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24 p.-7 fig., Epigenetic modifications play a vital role in the preservation of genome integrity and in the regulation of gene expression. DNA methylation, one of the key mechanisms of epigenetic control, impacts growth, development, stress response and adaptability of all organisms, including plants. The detection of DNA methylation marks is crucial for understanding the mechanisms underlying these processes and for developing strategies to improve productivity and stress resistance of crop plants. There are different methods for detecting plant DNA methylation, such as bisulfite sequencing, methylation-sensitive amplified polymorphism, genome-wide DNA methylation analysis, methylated DNA immunoprecipitation sequencing, reduced representation bisulfite sequencing, MS and immuno-based techniques. These profiling approaches vary in many aspects, including DNA input, resolution, genomic region coverage, and bioinformatics analysis. Selecting an appropriate methylation screening approach requires an understanding of all these techniques. This review provides an overview of DNA methylation profiling methods in crop plants, along with comparisons of the efficacy of these techniques between model and crop plants. The strengths and limitations of each methodological approach are outlined, and the importance of considering both technical and biological factors are highlighted. Additionally, methods for modulating DNA methylation in model and crop species are presented. Overall, this review will assist scientists in making informed decisions when selecting an appropriate DNA methylation profiling method., This publication is supported by COST (European Cooperation in Science and Technology) through grant CA19125 awarded to the COST Action 'Epigenetic mechanisms of crop adaptation to climate change' (EPI-CATCH), grant number CA19125 (https://www.epicatch.eu)".

14 p.-7 fig. Microspore embryogenesis is a biotechnological process that allows to rapidly obtain doubled haploid plants for breeding programs. The process is initiated by the application of stress treatment which reprograms microspores to embark on embryonic development. Typically, a part of the microspores undergoes cell death that reduces the efficiency of the process. Metacaspases (MCAs), a phylogenetically broad group of cysteine proteases, and autophagy, the major catabolic process in eukaryotes, are critical regulators of the balance between cell death and survival in various organisms. In this study we analyzed the role of MCAs and autophagy in cell death during stress-induced microspore embryogenesis in Brassica napus. We demonstrate that this cell death is accompanied by transcriptional upregulation of three BnMCA genes (BnMCA-Ia, BnMCA-IIa and BnMCA-IIi), increase in MCA proteolytic activity, and activation of autophagy. Accordingly, inhibition of autophagy and MCA activity, either individually or in combination, suppressed cell death and increased the number of proembryos, indicating that both components play a pro-cell death role and account for decreased efficiency of early embryonic development. Therefore, MCAs and/or autophagy can be used as new biotechnological targets to improve in vitro embryogenesis in Brassica species and doubled-haploid plant production in crop breeding and propagation programmes. The work was supported by the Spanish National Agency of Research (Agencia Estatal de Investigación, AEI) and European Regional Development Fund (ERDF/FEDER) [grant AGL2017- 82447-R to PST], and by the Swedish Foundation for Strategic Research and the research program “Crops for the future” [grants to PVB].

[EN] The present invention relates to the use of mammal kinase inhibitors, preferably human kinase inhibitors, to promote the induction of in vitro embryogenesis, a strategy never used in plants systems before. The results obtained indicated that these inhibitors have beneficial effects in both crop and forest plants in in vitro systems of microspore and somatic embryogenesis, [FR] La présente invention concerne l'utilisation d'inhibiteurs de kinase de mammifère, de préférence des inhibiteurs de kinase humaine, pour favoriser l'induction de l'embryogenèse in vitro, une stratégie jamais utilisée dans les systèmes de plantes avant. Les résultats obtenus indiquent que ces inhibiteurs ont des effets bénéfiques dans les plantes cultivées et forestières dans des systèmes in vitro de microspore et d'embryogenèse somatique, Consejo Superior de Investigaciones Científicas (España), A1 Solicitud de patente con informe sobre el estado de la técnica

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En el marco del proyecto SEFEAL-2, liderado por TRAGSA, se aplican protocolos de embriogénesis somática (ES) para desarrollar variedades de alcornoque de alta calidad y productividad. Al mismo tiempo se mejora la técnica de ES para abaratar los costes y permitir su aplicación a escala comercial. En el alcornoque, como en otras especies, el desarrollo comercial de la embriogénesis como técnica de multiplicación masiva se basa en el uso de biorreactores y medios líquidos agitados. El diseño del biorreactor, su sistema de cierre y el nivel de agitación determinan el grado de mezclado, el estrés hidrodinámico y el intercambio gaseoso, y por ello afectan tanto al crecimiento como al desarrollo de los cultivos embriogénicos. Mediante un ensayo factorial se testaron 3 tipos de envase y tres niveles de agitación. Los efectos sobre el intercambio gaseoso se estimaron a través de la tasa de transferencia de O2 (OTR) y su coeficiente volumétrico de transferencia de masa (KLa), y los efectos sobre el nivel de mezclado mediante el “shear force index” (SFI), un indicador de estrés hidrodinámico. El tipo de envase afectó básicamente al número total de agregados embriogénicos y a la frecuencia de formación de los agregados de mayor tamaño. El nivel de agitación tuvo mayores efectos que el tipo de envase tanto sobre el número como sobre el tamaño de los agregados. Para las condiciones ensayadas, que dieron lugar a valores de KLa comprendidos entre 0,11 h-1 y 1,47 h-1, la disponibilidad de oxígeno no pareció limitante. En cualquier caso, los efectos del tipo de envase y del nivel de agitación sobre los procesos de crecimiento y desarrollo de los materiales embriogénicos de alcornoque fueron complejos resultando muy significativa la interacción tipo de envase por nivel de agitación.

The development of reliable clonal propagation technologies is a requisite for performing Multi-Varietal Forestry (MVF). Somatic embryogenesis is considered the tissue culture based method more suitable for operational breeding of forest trees. Vegetative propagation is very difficult when tissues are taken from mature donors, making clonal propagation of selected trees almost impossible. We have been able to induce somatic embryogenesis in leaves taken from mature oak trees, including cork oak (Quercus suber). This important species of the Mediterranean ecosystem produces cork regularly, conferring to this species a significant economic value. In a previous paper we reported the establishment of a field trial to compare the growth of plants of somatic origin vs zygotic origin, and somatic plants from mature trees vs somatic plants from juvenile seedlings. For that purpose somatic seedlings were regenerated from five selected cork oak trees and from young plants of their half-sib progenies by somatic embryogenesis. They were planted in the field together with acorn-derived plants of the same families. After the first growth period, seedlings of zygotic origin doubled the height of somatic seedlings, showing somatic plants of adult and juvenile origin similar growth. Here we provide data on height and diameter increases after two additional growth periods. In the second one, growth parameters of zygotic seedlings were also significantly higher than those of somatic ones, but there were not significant differences in height increase between seedlings and somatic plants of mature origin. In the third growth period, height and diameter increases of somatic seedlings cloned from the selected trees did not differ from those of zygotic seedlings, which were still higher than data from plants obtained from somatic embryos from the sexual progeny. Therefore, somatic seedlings from mature origin seem not to be influenced by a possible ageing effect, and plants from somatic embryos tend to minimize the initial advantage of plants from acorns

Among the several applications of in vitro tissue culture techniques, the conservation of plant germplasm is one of the most widely used. The cork oak is one of the principal tree species in the Western Mediterranean región. Within this área, the Balearic Islands are considered to be a glacial refuge, and therefore a reservoir of genetic resources. A singular tree has been found in the small Minorca Island population. The haplotype of this tree is of Tyrrhenian origin, showing a past link between Minorca and Sardinia. Moreover, this tree do not bear a deletion within an ITS from ribosimic nuclear DNA, which is fairly common in many populations of this species, and indicates that ir may be the descendant of a very ancient population. This tree is currently in a precarious condition, and it has not produced acorns in the last years. Hence there is a clear need of vegetative propagation to conserve this genotype. We have previously developed methods to clone adult cork oak tres by somatic embryogenesis, and therefore the aim of the present work was to clone this singular tree. There braches from the corwn were collected in November 2004, and methods previously described were carried out. By February 2005 somatic embryogenesis was obtained from leaves of the tree with percentages on induction ranging from 17 to 54% depending on the branch, which may show a novel source of variation that requires further study. Spontaneously matured somatic embryos germinated at 46% in average, and the first somatic seedlings from the Alfavaret's cork oak tree were obtained. Therefore, this study shows one of the most relevant applications of somatic embryogenesis: the plant regeneration of valuable genotypes for the in situ and ex situ conservation of forest genetic resources.

La mejora y conservación de recursos genéticos en especies forestales lleva siglos de retraso con respecto a las especies agrícolas. Los recursos forestales se han considerado tradicionalmente como recursos �mineros�, en los que primaba la mera extracción dejando exclusivamente a la regeneración natural la labor de sostenibilidad en los montes y dehesas o montados. Hoy en día, el necesario desarrollo del medio rural obliga a la explotación racional de los recursos como medio de garantizar su sostenibilidad. Por ello se está empezando a extender el criterio de que las especies forestales se pueden y deben �cultivar� en determinados espacios. Las características biológicas de las especies forestales las hacen, a menudo, recalcitrantes a las técnicas de mejora y conservación de recursos genéticos tradicionalmente aplicadas a especies agrícolas. En particular, la propagación vegetativa se ha utilizado ampliamente en muchos cultivos leñosos como una herramienta muy poderosa para capturar todo el potencial genético de combinaciones genéticas valiosas. En especies forestales, en particular en las mediterráneas, esta posibilidad raramente se ha podido aplicar debido a la baja capacidad morfogénica de estas especies y la fuerte influencia de la maduración o cambio de fase. En los últimos años la biotecnología forestal ha tenido un desarrollo espectacular. En particular las técnicas de regeneración clonal de plantas basadas en técnicas de cultivo in vitro, fundamentalmente vía embriogénesis somática, se están ya aplicando por muchas empresas privadas e instituciones públicas a nivel semi-operativo con diversas especies, para la conservación de material selecto y el establecimiento de ensayos clonales. Nuestros grupos de trabajo están desarrollando protocolos de regeneración por embriogénesis somática en distintas especies forestales. En esta comunicación se presenta el estado actual de los conocimientos en dos especies típicamente mediterráneas, el alcornoque (Quercus suber L.) y el pino piñonero (Pinus pinea L.), destacando los principales cuellos de botella para su aplicación a gran escala.

18 p.-10 fig., Plant in vitro regeneration systems, like somatic embryogenesis, are essential in breeding; they permit to propagate elite genotypes, to produce doubled-haploids, and to convert gene editing or transformation events into plants. However, in many crop and forest species somatic embryogenesis is highly inefficient. We report a new strategy to improve in vitro embryogenesis using synthetic small molecule inhibitors of mammalian glycogen synthase kinase 3β (GSK-3β), never used in plants. These inhibitors increased in vitro embryo production in three different systems and species, microspore embryogenesis of Brassica napus and Hordeum vulgare, and somatic embryogenesis of Quercus suber. TDZD-8, representative compound of the molecules tested, inhibited GSK-3 activity in microspore cultures, and increased expression of embryogenesis- genes FUS3, LEC2 and AGL15. Plant GSK-3 kinase BIN2 is master regulator of brassinosteroid (BR) signalling. During microspore embryogenesis, BR biosynthesis and signalling genes CPD, GSK-3-BIN2, BES1 and BZR1 were upregulated and BAS1 catabolic gene was repressed, indicating activation of BR pathway. TDZD-8 increased expression of BR signalling elements, mimicking BR effects. The findings support that the small molecule inhibitors promoted somatic embryogenesis by activating the BR pathway, opening the way for new strategies using GSK-3β inhibitors that could be extended to other species., Work supported by the Spanish National Agency of Research (Agencia Estatal de Investigación, AEI) and European Regional Development Fund (ERDF/FEDER) [grants AGL2017-82447-R and PID2020-113018RB-I00 to PST; and SAF2016-76693-R and PID2019-105600RB-I00 to AM]

Some plant cells are able to rebuild new organs after tissue damage or in response to definite stress treatments and/or exogenous hormone applications. Whole plants can develop through de novo organogenesis or somatic embryogenesis. Recent findings have enlarged our understanding of the molecular and cellular mechanisms required for tissue reprogramming during plant regeneration. Genetic analyses also suggest the key role of epigenetic regulation during de novo plant organogenesis. A deeper understanding of plant regeneration might help us to enhance tissue culture optimization, with multiple applications in plant micropropagation and green biotechnology. In this review, we will provide additional insights into the physiological and molecular framework of plant regeneration, including both direct and indirect de novo organ formation and somatic embryogenesis, and we will discuss the key role of intrinsic and extrinsic constraints for cell reprogramming during plant regeneration., This research was funded by the Ministerio de Economía, Industria y Competitividad (MINECO)of Spain (grant numbers BIO2015-64255-R and RTI2018-096505-B-I00 to JMP-P, and AGL2017-82447-R to PST),the Conselleria d’Educació, Cultura i Sport of the Generalitat Valenciana (grant numbers IDIFEDER 2018/016and PROMETEO/2019/117 to JMP-P), and the European Regional Development Fund (ERDF) of the EuropeanCommission. SI is a research fellow of the Generalitat Valenciana (grant number ACIF/2018/220).

21 p.-5 fig., Microspore embryogenesis is a powerful biotechnological tool that is very useful in crop breeding for the rapid production of haploid and double-haploid embryos and plants. In this in vitro system, the haploid microspore is reprogrammed by the application of specific stress treatments. A high level of cell death after the stress is a major factor that greatly reduces embryogenesis yield at its initial stages. Autophagy is a degradation pathway that is present in all eukaryotes and plays key roles in a range of processes, including stress responses. Many proteases participate in autophagy and cell death; among them, cathepsins are the most abundant enzymes with a role in plant senescence and programmed cell death (PCD). Moreover, although plant genomes do not contain homologues of caspases, caspase 3-like activity (main executioner protease of animal cell death) has been detected in many plant PCD processes. Recent studies by our group in barley microspore cultures reported that the stress treatment required for inducing microspore embryogenesis (cold treatment), also produced reactive oxygen species (ROS) and cell death, concomitantly with the induction of autophagy, as well as cathepsin-like and caspase 3-like proteolytic activities. In the present study, we report new data on microspore embryogenesis of rapeseed that indicate, as in barley, activation of cell death and autophagy processes after the inductive stress. The results revealed that treatments modulating autophagy and proteases produced the same effect in the two plant systems, regardless of the stress applied, cold in barley or heat in rapeseed. Pharmacological treatments with small bioactive compounds that inhibit ROS, autophagy and specific cell death-proteases led to reduced cell death and an increased embryogenesis initiation rate in both, barley and rapeseed. Taken together, these findings open up new intervention pathways by modulating autophagy and proteases, which are very promising in terms of increasing the efficiency of in vitro microspore embryogenesis systems for biotechnological applications in crop breeding., Work supported by projects (AGL2014-52028-R and AGL2017-82447-R) funded by Spanish National Research Agency (AEI), Ministry of Economy and Competitiveness (MINECO) and European Regional Development Fund (ERDF/FEDER). Thanks are due to COST action TRANSAUTOPHAGY (CA15138), European Network of Multidisciplinary Research and Translation of Autophagy Knowledge. YPP was the recipient of a grant (PEJ15/BIO/AI-01S8) funded by Comunidad de Madrid and European Commission through ERDF/FEDER.

14 p.-7 fig., Microspore embryogenesis is a biotechnological process that allows to rapidly obtain doubled haploid plants for breeding programs. The process is initiated by the application of stress treatment which reprograms microspores to embark on embryonic development. Typically, a part of the microspores undergoes cell death that reduces the efficiency of the process. Metacaspases (MCAs), a phylogenetically broad group of cysteine proteases, and autophagy, the major catabolic process in eukaryotes, are critical regulators of the balance between cell death and survival in various organisms. In this study we analyzed the role of MCAs and autophagy in cell death during stress-induced microspore embryogenesis in Brassica napus. We demonstrate that this cell death is accompanied by transcriptional upregulation of three BnMCA genes (BnMCA-Ia, BnMCA-IIa and BnMCA-IIi), increase in MCA proteolytic activity, and activation of autophagy. Accordingly, inhibition of autophagy and MCA activity, either individually or in combination, suppressed cell death and increased the number of proembryos, indicating that both components play a pro-cell death role and account for decreased efficiency of early embryonic development. Therefore, MCAs and/or autophagy can be used as new biotechnological targets to improve in vitro embryogenesis in Brassica species and doubled-haploid plant production in crop breeding and propagation programmes., The work was supported by the Spanish National Agency of Research (Agencia Estatal de Investigación, AEI) and European Regional Development Fund (ERDF/FEDER) [grant AGL2017- 82447-R to PST], and by the Swedish Foundation for Strategic Research and the research program “Crops for the future” [grants to PVB].