: Homeopatía, Terapias complementarias, Usuario, Encuesta, Keywords: Homeopathy, Complementary therapies, User, Survey

Objective: To review the hoaxes’ characteristics spread through WhatsApp in Spain during COVID-19 lockdown and identify what kind of substances were promoted for consumption or application. Method: A phone number was activated to receive hoaxes via WhatsApp. A total of 2353 messages were collected, and among those 584 different hoaxes were identified and validated, between March 18 and April 18, 2020. From these 584 hoaxes, a sub-sample of 126 was selected, exclusively related to the object of study, and a content analysis table with fourteen registration fields was applied. Besides, the averages and medians of the quantitative fields were extracted. Results: Most of the messages received were texts (39%) and videos (30%). In the majority, the acting subject was presented as medical personnel (36.5%) or as an anonymous person (30.9%). The videos lasted between 2’ 40” and 18’ 18”, while the audios ranged between 1’ 35” and 7’ 48”. Regarding the gender of the informant, there was a significant difference, with 45.2% being male, and 13.5%, female and 41.3% non-identifiable. Conclusions: One out of five false health claims received was about prevention or cure of the coronavirus, based on the principle of medical authority, and basically promoting the use and consumption of substances of natural origin. Resumen: Objetivo: Examinar las características de los bulos difundidos por WhatsApp en España durante el confinamiento por la pandemia de COVID-19 e identificar qué tipo de sustancias se promovieron para su ingesta o uso. Método: Se habilitó un número de teléfono para recibir bulos por WhatsApp. Se recibieron 2353 mensajes, de los que se identificaron y validaron 584 bulos distintos, entre el 18 de marzo y el 18 de abril de 2020. De los 584 bulos, se seleccionó una submuestra de 126 relacionados con el objeto de estudio y se aplicó una ficha de análisis de contenido con 14 campos de registro. Además, se extrajeron las medias y medianas de los campos cuantitativos. Resultados: La mayor parte de los mensajes fueron cadenas de texto (39%) y vídeos (30%). En la mayoría, el sujeto se presentaba como personal médico (36,5%) o como persona anónima (30,9%). Los vídeos tuvieron una duración de entre 2’ 40” y 18’ 18”, mientras que los audios fueron de entre 1’ 35” y 7’ 48”. Con respecto al sexo, se apreció una gran diferencia: el 45,2% de los protagonistas fueron hombres, frente al 13,5% de mujeres; el 41,3% no era identificable. Conclusiones: Uno de cada cinco bulos recibidos fue sobre prevención o curación del coronavirus, argumentados sobre el principio de autoridad médica y básicamente promoviendo el uso y consumo de sustancias de origen natural.

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 synthases to produce changes in the qualitative profile of particular monoterpenes.

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 induction, proliferation, cryopreservation, maturation, germination, plant formation, and acclimatization are described.

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 could recover the phenotype of MEV-treated plants, including the normal growth and development of roots, and could partially restore the biosynthesis of photosynthetic pigments and, to a lesser extent, of the essential oils in plantlets treated with FSM. Spike lavender shoot apices were also used in 13 C-labeling experiments, where the plantlets were grown in the presence of [U– 13 C 6 ]glucose. GC-MS-analysis of 1,8-cineole and camphor indicated that the C 5 -precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) of both monoterpenes are predominantly biosynthesized via the methylerythritol phosphate (MEP) pathway. However, on the basis of the isotopologue profiles, a minor contribution of the MVA pathway was evident that was increased in transgenic spike lavender plants overexpressing the 3-hydroxy-3-methylglutaryl CoA reductase (HMGR), the first enzyme of the MVA pathway. Together, these findings provide evidence for a transport of MVA-derived precursors from the cytosol to the plastids in leaves of 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 constitutively expressing the Arabidopsis thaliana DXR gene. Although two out of the seven transgenic T0 plants analyzed accumulated more essential oils than the controls, this is hardly imputable to the DXR transgene effect since a clear correlation between transcript accumulation and monoterpene production could not be established. Furthermore, these increased essential oil phenotypes were not maintained in their respective T1 progenies. Similar results were obtained when total chlorophyll and carotenoid content in both T0 transgenic plants and their progenies were analyzed. Our results then demonstrate that DXR enzyme does not play a crucial role in the synthesis of plastidial monoterpene precursors, suggesting that the control flux of the MEP pathway in spike lavender is primarily exerted by the DXS enzyme, This work has been funded by the Regional Government of Valencia (PROMETEO/2009/075, Generalitat Valenciana); the Spanish Ministerio de Educacion y Cultura, FPU fellowship to I. M-P (AP2007/04606); and Fundacion Ramon Areces, fellowship to I. M-P.

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 DXS (1-deoxy-d-xylulose-5-P synthase), coding for the first enzyme of the methyl-d-erythritol-4-phosphate pathway, and LIS genes. Both essential oil yield and linalool content in double DXS-LIS transgenic plants were lower than that of their parentals, which could be due to co-suppression effects linked to the structures of the constructs used.

[EN] The potential of haploid tissues for genetic studies in conifers is hampered by the lack of abundant and homogeneous plant material suitable for DNA isolation. In this work we have determined factors promoting haploid callus induction and proliferation from megagametophytes of Oria 6, a genotype of Pinus pinaster Aiton (maritime pine) from the natural population Sierra de Oria (Almeria, Spain), selected based on its response to extreme drought conditions. The generation of haploid callus was restricted to megagametophytes isolated from light-brown cones with no dehydrated seeds collected in September. Culture medium composition did not significantly affect callus induction, but a modified Murashige and Skoog medium with 2,4-dichlorophenoxyacetic acid and 6-benzyladenine favored further multiplication. The ploidy status of the callus lines was determined by flow cytometry and seven polymorphic microsatellites. A total of sixteen haploid callus lines were established and one of these is being used as a source of DNA for massive sequencing of maritime pine genome., Research funds were provided by Spanish National projects (BIO2007-29814 and BIO2010-12302-E) and Generalitat Valenciana (Prometeo/2009/075). The research leading to these results has also received funding from the European Union’s Seventh Framework Programme (FP7/2007–2013) under Grant Agreement No. 289841 (ProCoGen). We thank M. A. Morcillo for technical assistance, Dr. Jose´ Juarez (Instituto Valenciano de Investigaciones Agrarias, IVIA, Spain) and Dr. Vesselina Nikolova (Vegetable Crops Research Institute, Plovdiv, Bulgaria) for their support in the flow cytometry and karyological analyses, respectively.

Reverse genetics, defined as ectopic candidate gene expression or silencing, has become an indispensable tool for functional dissection of traits of interest in forest trees. In maritime pine as in other conifers, long generation time and long life span, high genetic loads as well as high genetic redundancy are major obstacles to perform standard genetic approaches including association genetics. Validating marker associations with specific properties before transferring into breeding selection models is still challenging. Stable Agrobacterium-mediated genetic transformation of maritime pine was first reported by Trontin et al. (2002) and developed in both France (FCBA, INRA) and Portugal (IBET) with sufficient refinement (reviewed in Trontin et al. 2007) to envisage practical application in reverse genetics as an attractive alternative to association studies. As a result, the technology has been recently implemented in French (GenoQB, 2006-2009) and transnational (Sustainpine, 2010-2013) or European initiatives (ProCoGen 2012-2016). Somatic embryogenesis (SE) was revealed as a key tissue culture system for achieving genetic transformation, easy cryopreservation of transgenic tissue and efficient transgenic plant regeneration in maritime pine. Much consideration is given in France, Portugal and Spain to apply SE as a clonal propagation system for maritime pine improvement and deployment strategies in multivarietal forestry (MVF, Park YS, reviewed in Klimaszewska et al. 2007). By using this technology it is expected that we will obtain greater genetic gain, high flexibility in deployment of tested embryogenic varieties and easy balancing of genetic gain and diversity in plantations. Both SE and Agrobacterium-mediated transformation methods developed at FCBA, INRA or IBET are being successfully transferred to different partners through running the Sustainpine project (http://www.scbi.uma.es/sustainpine/).

La ciencia ciutadana forma part d’una tendencia cientifica i social mes amplia que promou el dialeg bidireccional i la implicacio dels cientifics i el public a forca d’involucrar la ciutadania en el proces d’investigacio. Aquest article examina els esforcos de CONCISE (un projecte internacional en el qual participen Espanya, Italia, Portugal, Polonia i Eslovaquia) per a comprendre, mitjancant consultes publiques, com adquireix i utilitza la informacio cientifica la ciutadania. Les consultes, celebrades en 2019, van incloure prop de 500 persones. CONCISE intenta posar la participacio de la ciutadania en el centre del proces d’investigacio demanant-li suggeriments sobre com millorar la comunicacio cientifica i implicant-la en el treball de divulgacio.